U.S. patent application number 11/019711 was filed with the patent office on 2006-01-12 for proteins and nucleic acids encoding same.
This patent application is currently assigned to Ramesh Kekuda. Invention is credited to John P. II Alsobrook, David W. Anderson, Ferenc L. Boldog, Catherine E. Burgess, Shlomit R. Edinger, Andrew Eisen, Karen Ellerman, Linda Gorman, William M. Grosse, Xiaojia (Sasha) Guo, Ramesh Kekuda, Denise M. Lepley, Li Li, Xiaohong Liu, Uriel M. Malyankar, Charles E. Miller, Muralidhara Padigaru, Meera Patturajan, Mark E. Rothenberg, Paul Sciore, Suresh G. Shenoy, Kimberly A. Spytek, David J. Stone, Raymond J. JR. Taupier, Velizar T. Tchernev, Corine A.M. Vernet.
Application Number | 20060009634 11/019711 |
Document ID | / |
Family ID | 31999943 |
Filed Date | 2006-01-12 |
United States Patent
Application |
20060009634 |
Kind Code |
A1 |
Kekuda; Ramesh ; et
al. |
January 12, 2006 |
Proteins and nucleic acids encoding same
Abstract
Disclosed herein are nucleic acid sequences that encode novel
polypeptides. Also disclosed are polypeptides encoded by these
nucleic acid sequences, and antibodies, which
immunospecifically-bind to the polypeptide, as well as derivatives,
variants, mutants, or fragments of the aforementioned polypeptide,
polynucleotide, or antibody. The invention further discloses
therapeutic, diagnostic and research methods for diagnosis,
treatment, and prevention of disorders involving any one of these
novel human nucleic acids and proteins.
Inventors: |
Kekuda; Ramesh; (Danbury,
CT) ; Alsobrook; John P. II; (Madison, CT) ;
Tchernev; Velizar T.; (Branford, CT) ; Liu;
Xiaohong; (Branford, CT) ; Spytek; Kimberly A.;
(New Haven, CT) ; Patturajan; Meera; (Branford,
CT) ; Grosse; William M.; (Branford, CT) ;
Lepley; Denise M.; (Branford, CT) ; Burgess;
Catherine E.; (Wethersfield, CT) ; Vernet; Corine
A.M.; (Branford, CT) ; Li; Li; (Branford,
CT) ; Gorman; Linda; (Branford, CT) ; Edinger;
Shlomit R.; (New Haven, CT) ; Sciore; Paul;
(North Haven, CT) ; Ellerman; Karen; (Branford,
CT) ; Malyankar; Uriel M.; (Branford, CT) ;
Rothenberg; Mark E.; (Clinton, CT) ; Stone; David
J.; (Guilford, CT) ; Boldog; Ferenc L.; (North
Haven, CT) ; Guo; Xiaojia (Sasha); (Branford, CT)
; Shenoy; Suresh G.; (Branford, CT) ; Anderson;
David W.; (Branford, CT) ; Padigaru; Muralidhara;
(Branford, CT) ; Taupier; Raymond J. JR.; (East
Haven, CT) ; Miller; Charles E.; (Guilford, CT)
; Eisen; Andrew; (Rockville, MD) |
Correspondence
Address: |
Jenell Lawson;Intellectual Property
CuraGen Corporation
555 Long Wharf Drive
New Haven
CT
06551
US
|
Assignee: |
Kekuda; Ramesh
Alsobrook; John II
Tchernev; Velizar
Liu; Xiaohong
Spytek; Kimberly
Patturajan; Meera
Grosse; William
Lepley; Denise
Burgess; Catherine
Vernet; Corine
Li; Li
Gorman; Linda
Edinger; Shlomit
Sciore; Paul
Ellerman; Karen
Malyankar; Uriel
Rothenberg; Mark
Stone; David
Boldog; Ferenc
Guo; Xiaojia
Shenoy; Suresh
Anderson; David
Padigaru; Muralidhara
Taupier; Raymond
Miller; Charles
Eisen; Andrew
|
Family ID: |
31999943 |
Appl. No.: |
11/019711 |
Filed: |
December 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10037417 |
Jan 4, 2002 |
6903201 |
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11019711 |
Dec 21, 2004 |
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60260018 |
Jan 5, 2001 |
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60260360 |
Jan 8, 2001 |
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60272411 |
Feb 28, 2001 |
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60272817 |
Mar 2, 2001 |
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60291186 |
May 15, 2001 |
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60318700 |
Sep 12, 2001 |
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60318405 |
Sep 10, 2001 |
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60305060 |
Jul 12, 2001 |
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60303231 |
Jul 5, 2001 |
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Current U.S.
Class: |
536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
536/023.5 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Claims
1-49. (canceled)
50. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) SEQ ID NO: 46; (b) a
mature form of the amino acid sequence of SEQ ID NO: 46; (c) a
variant of the amino acid sequence of SEQ ID NO: 46, wherein one or
more amino acid residues in said variant differ from SEQ ID NO: 46,
provided that said variant differs in no more than 15% of amino
acid residues from SEQ ID NO: 46; (d) a variant of the amino acid
sequence of SEQ ID NO: 46, wherein the amino acid sequence of said
variant comprises one or more conservative amino acid substitutions
to SEQ ID NO: 46.
51. The isolated polypeptide of claim 50 comprising an amino acid
sequence of SEQ ID NO: 46.
52. The isolated polypeptide of claim 50 comprising a mature form
of the amino acid sequence of SEQ ID NO: 46.
53. The isolated polypeptide of claim 50 comprising a variant of
the amino acid sequence of SEQ ID NO: 46, wherein the amino acid
sequence of said variant comprises one or more conservative amino
acid substitutions to SEQ ID NO: 46.
54. a pharmaceutical composition comprising the polypeptide of
claim 1 and a pharmaceutically acceptable carrier.
55. An antibody that binds immunospecifically to the polypeptide of
claim 1.
56. The antibody of claim 55, wherein said antibody is a monoclonal
antibody.
57. The antibody of claim 55, wherein said antibody is a humanized
antibody.
58. A pharmaceutical composition comprising the antibody of claim
55 and a pharmaceutically acceptable carrier.
59. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding a polypeptide selected from the group consisting
of: (a) a polypeptide of SEQ ID NO: 2, 6, or 36; (b) a mature form
of a polypeptide of SEQ ID NO: 2, 6, or 36; (b) a polypeptide
consisting of an amino acid sequence having one or more
conservative amino acid substitutions to SEQ ID NO: 2, 6, or 36. or
a complement of said nucleotide sequence.
60. The isolated nucleic acid molecule of claim 59 comprising
nucleotides 1-1083 of SEQ ID NO: 1.
61. The isolated nucleic acid molecule of claim 59 comprising
nucleotides 1-10794 of SEQ ID NO: 5.
62. The isolated nucleic acid molecule of claim 59 comprising
nucleotides 141-4580 of SEQ ID NO: 35.
63. A vector comprising the nucleic acid molecule of claim 59.
64. The vector of claim 63 further comprising a promoter
operably-linked to said nucleic acid molecule.
65. A cell comprising the vector of claim 63.
66. A pharmaceutical composition comprising the nucleic acid
molecule of claim 59, and a pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application claims priority from U.S. Ser. No.
60/260,018, filed Jan. 5, 2001; U.S. Ser. No. 60/260,360, filed
Jan. 8, 2001; U.S. Ser. No. 60/272,411 filed Feb. 28, 2001; U.S.
Ser. No. 60/272,817 filed Mar. 2, 2001; U.S. Ser. No. 60/291,186,
filed May 15, 2001; U.S. Ser. No. 60/303,231, filed Jul. 5, 2001;
U.S. Ser. No. 60/305,060 filed Jul. 12, 2001; U.S. Ser. No.
60/318,405, filed Sep. 10, 2001; U.S. Ser. No. 60/318,700 filed
Sep. 12, 2001; each of which is incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The invention generally relates to nucleic acids and
polypeptides encoded thereby.
BACKGROUND OF THE INVENTION
[0003] The invention generally relates to nucleic acids and
polypeptides encoded therefrom. More specifically, the invention
relates to nucleic acids encoding cytoplasmic, nuclear, membrane
bound, and secreted polypeptides, as well as vectors, host cells,
antibodies, and recombinant methods for producing these nucleic
acids and polypeptides.
SUMMARY OF THE INVENTION
[0004] The invention is based in part upon the discovery of nucleic
acid sequences encoding novel polypeptides. The novel nucleic acids
and polypeptides are referred to herein as NOVX, or NOV1, NOV2,
NOV3, NOV4, NOV5, NOV6, NOV7, NOV8, NOV9, NOV10, NOV11, NOV12,
NOV13, and NOV14 nucleic acids and polypeptides. These nucleic
acids and polypeptides, as well as derivatives, homologs, analogs
and fragments thereof, will hereinafter be collectively designated
as "NOVX" nucleic acid or polypeptide sequences.
[0005] In one aspect, the invention provides an isolated NOVX
nucleic acid molecule encoding a NOVX polypeptide that includes a
nucleic acid sequence that has identity to the nucleic acids
disclosed in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197. In some
embodiments, the NOVX nucleic acid molecule will hybridize under
stringent conditions to a nucleic acid sequence complementary to a
nucleic acid molecule that includes a protein-coding sequence of a
NOVX nucleic acid sequence. The invention also includes an isolated
nucleic acid that encodes a NOVX polypeptide, or a fragment,
homolog, analog or derivative thereof. For example, the nucleic
acid can encode a polypeptide at least 80% identical to a
polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28,
40, 42, 44, 46, and 198. The nucleic acid can be, for example, a
genomic DNA fragment or a cDNA molecule that includes the nucleic
acid sequence of any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and
197.
[0006] Also included in the invention is an oligonucleotide, e.g.,
an oligonucleotide which includes at least 6 contiguous nucleotides
of a NOVX nucleic acid (e.g., SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and
197) or a complement of said oligonucleotide.
[0007] Also included in the invention are substantially purified
NOVX polypeptides (SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, and 198). In
certain embodiments, the NOVX polypeptides include an amino acid
sequence that is substantially identical to the amino acid sequence
of a human NOVX polypeptide.
[0008] The invention also features antibodies that
immunoselectively bind to NOVX polypeptides, or fragments,
homologs, analogs or derivatives thereof.
[0009] In another aspect, the invention includes pharmaceutical
compositions that include therapeutically- or
prophylactically-effective amounts of a therapeutic and a
pharmaceutically-acceptable carrier. The therapeutic can be, e.g.,
a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific
for a NOVX polypeptide. In a further aspect, the invention
includes, in one or more containers, a therapeutically- or
prophylactically-effective amount of this pharmaceutical
composition.
[0010] In a further aspect, the invention includes a method of
producing a polypeptide by culturing a cell that includes a NOVX
nucleic acid, under conditions allowing for expression of the NOVX
polypeptide encoded by the DNA. If desired, the NOVX polypeptide
can then be recovered.
[0011] In another aspect, the invention includes a method of
detecting the presence of a NOVX polypeptide in a sample. In the
method, a sample is contacted with a compound that selectively
binds to the polypeptide under conditions allowing for formation of
a complex between the polypeptide and the compound. The complex is
detected, if present, thereby identifying the NOVX polypeptide
within the sample.
[0012] The invention also includes methods to identify specific
cell or tissue types based on their expression of a NOVX.
[0013] Also included in the invention is a method of detecting the
presence of a NOVX nucleic acid molecule in a sample by contacting
the sample with a NOVX nucleic acid probe or primer, and detecting
whether the nucleic acid probe or primer bound to a NOVX nucleic
acid molecule in the sample.
[0014] In a further aspect, the invention provides a method for
modulating the activity of a NOVX polypeptide by contacting a cell
sample that includes the NOVX polypeptide with a compound that
binds to the NOVX polypeptide in an amount sufficient to modulate
the activity of said polypeptide. The compound can be, e.g., a
small molecule, such as a nucleic acid, peptide, polypeptide,
peptidomimetic, carbohydrate, lipid or other organic (carbon
containing) or inorganic molecule, as further described herein.
[0015] Also within the scope of the invention is the use of a
therapeutic in the manufacture of a medicament for treating or
preventing disorders or syndromes including, e.g., Von
Hippel-Lindau (VHL) syndrome, tuberous sclerosis, hypercalceimia,
Lesch-Nyhan syndrome, multiple sclerosis, Corneal dystrophy,
Thiel-Behnke type; Dubin-Johnson syndrome; Retinol binding protein,
deficiency of; SEMD, Split hand/foot malformation, type 3;
Tolbutamide poor metabolizer; Urofacial syndrome; Warfarin
sensitivity; Wolman disease, Combined factor V and VIII deficiency;
Cone-rod retinal dystrophy-1; myasthenia gravis, endometriosis,
pancreatitis, hyperparathyroidism, hypoparathyroidism, xerostomia,
actinic keratosis, acne, hair growth/loss, allopecia, pigmentation
disorders, endocrine disorders, tonsillitis, cystitis,
incontinence, fatty acid transport of skin, oral mucosa, uveitis
and corneal fibroblast proliferation, amyotrophic lateral
sclerosis, acute pancreatitis, cerebral cryptococcosis, colitis,
thyroiditis, cirrhosis, Alzheimer's disease, stroke, Parkinson's
disease, Huntington's disease, cerebral palsy, epilepsy,
ataxia-telangiectasia, behavioral disorders, addiction, anxiety,
pain, neurodegeneration; Pakistani type; Spinocerebellar ataxia,
infantile-onset, with sensory neuropathy, neuroprotection, muscular
dystrophy, leukodystrophies, Leukemia, T-cell acute lymphocytic;
Colorectal cancer; Leukemia/lymphoma, B-cell, 2; Lymphoma/leukemia,
Osteosarcoma; cancer, lymphedema, Cholesteryl ester storage
disease; diabetes, obesity, fertility, growth and reproductive
disorders, pregnancy, hypertensive toxemia, pre-eclampsia/eclampsia
(gestational proteinuric hypertension), glomerular endotheliosis,
cholestasis, and pruritic urticarial papules and plaques of
pregnancy autoimmune disease, lupus erythematosus, tuberous
sclerosis, scleroderma, B-cell, variant; Protoporphyria,
erythropoietic; Protoporphyria, erythropoietic, recessive, with
liver failure; skin psoriasis, allergic encephalomyelitis, various
forms of arthritis, cancer such as AML, bacterial infections, graft
versus host disease (GVHD), lymphaedema renal artery stenosis,
interstitial nephritis, glomerulonephritis, polycystic kidney
disease, systemic renal tubular acidosis, IgA nephropathy, asthma,
emphysema, scleroderma, allergy, ARDS, cardiomyopathy,
atherosclerosis, hypertension, congenital heart defects, aortic
stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal
defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis,
ventricular septal defect (VSD), valve diseases, transplantation,
ulcers, and/or other pathologies and disorders of the like.
[0016] The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX
polypeptide, or a NOVX-specific antibody, or biologically-active
derivatives or fragments thereof.
[0017] For example, the compositions of the present invention will
have efficacy for treatment of patients suffering from the diseases
and disorders disclosed above and/or other pathologies and
disorders of the like. The polypeptides can be used as immunogens
to produce antibodies specific for the invention, and as vaccines.
They can also be used to screen for potential agonist and
antagonist compounds. For example, a cDNA encoding NOVX may be
useful in gene therapy, and NOVX may be useful when administered to
a subject in need thereof. By way of non-limiting example, the
compositions of the present invention will have efficacy for
treatment of patients suffering from the diseases and disorders
disclosed above and/or other pathologies and disorders of the
like.
[0018] The invention further includes a method for screening for a
modulator of disorders or syndromes including, e.g., the diseases
and disorders disclosed above and/or other pathologies and
disorders of the like. The method includes contacting a test
compound with a NOVX polypeptide and determining if the test
compound binds to said NOVX polypeptide. Binding of the test
compound to the NOVX polypeptide indicates the test compound is a
modulator of activity, or of latency or predisposition to the
aforementioned disorders or syndromes.
[0019] 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 including, e.g., the
diseases and disorders disclosed above and/or other pathologies and
disorders of the like by administering a test compound to a test
animal at increased risk for the aforementioned disorders or
syndromes. The test animal expresses a recombinant polypeptide
encoded by a NOVX nucleic acid. Expression or activity of NOVX
polypeptide is then measured in the test animal, as is expression
or activity of the protein in a control animal which
recombinantly-expresses NOVX polypeptide and is not at increased
risk for the disorder or syndrome. Next, the expression of NOVX
polypeptide in both the test animal and the control animal is
compared. A change in the activity of NOVX polypeptide in the test
animal relative to the control animal indicates the test compound
is a modulator of latency of the disorder or syndrome.
[0020] In yet another aspect, the invention includes a method for
determining the presence of or predisposition to a disease
associated with altered levels of a NOVX polypeptide, a NOVX
nucleic acid, or both, in a subject (e.g., a human subject). The
method includes measuring the amount of the NOVX polypeptide in a
test sample from the subject and comparing the amount of the
polypeptide in the test sample to the amount of the NOVX
polypeptide present in a control sample. An alteration in the level
of the NOVX polypeptide in the test sample as compared to the
control sample indicates the presence of or predisposition to a
disease in the subject. Preferably, the predisposition includes,
e.g., the diseases and disorders disclosed above and/or other
pathologies and disorders of the like. Also, the expression levels
of the new polypeptides of the invention can be used in a method to
screen for various cancers as well as to determine the stage of
cancers.
[0021] 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 NOVX
polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a
subject (e.g., a human subject), in an amount sufficient to
alleviate or prevent the pathological condition. In preferred
embodiments, the disorder, includes, e.g., the diseases and
disorders disclosed above and/or other pathologies and disorders of
the like.
[0022] In yet another aspect, the invention can be used in a method
to identity the cellular receptors and downstream effectors of the
invention by any one of a number of techniques commonly employed in
the art. These include but are not limited to the two-hybrid
system, affinity purification, co-precipitation with antibodies or
other specific-interacting molecules.
[0023] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0024] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides novel nucleotides and
polypeptides encoded thereby. Included in the invention are the
novel nucleic acid sequences and their encoded polypeptides. The
sequences are collectively referred to herein as "NOVX nucleic
acids" or "NOVX polynucleotides" and the corresponding encoded
polypeptides are referred to as "NOVX polypeptides" or "NOVX
proteins." Unless indicated otherwise, "NOVX" is meant to refer to
any of the novel sequences disclosed herein. Table A provides a
summary of the NOVX nucleic acids and their encoded polypeptides.
TABLE-US-00001 TABLE A Sequences and Corresponding SEQ ID Numbers
SEQ ID NO NOVX Internal (nucleic SEQ ID NO Assignment
Identification acid) (polypeptide) Homology 1a CG55974-01 1 2 Human
laminin alpha 5-like 1b CG102167-01 3 4 Human laminin alpha 5-like
1c CG55974-02 5 6 Human leminin alpha 5-like 1d 164875783 7 8 Human
laminin alpha 5-like 2a CG55999-01 9 10 Human Hurpin/PI 13-like 2b
CG55999-02 11 12 Human Hurpin/PI 13-like 2c CG55999-05 13 14 Human
Hurpin/PI 13-like 2d CG55999-06 15 16 Human Hurpin/PI 13-like 2e
166485357 197 198 Human Hurpin/PI 13-like 3a CG56019-01 17 18 Set
Binding Factor (SBF1) - like 3b CG56019-02 19 20 Set Binding Factor
(SBF1) - like 4 CG55692-01 21 22 TSPAN-1-like 5 CG56073-01 23 24
Fatty Acid-Binding protein, Epidermal-like 6 CG50261-02 25 26
Uncoupling Protein 1-like 7a CG56077-01 27 28 Leucine-Rich Glioma-
Inactivated protein-like 7b CG56077-02 29 30 Leucine-Rich Glioma-
Inactivated Protein-like 8 AL163195_da1 31 32 RNase-like 9
CG56069-01 33 34 Insulin like growth factor binding protein-like 10
SC133419534_A 35 36 Novel pregnancy zone protein precursor --like
11 SC139725617_A 37 38 Transmembrane Receptor UNC5H2-like 12a
SC134999661_A 39 40 Thymosin-like 13 AC025256_da7 41 42
Neuromodulin-like 14a CG56075-01 43 44 Prostatin Precursor-like 14b
CG56075-01 45 46 Prostatin Precursor-like
[0026] NOVX nucleic acids and their encoded polypeptides are useful
in a variety of applications and contexts. The various NOVX nucleic
acids and polypeptides according to the invention are useful as
novel members of the protein families according to the presence of
domains and sequence relatedness to previously described proteins.
Additionally, NOVX nucleic acids and polypeptides can also be used
to identify proteins that are members of the family to which the
NOVX polypeptides belong.
[0027] NOV1 is homologous to a Human laminin alpha 5-like family of
proteins. Thus, the NOV1 nucleic acids, polypeptides, antibodies
and related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example;
Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke,
tuberous sclerosis, hypercalceimia, Parkinson's disease,
Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan
syndrome, multiple sclerosis, ataxia-telangiectasia,
leukodystrophies, behavioral disorders, addiction, anxiety, pain,
neurodegeneration; Cholesteryl ester storage disease; Corneal
dystrophy, Thiel-Behnke type; Dubin-Johnson syndrome; Leukemia,
T-cell acute lymphocytic; Retinol binding protein, deficiency of;
SEMD, Pakistani type; Spinocerebellar ataxia, infantile-onset, with
sensory neuropathy; Split hand/foot malformation, type 3;
Tolbutamide poor metabolizer; Urofacial syndrome; Warfarin
sensitivity; Wolman disease, neuroprotection, fertility, diabetes,
autoimmune disease, renal artery stenosis, interstitial nephritis,
glomerulonephritis, polycystic kidney disease, systemic lupus
erythematosus, renal tubular acidosis, IgA nephropathy,
cardiomyopathy, atherosclerosis, hypertension, congenital heart
defects, aortic stenosis, atrial septal defect (ASD),
atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary
stenosis, subaortic stenosis, ventricular septal defect (VSD),
valve diseases, tuberous sclerosis, scleroderma, obesity,
transplantation, ulcers, systemic lupus erythematosus, autoimmune
disease, asthma, emphysema, scleroderma, allergy, ARDS, or other
pathologies or conditions.
[0028] NOV2 is homologous to the Human Hurpin/PI 13-like family of
proteins. Thus NOV2 nucleic acids, polypeptides, antibodies and
related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in Colorectal
cancer; Combined factor V and VIII deficiency; Cone-rod retinal
dystrophy-1; Leukemia/lymphoma, B-cell, 2; Lymphoma/leukemia,
B-cell, variant; Protoporphyria, erythropoietic; Protoporphyria,
erythropoietic, recessive, with liver failure; Obesity, autosomal
dominant; Osteosarcoma; cancer, skin psoriasis, and/or other
pathologies and disorders.
[0029] NOV3 is homologous to a family of Set Binding Factor
(SBF1)-like proteins. Thus, the NOV3 nucleic acids and
polypeptides, antibodies and related compounds according to the
invention will be useful in therapeutic and diagnostic applications
implicated in, for example: Von Hippel-Lindau (VHL) syndrome,
Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia,
Parkinson's disease, Huntington's disease, cerebral palsy,
epilepsy, Lesch-Nyhan syndrome, multiple sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders,
addiction, anxiety, pain, neurodegeneration; Cholesteryl ester
storage disease; Corneal dystrophy, Thiel-Behnke type;
Dubin-Johnson syndrome; Leukemia, T-cell acute lymphocytic; Retinol
binding protein, deficiency of; SEMD, Pakistani type;
Spinocerebellar ataxia, infantile-onset, with sensory neuropathy;
Split hand/foot malformation, type 3; Tolbutamide poor metabolizer;
Urofacial syndrome; Warfarin sensitivity; Wolman disease, and/or
other pathologies.
[0030] NOV4 is homologous to the TSPAN-1-like family of proteins.
Thus, NOV4 nucleic acids, polypeptides, antibodies and related
compounds according to the invention will be useful in therapeutic
and diagnostic applications implicated in, for example:
adrenoleukodystrophy, congenital adrenal hyperplasia, hemophilia,
hypercoagulation, idiopathic thrombocytopenic purpura, autoimmune
disease, allergies, asthma, immunodeficiencies, transplantation,
graft versus host disease, Von Hippel-Lindau (VHL) syndrome,
Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia,
Parkinson's disease, Huntington's disease, cerebral palsy,
epilepsy, Lesch-Nyhan syndrome, multiple sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders,
addiction, anxiety, pain, neuroprotection, arthritis, tendonitis,
fertility, atherosclerosis, aneurysm, hypertension, fibromuscular
dysplasia, stroke, scleroderma, obesity, myocardial infarction,
embolism, cardiovascular disorders, bypass surgery, cirrhosis,
inflammatory bowel disease, diverticular disease, Hirschsprung's
disease, Crohn's Disease, appendicitis, ulcers, diabetes, renal
artery stenosis, interstitial nephritis, glomerulonephritis,
polycystic kidney disease, systemic lupus erythematosus, renal
tubular acidosis, IgA nephropathy, laryngitis, emphysema, ARDS,
lymphedema, muscular dystrophy, myasthenia gravis, endometriosis,
pancreatitis, hyperparathyroidism, hypoparathyroidism, growth and
reproductive disorders, xerostomia, psoriasis, actinic keratosis,
acne, hair growth/loss, allopecia, pigmentation disorders,
endocrine disorders, tonsillitis, cystitis, incontinence, and/or
other pathologies.
[0031] NOV5 is homologous to the Fatty Acid-Binding Protein,
Epidermal-like family of proteins. Thus NOV5 nucleic acids,
polypeptides, antibodies and related compounds according to the
invention will be useful in therapeutic and diagnostic applications
implicated in, fatty acid transport of skin, oral mucosa, and/or
other disorders and conditions.
[0032] NOV6 is homologous to the Uncoupling Protein 1-like family
of proteins. Thus NOV6 nucleic acids, polypeptides, antibodies and
related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example:
obesity, hyperphagia, and/or other pathologies/disorders.
[0033] NOV7 is homologous to members of the Leucine-Rich
Glioma-Inactivated Protein-like family of proteins. Thus, the NOV7
nucleic acids, polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and
diagnostic applications implicated in, for example; uveitis and
corneal fibroblast proliferation, allergic encephalomyelitis,
amyotrophic lateral sclerosis, acute pancreatitis, cerebral
cryptococcosis, autoimmune disease including Type I diabetes
mellitus (DM), experimental allergic encephalomyelitis (EAE),
systemic lupus erythematosus (SLE), colitis, thyroiditis and
various forms of arthritis, cancer such as AML, bacterial
infections, and/or other pathologies/disorders.
[0034] NOV8 is homologous to the RNase-like family of proteins.
Thus, NOV8 nucleic acids and polypeptides, antibodies and related
compounds according to the invention will be useful in therapeutic
and diagnostic applications implicated in, for example; Diabetes,
Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Obesity,
Hyperthyroidism and Hypothyroidism and Cancers including, but no
limited to Thyroid and Pancreas, and/or other
pathologies/disorders.
[0035] NOV9 is homologous to the Insulin like growth factor binding
protein-like family of proteins. Thus, NOV9 nucleic acids and
polypeptides, antibodies and related compounds according to the
invention will be useful in therapeutic and diagnostic applications
implicated in diabetes, obesity, Von Hippel-Lindau (VHL) syndrome,
Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia,
Parkinson's disease, Huntington's disease, cerebral palsy,
epilepsy, Lesch-Nyhan syndrome, multiple sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders,
addiction, anxiety, pain, neuroprotection, cirrhosis,
transplantation, hemophilia, hypercoagulation, idiopathic
thrombocytopenic purpura, autoimmume disease, allergies,
immunodeficiencies, graft versus host disease (GVHD), lymphaedema,
and/or other pathologies or disorders.
[0036] NOV10 is homologous to the Pregnancy Zone Protein
Precursor-like family of proteins. Thus, NOV10 nucleic acids and
polypeptides, antibodies and related compounds according to the
invention will be useful in pregnancy, hypertensive toxemia,
pre-eclampsia/eclampsia (gestational proteinuric hypertension),
glomerular endotheliosis, cholestasis, and pruritic urticarial
papules and plaques of pregnancy, and/or other pathologies or
disorders.
[0037] NOV11 is homologous to the Transmembrane Receptor
UNC5H2-like family of proteins. Thus, NOV11 nucleic acids and
polypeptides, antibodies and related compounds according to the
invention will be useful in therapeutic and diagnostic applications
implicated in various pathologies or disorders.
[0038] NOV12 is homologous to the Thymosin-like family of proteins.
Thus, NOV12 nucleic acids and polypeptides, antibodies and related
compounds according to the invention will be useful in therapeutic
and diagnostic applications implicated in, for example;
osteoporosis, osteoarthritis, cardiac hypertrophy, atherosclerosis,
hypertension, restenosis, and/or other pathologies/disorders.
[0039] NOV13 is homologous to the Neuromodulin-like family of
proteins. Thus, NOV13 nucleic acids and polypeptides, antibodies
and related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in various
pathologies/disorders.
[0040] NOV14 is homologous to the Prostatin Precursor-like family
of proteins. Thus, NOV14 nucleic acids and polypeptides, antibodies
and related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart
defects, Aortic stenosis, Atrial septal defect (ASD),
Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD),
valve diseases, Tuberous sclerosis, Scleroderma, Obesity,
Transplantation, and/or other pathologies/disorders.
[0041] The NOVX nucleic acids and polypeptides can also be used to
screen for molecules, which inhibit or enhance NOVX activity or
function. Specifically, the nucleic acids and polypeptides
according to the invention may be used as targets for the
identification of small molecules that modulate or inhibit, e.g.,
neurogenesis, cell differentiation, cell proliferation,
hematopoiesis, wound healing and angiogenesis.
[0042] Additional utilities for the NOVX nucleic acids and
polypeptides according to the invention are disclosed herein.
[0043] NOV1
[0044] NOV1 includes three novel human laminin alpha 5-like
proteins disclosed below. The disclosed sequences have been named
NOV1a, NOV1b, and NOV1c.
[0045] NOV1a
[0046] A disclosed NOV1a nucleic acid of 10809 nucleotides (also
referred to as CG55974-01) encoding a human laminin alpha 5-like
protein is shown in Table 1A. An open reading frame was identified
beginning with an ATG initiation codon at nucleotides 1-3 and
ending with a TAG codon at nucleotides 10801-10803. A putative
untranslated region downstream from the termination codon is
underlined in Table 1A. The start and stop codons are in bold
letters. TABLE-US-00002 TABLE 1A NOV1a nucleotide sequence (SEQ ID
NO:1).
ATGGCGAAGCGGCTCTGCGCGGGGAGCGCACTGTGTGTTCGCGGCCCCCGGGGCCCCGCGCCGCTGCTGCTG
CACCCGCCCTACTTCAACCTGGCCGAGGGCGCCCGCATCGCCGCCTCCGCGACCTGCGGAGAGGAGGCCCCG
GCGCGCGGCTCCCCGCGCCCCACCGAGGACCTTTACTGCAAGCTGGTAGGGGGCCCCGTGGCCGGCGGCGAC
CCCAACCAGACCATCCAGGGCCAGTACTGTGACATCTGCACGGCTGCCAACAGCAACAAGGCACACCCCGCG
AGCAATGCCATCGATGGCACGGAGCGCTGGTGCCAGAGTCCACCGCTGTCCCGCGGCCTGGAGTACAACGAG
GTCAACGTCACCCTGGACCTGGGCCAGGTCTTCCACGTGGCCTACGTCCTCATCAAGTTTGCCAACTCACCC
CGGCCGGACCTCTGGGTGCTGGAGCGGTCCATGGACTTCGGCCGCACCTACCAGCCCTGGCAGTTCTTTGCC
GCCTCCAAGAGGGACTGTCTGGAGCGGTTCGGGCCACAGACGCTGGAGCGCATCACACGGGACGACGCGGCC
ATCTGCACCACCGAGTACTCACGCATCGTGCCCCTGGAGAACGGAGAGATCGTGGTGTCCCTGGTGAACGGA
CGTCCGGGCGCCATGAATTTCTCCTACTCGCCGCTGCTACGTGAGTTCACCAAGGCCACCAACGTCCGCCTG
CGCTTCCTGCGTACCAACACGCTGCTGGGCCATCTCATGGGGAAGGCGCTGCGGGACCCCACGGTCACCCGC
CGGTATTATTACAGCATCAAGGATATCAGCATCGGAGGCCGCTGTGTCTGCCACGGCCACGCGGATGCCTGC
GATGCCAAAGACCCCACGGACCCGTTCAGGCTGCAGTGCACCTGCCAGCACAACACCTGCGGGGGCACCTGC
GACCGCTGCTGCCCCGGCTTCAATCAGCAGCCGTGGAAGCCTGCGACTGCCAACAGTGCCAACGAGTGCCAG
TGTGAGTGCTACGGCCATGCCACCGACTGTTACTACGACCCTGAGGTGGACCGGCGCCGCGCCAGCCAGAGC
CTGGATGGCACCTATCAGGGTGGGGGTGTCTGTATCGACTGCCAGCACCACACCACCGGCGTCAACTGTGAG
CGCTGCCTGCCCGGCTTCTACCGCTCTCCCAACCACCCTCTCGACTCGCCCCACGTCTGCCGCGGCTGCAAC
TGCGAGTCCGACTTCACGGATGGCACCTGCGAGGACCTGACGGGTCGATGCTACTGCCGGCCCAACTTCTCT
GGGGAGCGGTGTGACGTGTGTGCCGAGGGCTTCACGGGCTTCCCAAGCTGCTACCGTGAGCACCTGCCAGGG
AATGACACCAGGGAGCAGGTGCTGCCAGCCGGCCAGATTGTGAGTTGTGACTGCAGCGCGGCAGGGACCCAG
GGCAACGCCTGCCGGAAGGACCCAAGGGTGGGACGCTGTCTGTGCAAACCCAACTTCCAAGGCACCCATTGT
GAGCTCTGCGCGCCAGGGTTCTACGGCCCCGGCTGCCCTGCCAGTGTTCCAGCCCTGGAGTGGCCGATGACC
GCTGTGACCCTGACACAGGCCAGTGCAGGTGCCGAGTGGGCTTCGAGGGGGCCACATGTGATCGCTGTGCCC
CCGGCTACTTTCACTTCCCTCTCTGCCAGTCACCCGCTCCGCTCTGCAGTGTGTGGCTGCAGCCCTGCAGGA
ACCTTGCCCGAGGGCTGCGATGAGGCCGGCCGCTGCCTATGCCAGCCTGAGTTTGCTGGACCTCATTGTGAC
CGGTCCCGCCCTGGCTACCATGGTTTCCCCAACTGCGCAGCATGCACCTGCGACCCTCGGGGAGCCCTGGAC
CAGCTCTGTGGGGCGGGAGGTTTGTGCCGCTGCCGCCCCGGCAACACAGGCACTGCCTGCCAGGAATGCAGC
CCCGGCTTTCACGGCTTCCCCAGCTGTCCTGCCACTGCTCTGCTGAAGGCTCCCTGCACGCAGCCTGTGACC
CCCGGAGTGGGCAGTGCAGCTGCCGGCCCCGTGCGGGGCTGCGGTGTGACACATGTGTGCCCGGTGCCTACA
ACTTCCCCTACTGCGAAGCCTCTCTTCACAGCTGGCTCTTGCCACCCTGCCGGTCTGGCCCCAGTGGATCCT
GCCCTTCCTGAGGTGAGCCCACCCTGTATGTGCCGGGCTCACGTGGAGGGGCCGAGCTGTGACCGCTGCAAA
CCTGCGTTCTGGGGACTGAGCCCCAGCAACCCCGAGGGCTGTACCCGTTGCAGCTGCGACCTCAGGGGCACA
CTGGGTGGAGTTGCTGAGTGCCAGGGCACCGGCCAGTGCTTCTGCAAGCCCCACGTGTGCGGCCAGGCCTGC
GCGTCCTGCAAGGATGGCTTCTTTGGACTGGATCAGGCTGACTATTTTGGCTGCCGCAGTTGCCGGTGTGAC
ATTGGCGGTGCACTGGGCCAGAGCTGTGAACCGAGGACGGGCGTCTGCCGGTGCCGCCCCAACACCCAGGGC
CCCACCTGCAGCGAGCCTGCGAGGGACCACTACCTCCCGGACCTGCACCACCTGCGCCTGGAGCTGGAGCAG
GCTGCCACACCTGAGGCTCACGCCGTGCGCTTTGGCTTCAACCCCCTCGAGTTCGAGAACTTCAGCTGGAGG
GGCTACGCGCAGATGGCACCTGTCCAGCCCAGGATCGTGGCCAGGCTGAACCTGACCTCCCCTGACCTTTTC
TGGCTCGTCTTCCGATACGTCAACCGGGGGGCCATGAGTGTGAGCGGGCGGGTCTCTGTGCGAGAGGAGGGC
AGGTCGGCCACCTGCGCCAACTGTACAGCACAGAGTCAGCCCGTGGCCTTCCCACCCAGCACGGAGCCTGCC
TTCATCACCGTGCCCCAGAGGGGCTTCGGAGAGCCCTTTGTGCTGAACCCTGGCACCTGGGCCCTGCGTGTG
GAGGCCGAAGGGGTGCTCCTGGACTACGTGGTTCTGCTGCCTAGCGCATACTACGAGGCGGCGCTCCTGCAG
CTGCGGGTGACTGAGGCCTGCACATACCGTCCCTCTGCCCAGCAGTCTCCCCCCAGCTGCCTCCTCTACACA
CACCTCCCCCTGGATGGCTTCCCCTCGGCCGCCGGGCTGGAGGCCCTGTGTCGCCAGGACAACAGCCTGCCC
CGGCCCTGCCCCACGGAGCAGCTCAGCCCGTCGCACCCGCCACTGATCACCTGCACGGGCAGTGATGTGGAC
GTCCAGCTTCAAGTGGCAGTGCCACAGCCAGGCCGCTATGCCCTAGTGGTGGAGTACGCCAATGAGGATGCC
CGCCAGGAGGTGGGCGTGGCCGTGCACACCCCACAGCGGGCCCCCCAGCAGGGGCTGCTCTCCCTGCACCCC
TGCCTGTACAGCACCCTGTGCCGGGGCACTGCCCGGGATACCCAGGACCACCTGGCTGTCTTCCACCTGGAC
TCGGAGGCCAGCGTGAGGCTCACAGCCGAACAGGCACGCTTCTTCCTGCACGGGGTCACTCTGGTGCCCATT
GAGGAGTTCAGCCCGGAGTTCGTGGAGCCCCGGGTCAGCTGCATCAGCAGCCACGGCGCCTTTGGCCCCAAC
AGTGCCGCCTGTCTGCCCTCGCGCTTCCCAAAGCCGCCCCAGCCCATCATCCTCAGGGACTGCCAGGTGATC
CCGCTGCCGCCCGGCCTCCCGCTGACCCACGCGCAGGATCTCACTCCAGCCATGTCCCCAGCTGGACCCCGA
CCTCGGCCCCCCACCGCTGTGGACCCTGATGCAGAGCCCACCCTGCTGCGTGAGCCCCAGGCCACCGTGGTC
TTCACCACCCATGTGCCCACGCTGGGCCGCTATGCCTTCCTGCTGCACGGCTACCAGCCAGCCCACCCCACC
TTCCCCGTGGAAGTCCTCATCAACGCCGGCCGCGTGTGGCAGGGTCACGCCAACGCCAGCTTCTGTCCACAT
GGCTACGGCTGCCGCACCCTGGTGGTGTGTGAGGGCCAGGCCCTGCTGGACGTGACCCACAGCGAGCTCACT
GTGACCGTGCGTGTGCCCAAGGGCCGGTGGCTCTGGCTGGATTATGTACTCGTGGTCCCTGAGAACGTCTAC
AGCTTTGGCTACCTCCGGGAGGAGCCCCTGGATAAATCCTATGACTTCATCAGCCACTGCGCAGCCCAGGGC
TACCACATCAGCCCCAGCAGCTCATCCCTGTTCTGCCGAAACGCTGCTGCTTCCCTCTCCCTCTTCTATAAC
AACGGAGCCCGTCCATGTGGCTGCCACGAAGTAGGTGCTACAGGCCCCACGTGTGAGCCCTTCGGGGGCCAG
TGTCCCTGCCATGCCCATGTCATTGGCCGTGACTGCTCCCGCTGTGCCACCGGATACTGGGGCTTCCCCAAC
TGCAGGGCCTGTGACTGCGCTGCCCGCCTCTGTGACGAGCTCACGGGCCAGTGCATCTGCCCGCCACGCACC
ATCCCGCCCGACTGCCTGCTGTGCCAGCCCCAGACCTTTGGCTGCCACCCCCTGGTCGGCTGTGAGGAGTGT
AACTGCTCAGGGCCCGGCATCCAGGAGCTCACAGACCCTACCTGTGACACAGACAGCGGCCAGTGCAGGTGC
AGACCCAACGTGACTGGGCGCCGCTGTGATACCTGCTCTCCGGGCTTCCATGGCTACCCCCGCTGCCGCCCC
TGTGACTGTCACGAGGCGGGCACTGCGCCTGGCGTGTGTGACCCCCTCACAGGGCAGTGCTACTGTAAGCAG
AACGTGCAGGGCCCCAAATGTGACCAGTGCAGCCTTGGGACCTTCTCACTGGATGCTGCCAACCCCAAAGGT
TGCACCCGCTGCTTCTGCTTTGGGGCCACGGAGCGCTGCCGGAGCTCGTCCTACACCCGCCAGGAGTTCGTG
GATATGGAGGGATGGGTGCTGCTGAGCACTGACCGGCAGGTGGTGCCCCACGAGCGGCAGCCAGGGACGGAG
ATGCTCCGTGCAGACCTGCGGCACGTGCCTGAGGCTGTGCCCGAGGCTTTCCCCGAGCTGTACTGGCAGGCC
CCACCCTCCTACCTGGGGGACCGGGTAAGCTCCTACGGTGGGACCCTCCGTTATGAACTGCACTCAGAGACC
CAGCGGGGAGATGTCTTTGTCCCCATGGAGAGCAGGCCGGATGTGGTGCTGCAGGGCAACCAGATGAGCATC
ACATTCCTGGAGCCGGCATACCCCACGCCTGGCCACGTTCACCGTGGGCAGCTGCAGCTGGTGGAGGGGAAC
TTCCGGCATAGGGAGACGCGCAACACTGTGTCCCGCGAGGAGCTCATGATGGTGCTGGCCAGCCTGGAGCAG
CTGCAGATCCGTGCCCTCTTCTCACAGATCTCCTCGGCTGTCTTCCTGCGCAGGGTGGCACTGGAGGTGGCC
AGCCCAGCAGGCCAGGGGGCCCTGGCCAGCAATGTGGAGCTGTGCCTGTGCCCCGCCAGCTACCGGGGGGAC
TCATGCCAGGAATGTGCCCCCGGCTTCTATCGGGACGTCAAAGGTCTCTTCCTGGGCCGATGTGTCCCTTGT
CAGTGCCATGGACACTCAGACCGCTGCCTCCCTGGCTCTGGCGTCTGTGTGTGCCAGCACAACACCGAAGGG
GCCCACTGTGAGCGCTGCCAGGCTGGCTTCGTGAGCAGCAGGGACGACCCCAGCGCCCCCTGTGTCAGCTGC
CCCTGCCCCCTCTCAGTGCCTTCCAACAGGTGTGCGCCCGGATTCTTTGGGAACCCACTGGTGCTGGGCAGC
TCCTGCCAGCCATGCGACTGCAGCGGCAACGGTGACCCCAACTTGCTCTTCAGCGACTGCGACCCCCTGACG
GGCGCCTGCCGTGGCTGCCTGCGCCACACCACTGGGCCCCGCTGCGAGATCTGTGCCCCCGGCTTCTACGGC
AACGCCCTGCTGCCCGGCAACTGCACCCGTTGCGACTGTAGCCCATGTGGGACAGAGGCCTGCGACCCCCAC
AGCGGGCACTGCCTGTGCAAGGCGGGCGTGACTGGGCGGCGCTGTGACCGCTGCCAGGAGGGACATTTTGGT
TTCGATGGCTGCGGGGGCTGCCGCCCGTGTGCTTGTGGACCGGCCGCCGAGGGCTCCGAGTGCCACCCCCAG
AGCGCACAGTGCCACTGCCGACCAGGGACCATGGGACCCCAGTGCCGCGAGTGTGCCCCTGGCTACTGGGGG
CTCCCTGAGCAGGGCTGCAGGCGTTGCCAGTGCCCTGGGGGCCGCTGTGACCCTCACACGGGCCGCTGCAAC
TGCCCCCCGGGGCTCAGCGGGGAGCGCTGCGACACCTGCAGCCAGCAGCATCAGGTGCCTGTTCCAGGCGGG
CCTGTGGGCCACAGCATCCACTGTGAAGTGTGTGACCACTGTGTGGTCCTGCTCCTGGATGACCTGGAACGG
GCCGGCGCCCTCCTCCCCGCCATTCACGAGCAACTGCGTGGCATCAATGCCAGCTCCATGGCCTGGGCCCGT
CTGCACAGGCTGAACGCCTCCATCGCTGACCTGCAGGTACTGAGCGTCCTGGCCTTCCCTCCCCAACCCGGG
CCAGTGCAGGCCTTCACCTTTCGCCTCCCACAGAGCCAGCTCCGGAGCCCCCTGGGCCCCCGCCATGAGACG
GCACAGCAGCTGGAGGTGCTGGAGCAGCAGAGCACAAGCCTTCCTCCACAGGCCGTGGGGACCCGAGACCAG
GCGAGCCAATTGCTGGCCGGCACCGAGGCCACACTGGGCCATGCGAAGACGCTGTTGGCGGCCATCCGGGCT
GTGGACCGCACCCTGAGCGAGCTCATGTCCCAGACGCGCCACCTGGGGCTGGCCAATGCCTCGGCTCCATCA
GGTGAGCAGCTGCTCCGGACACTGGCCGAGGTGGAGCGGCTGCTCTGGGAGATGCGGGCCCGGGACCTGGGG
GCCCCGCAGGCAGCAGCTGAGGCTGAGTTGGCTGCAGCACAGAGAGTGCTGGCCCGGGTGCAGGAGCAGCTG
AGCAGCCTCTGGGAGGAGAACCAGCCACTGGCCACACAAACCCGCGACCGGCTGGCCCAGCACGAGGCCGGC
CTCATGGACCTGCGAGAGGCTTTGAACCGGGCAGTGGACGCCACACGGGAGGCCCAGGAGCTCAACAGCCGC
AACCAGGAGCGCCTGGAGGAAGCCCTGCAAAGGAAGCAGGAGCTGTCCCGGGACAATGCCACCCTGCAGGCC
ACTCTGCATGCGGCTAGGGACACCCTGGCCAGCGTCTTCAGATTGCTGGAGGGGCTAAGTCCACTCAAATTC
CAGGAGCTGGAGCGCCTCGCCGCCAGCCTGGATGGGGCTCGGACCCCACTGCTGCAGAGGATGCAGACCTTC
TCCCCGGCGGGCAGCAAGCTGCGTCTAGTGGAGGCCGCCGAGGCCCACGCACAGCAGCTGGGCCAGCTGGCA
CTCAATCTGTCCATCATCCTGGACGTCAACCAGGACCGCCTCACCCAGAGGGCCATCGAGGCCTCCAACGCC
TACAGCCGCATCCTGCAGGCCGTGCAGGCTGCCGAGGATGCTGCTGGCCAGGCCCTGCAGCAGGCGGACCAC
ACGTGGCAGACGGTGGTGCGGCAGGGCCTGGTGGACCGAGCCCAGCAGCTCCTGGCCAACAGCACTGCACTA
GAAGAGGCCATGCTCCAGGAACAGCAGAGGCTGGGCCTTGGTGAGTGCTGGGCTCCGATGGGGGCCCTTAGG
CCTGCTGGGACCCAGCTCCGAGATGTCCGGGCCAAGAAGGACCAGCTGGAGGCGCACATCCAGGCGGCGCAG
GCCATGCTTGCCATGGACACAGGTGAGACAAGCAAGAAGATCGCACATGCCAAGGCTGTGGCTGCTGAAGCC
CAGGACACCGCCACCCGTGTGCAGTCCCAGCTGCAGGCCATGCAGGAGAATGTGCAGCGGTGGCAGGGCCAG
TACGAGGGCCTGCGGGGCCAGGACCTGGGCCAGGCAGTGCTTGACGCAGGCTCTGCAGTGTCCACCCTGGAG
AAGACGCTGCCCCAGCTGCTGGCCAAGCTGAGCATCCTGGAGAACCGTCGGGTGCACAACGCCAGCCTGGCC
CTGTCCGCCAGCATTGGCCGCGTGCGAGAGCTCATTGCCCAGGCCCGGGGGGCTGCCAGTAAGGTGGTCAAG
GTGCCCATGAAGTTCAACGGGCGCTCAGGGGTGCAGCTGCGCACCCCACGGGATCTTGCCGACCTTGCTGCC
TACACTGCCCTCAAGTTCTACCTGCAGGGCCCAGAGCCTGAGCCTGGGCAGGGTACCGAGGATCGCTTTGTG
ATGTACATGGGCAGCCGCCAGGCCACTGGGGACTACATGGGTGTGTCTCTGCGTGACAAGAAGGTGCACTGG
GTGTATCAGCTGGGTGAGGCGGGCCCTGCAGTCCTAAGCATCGATGAGGACATTGGGGAGCAGTTCGCAGCT
GTCAGCCTGGACAGGACTCTCCAGTTTGGCCACATGTCCGTCACAGTGGACAGACAGATGATCCAGGAAACC
AAGGGTGACACGGTGGCCCCTGGGGCAGAGGGGCTGCTCAACCTGCGGCCAGACGACTTCGTCTTCTACGTC
GGGGGGTACCCCAGTACCTTCACGCCCCCTCCCCTGCTTCGCTTCCCCGGCTACCGGGGCTGCATCGAGATG
GACACGCTGAATGAGGAGGTGGTCAGCCTCTACAACTTCGAGAGGACCTTCCAGCTGGACACGGCTGTGGAC
AGGCCTTGTGCCCGGTCCAAGTCGACCGGGGACCCGTGGCTCACGGACGGCTCCTACCTGGACGGCACCGGC
TTCGCCCGCATCAGCTTCGACAGTCAGATCAGCACCACCAAGCGCTTCGAGCAGGAGCTGCGGCTCGTGTCC
TACAGCGGGGTGCTCTTCTTCCTGAAGCAGCAGAGCCAGTTCCTGTGCTTGGCCGTGCAAGAAGGCAGCCTC
GTGCTGTTGTATGACTTTGGGGCTGGCCTGAAAAAGGCCGTCCCACTGCAGCCCCCACCGCCCCTGACCTCG
GCCAGCAAGGCGATCCAGGTGTTCCTGCTGGGGGGCAGCCGCAAGCGTGTGCTGGTGCGTGTGGAGCGGGCC
ACGGTGTACAGCGTGGAGCAGGACAATGATCTGGAGCTGGCCGACGCCTACTACCTGGGGGGCGTGCCGCCC
GACCAGCTGCCCAGCCTGCGACGGCTCTTCCCCACCGGAGGCTCAGTCCGTGGCTGCGTCAAAGGCATCAAG
GCCCTGGGCAAGTATGTGGACCTCAAGCGGCTGAACACGACAGGCGTGAGCGCCGGCTGCACCGCCGACCTG
CTGGTGGGGCGCGCCATGACTTTCCATGGCCACGGCTTCCTTCGCCTGGCGCTCTCGAACGTGGCACCGCTC
ACTCGCAACGTCTACTCCGGCTTCGGCTTCCACAGCGCCCAGGACAGTGCCCTGCTCTACTACCGGGCGTCC
CCGGTGAGACCTCACCAGGTGTCCCTGCAGCAGGGCCGTGTGAGCCTACAGCTCCTGAGGACTGAAGTGAAA
ACTCAAGCGGGCTTCGCCGATGGTGCCCCCCATTACGTCGCCTTCTACAGCAATGCCACGGGGGTCTGGCTG
TATGTCGATGACCAGCTCCAGCAGATGAAGCCCCACCGGGGACCACCCCCCGAGCTCCAGCCGCAGCCTGAG
GGGCCCCCGAGGCTCCTCCTGGGAGGCCTGCCTGAGTCTGGCACCATTTACAACTTCAGTGGCTGCATCAGC
AACGTCTTCGTGCAGCGGCTCCTGGGCCCACAGCGCGTATTTGATCTGCAGCAGAACCTGGGCAGCGTCAAT
GTGAGCACGGGCTGTGCACCCGCCCTGCAAGCCCAGACCCCGGGCCTGGCGCCTAGACAGGCCTCCCGCCGC
AGCCGTCAGCCCGCCCGGCATCCTGCCTGCATGCTGCCCCCACACCTCAGGACCACCCGAGACTCCTACCAG
TTTGGGGGTTCCCTGTCCAGTCACCTGGAGTTTGTGGGCATCCTGGCCCGACATAGGAACGTCTCCGTGCGC
TGGGAGAAGAACCGGATCCTGCTGGTGACGGACGGGGCCCGGGCCTGGAGCCAGGAGGGGCCGCACCGGCAG
CACCAGGGGGCAGAGCACCCCCAGCCCCACACCCTCTTTGTGGGCGGCCTCCCGGCCAGCAGCCACAGCTCC
AAACTTCCGGTGACCGTCGGGTTCAGCGGCTGTGTGAAGAGACTGAGGCTGCACGGGAGGCCCCTGGGGGCC
CCCACACGGATGGCAGGGGTCACACCCTGCATCTTGGGCCCCCTGGAGGCGGGCCTGTTCTTCCCAGGCAGC
GGGGGAGTTATCACTTTAGGTCTGCCAGGAGCTACACTGCCTGATGTGGGCCTGGAACTGGAGGTGCGGCCC
CTGGCAGTCACCGGACTGATCTTCCACTTGGGCCAGGCCCGGACGCCCCCCTACTTGCAGTTGCAGGTGCTA
CCCCGCCAGGTCCTGCTGCGGGCGGATGACGGAGCAGGGGAGTTCTCCACGTCAGTGACCCGCCCCTCAGTG
CTGTGTGATGGCCAGTGGCACCGGCTAGCGGTGATGAAAAGCGGGAATGTGCTCCGGCTGGAGGTGGACGCG
CAGAGCAACCACACCGTGGGCCCCTTGCTGGCGGCTGCAGCTGGTGCCCCAGCCCCTCTGTACCTCGGGGGC
CTGCCTGAGCCCATGGCCGTGCAGCCCTGGCCCCCCGCCTACTGCGGCTGCATGAGGAGGCTGGCGGTGAAC
CGGTCCCCCGTCGCCATGACTCGCTCTGTGGAGGTCCACGGGGCAGTGGGGGCCAGTGGCTGCCCAGCCGCC
TAGAATAAA
[0047] In a search of public sequence databases, the NOV1a nucleic
acid sequence, located on chromsome 20 is 80% identical to a
gb:GENBANK-ID:MMU37501|acc:U37501.1 Mus musculus laminin alpha 5
chain (Lama5) mRNA, and 61% identical to a
gb:GENBANK-ID:DROLAMZ|acc:L07288.1 Drosophila melanogaster laminin
A (Lam-A) mRNA. Public nucleotide databases include all GenBank
databases and the GeneSeq patent database.
[0048] In all BLAST alignments herein, the "E-value" or "Expect"
value is a numeric indication of the probability that the aligned
sequences could have achieved their similarity to the BLAST query
sequence by chance alone, within the database that was searched.
For example, the probability that the subject ("Sbjct") retrieved
from the NOV1 BLAST analysis, e.g., Mus musculus laminin alpha 5
chain (Lama5) mRNA, matched the Query NOV1 sequence purely by
chance is 0.0. The Expect value (E) is a parameter that describes
the number of hits one can "expect" to see just by chance when
searching a database of a particular size. It decreases
exponentially with the Score (S) that is assigned to a match
between two sequences. Essentially, the E value describes the
random background noise that exists for matches between
sequences.
[0049] The Expect value is used as a convenient way to create a
significance threshold for reporting results. The default value
used for blasting is typically set to 0.0001. In BLAST 2.0, the
Expect value is also used instead of the P value (probability) to
report the significance of matches. For example, an E value of one
assigned to a hit can be interpreted as meaning that in a database
of the current size one might expect to see one match with a
similar score simply by chance. An E value of zero means that one
would not expect to see any matches with a similar score simply by
chance. See, e.g.,
http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a
string of X's or N's will result from a BLAST search. This is a
result of automatic filtering of the query for low-complexity
sequence that is performed to prevent artifactual hits. The filter
substitutes any low-complexity sequence that it finds with the
letter "N" in nucleotide sequence (e.g., "NNNNNNNNNN") or the
letter "X" in protein sequences (e.g., "XXXXXXXXX"). Low-complexity
regions can result in high scores that reflect compositional bias
rather than significant position-by-position alignment. (Wootton
and Federhen, Methods Enzymol 266:554-571, 1996).
[0050] The disclosed NOV1 a polypeptide (SEQ ID NO:2) encoded by
SEQ ID NO:1 has 3600 amino acid residues and is presented in Table
1B using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV1a has a signal peptide and is
likely to be localized extracellularly with a certainty of 0.8200.
In other embodiments, NOV1a may also be localized to the lysosome
(lumen) with acertainty of 0.1900, the endoplasmic reticulum
(membrane) with a certainty of 0.1000 or in the endoplasmic
reticulum (lumen) with a certainty of 0.1000. The most likely
cleavage site for a NOV1a peptide is between amino acids 14 and 15,
at: CVR-GP. TABLE-US-00003 TABLE 1B Encoded NOV1a protein sequence
(SEQ ID NO:2).
MAKRLCAGSALCVRGPRGPAPLLLHPPYFNLAEGARIAASATCGEEAPARGSPRPTEDLYCKLV
GGPVAGGDPNQTIQGQYCDICTAANSNKAHPASNAIDGTERWWQSPPLSRGLEYNEVNVTLDLG
QVFHVAYVLIKFANSPRPDLWVLERSMDFGRTYQPWQFFAASKRDCLERFGPQTLERITRDDAA
ICTTEYSRIVPLENGEIVVSLVNGRPGAMNFSYSPLLREFTKATNVRLRFLRTNTLLGHLMGKA
LRDPTVTRRYYYSTKDISIGGRCVCHGHADACDAKDPTDPFRLQCTCQHNTCGGTCDRCCPGFN
QQPWKPATANSANECQCECYGHATDCYYDPEVDRRRASQSLDGTYQGGGVCIDCQHHTTGVNCE
RCLPGFYRSPNHPLDSPHVCRGCNCESDFTDGTCEDLTGRCYCRPNFSGERCDVCAEGFTGFPS
CYREHLPGNDTREQVLPAGQIVSCDCSAAGTQGNACRKDPRVGRCLCKPNFQGTHCELCAPGFY
GPGCPASVPALEWPMTAVTLTQASAGAEWASRGPHVIAVPPATFTSLSASHPLRSAVCGCSPAG
TLPEGCDEAGRCLCQPEFAGPHCDRCRPGYHGFPNCAACTCDPRGALDQLCGAGGLCRCRPGYT
GTACQECSPGFHGFPSCPATALLKAPCTQPVTPGVGSAAAGPVRGCGVTHVCPVPTTSPTAKPL
FTAGSCHPAGLAPVDPALPEVSPPCMCRAHVEGPSCDRCKPGFWGLSPSNPEGCTRCSCDLRGT
LGGVAECQGTGQCFCKPHVCGQACASCKDGFFGLDQADYFGCRSCRCDIGGALGQSCEPRTGVC
RCRPNTQGPTCSEPARDHYLPDLHHLRLELEEAATPEGHAVRFGFNPLEFENFSWRGYAQMAPV
QPRIVARLNLTSPDLFWLVFRYVNRGAMSVSGRVSVREEGRSATCANCTAQSQPVAFPPSTEPA
FITVPQRGFGEPFVLNPGTWALRVEAEGVLLDYVVLLPSAYYEAALLQLRVTEACTYRPSAQQS
PPSCLLYTHLPLDGFPSAAGLEALCRQDNSLPRPCPTEQLSPSHPPLITCTGSDVDVQLQVAVP
QPGRYALVVEYANEDARQEVGVAVHTPQRAPQQCLLSLHPCLYSTLCRGTARDTQDHLAVFHLD
SEASVRLTAEQARFFLHGVTLVPIEEFSPEFVEPRVSCISSHGAFGPNSAACLPSRFPKPPQPI
ILRDCQVIPLPPGLPLTHAQDLTPAMSPAGPRPRPPTAVDPDAEPTLLREPQATVVFTTHVPTL
GRYAFLLHGYQPAHPTFPVEVLINAGRVWQGHANASFCPHGYGCRTLVVCEGQALLDVTHSELT
VTVRVPKGRWLWLDYVLVVPENVYSFGYLREEPLDKSYDFISHCAAQGYHISPSSSSLFCRNAA
ASLSLFYNNGARPCGCHEVGATGPTCEPFGGQCPCHAHVIGRDCSRCATGYWGFPNCRACDCGA
RLCDELTGQCICPPRTIPPDCLLCQPQTFGCHPLVGCEECNCSGPGIQELTDPTCDTDSGQCRC
RPNVTGRRCDTCSPGFHGYPRCRPCDCHEAGTAPGVCDPLTGQCYCKENVQGPKCDQCSLGTFS
LDAANPKGCTRCFCFGATERCRSSSYTRQEFVDMEGWVLLSTDRQVVPHERQPGTENLRADLRH
VPEAVPEAFPELYWQAPPSYLGDRVSSYGGTLRYELHSETQRGDVFVPMESRPDVVLQGNQMSI
TFLEPAYPTPGHVHRGQLQLVEGNFRHTETRNTVSREELMMVLASLEQLQIRALFSQISSAVFL
RRVALEVASPAGQGALASNVELCLCPASYRGDSCQECAPGFYRDVKGLFLGRCVPCQCHGHSDR
CLPGSGVCVCQHNTEGAHCERCQAGFVSSRDDPSAPCVSCPCPLSVPSNRCAPGFFGNPLVLGS
SCQPCDCSGNGDPNLLFSDCDPLTGACRGCLRHTTGPRCEICAPGFYGNALLPGNCTRCDCTPC
GTEACDPHSGHCLCKAGVTGRRCDRCQEGHFGFDGCGGCRPCACGPAAEGSECHPQSGQCHCRP
GTMGPQCRECAPGYWGLPEQGCRRCQCPGGRCDPHTGRCNCPPGLSGERCDTCSQQHQVPVPGG
PVGHSIHCEVCDHCVVLLLDDLERAGALLPAIHEQLRGINASSMAWARLHRLNASIADLQVLSV
LAFPPQPGPVQAFTFRLPQSQLRSPLGPRHETAQQLEVLEQQSTSLPPQAVGTRDQASQLLAGT
EATLGHAKTLLAAIRAVDRTLSELMSQTGHLGLANASAPSGEQLLRTLAEVERLLWENRARDLG
APQAAAEAELAAAQRVLARVQEQLSSLWEENQALATQTRDRLAQHEAGLMDLREALNRAVDATR
EAQELNSRNQERLEEALQRKQELSRDNATLQATLHAARDTLASVFRLLEGLSPLKFQELERLAA
SLDGARTPLLQRMQTFSPAGSKLRLVEAAEAHAQQLGQLALNLSIILDVNQDRLTQRAIEASNA
YSRILQAVQAAEDAAGQALQQADHTWQTVVRQGLVDRAQQLLANSTALEEAMLQEQQRLGLGEC
WAPMGALRPAGTQLRDVRAKKDQLEAHIQAAQAMLAMDTGETSKKIAHAKAVAAEAQDTATRVQ
SQLQAMQENVERWQGQYEGLRGQDLGQAVLDAGSAVSTLEKTLPQLLAKLSILENRGVHNASLA
LSASIGRVRELIAQARGAASKVVKVPMKFNGRSGVQLRTPRDLADLAAYTALKFYLQGPEPEPG
QGTEDRFVMYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSIDEDIGEQFAAVSLDRTLQ
FGHMSVTVERQMIQETKGDTVAPGAEGLLNLRPDDFVFYVGGYPSTFTPPPLLRFPGYRGCIEM
DTLNEEVVSLYNFERTFQLDTAVDRPCARSKSTGDPWLTDGSYLDGTGFARISFDSQISTTKRF
EQELRLVSYSGVLFFLKQQSQFLCLAVQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVF
LLGGSRKRVLVRVERAPVYSVEQDNDLELADAYYLGGVPPDQLPSLRRLFPTGGSVRGCVKGIK
ALGKYVDLKRLNTTGVSAGCTADLLVGRAMTFHGHGFLRLALSNVAPLTGNVYSGFGFHSAQDS
ALLYYRASPVRPHQVSLQQGRVSLQLLRTEVKTQAGFADGAPHYVAFYSNATGVWLYVDDQLQQ
MKPHRGPPPELQPQPEGPPRLLLGGLPESGTIYNFSGCISNVFVQRLLGPQRVFDLQQNLGSVN
VSTGCAPALQAQTPGLGPRQASRRSRQPARHPACMLPPHLRTTRDSYQFGGSLSSHLEFVGILA
RHRNVSVRWEKNRILLVTDGARAWSQEGPHRQHQGAEHPQPHTLFVGGLPASSHSSKLPVTVGF
SGCVKRLRLHGRPLGAPTRMAGVTPCILGPLEAGLFFPGSGGVITLGLPGATLPDVGLELEVRP
LAVTGLIFHLGQARTPPYLQLQVLPRQVLLRADDGAGEFSTSVTRPSVLCDGQWHRLAVMKSGN
VLRLEVDAQSNHTVGPLLAAAAGAPAPLYLGGLPEPMAVQPWPPAYCGCMRRLAVNRSPVAMTR
SVEVHGAVGASGCPAA
[0051] A search of sequence databases reveals that the NOV1a amino
acid sequence has 2566 of 3652 amino acid residues (70%) identical
to, and 2823 of 3652 amino acid residues (77%) similar to, the 3652
amino acid residue ptnr:>ptnr: SWISSNEW-ACC:T10053 laminin alpha
5 chain from mouse (E=0.0). Public amino acid databases include the
GenBank databases, SwissProt, PDB and PIR.
[0052] NOV1a is expressed in at least the following tissues: brain,
Prostate, ovary, kidney, melanocyte+heart+uterus, breast, head and
neck, stomach, genitourinary tract, pancreas, lung+testis+b-cell,
dorsal root ganglia. This information was derived by determining
the tissue sources of the sequences that were included in the
invention including but not limited to SeqCalling sources, Public
EST sources, Literature sources, and/or RACE sources.
[0053] NOV1b
[0054] A disclosed NOV1b nucleic acid of 3126 nucleotides (also
referred to as CG102167-01) encoding a novel human laminin alpha
5-like protein is shown in Table 1C. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
121-123 and ending with a TGA termination codon at nucleotides
2845-2847. The start and stop codons are in bold letters in Table
1C, and the 5' and 3' untranslated regions are underlined.
TABLE-US-00004 TABLE 1C NOV1b nucleotide sequence (SEQ ID NO:3).
TCAGGGGTGCAGCTGCGCACCCCACGGGATCTTGCCGACCTTGCTGCCTACACTGCCCTCAAGTTCTACCTG
CAGGGCCCAGAGCCTGAGCCTGGGCAGGGTACCGAGGATCGCTTTGTGATGTACATGGGCAGCCGCCAGGCC
ACTGGGGACTACATGGGTGTGTCTCTGCGTGACAAGAAGGTGCACTGGGTGTATCAGCTGGGTGAGGCGGGC
CCTGCAGTCCTAAGCATCGATGAGGACATTGGGGAGCAGTTCGCAGCTGTCAGCCTGGACAGGACTCTCCAG
TTTGGCCACATGTCCGTCACAGTGGAGAGACAGATGATCCAGGAAACCAAGGGTGACACGGTGGCCCCTGGG
GCAGAGGGGCTGCTCAACCTGCGGCCAGACGACTTCGTCTTCTACGTCGGGGGGTACCCCAGTACCTTCACG
CCCCCTCCCCTGCTTCGCTTCCCCGGCTACCGGGGCTGCATCGAGATGGACACGCTGAATGAGGAGGTGGTC
AGCCTCTACAACTTCGAGAGGACCTTCCAGCTGGACACGGCTGTGGACAGGCCTTGTGCCCGCTCCAAGTCG
ACCGGGGACCCGTGGCTCACGGACGGCTCCTACCTGGACGGCACCGGCTTCGCCCGCATCAGCTTCGACAGT
CAGATCAGCACCACCAAGCGCTTCGAGCAGGAGCTGCGGCTCGTGTCCTACAGCGGGGTGCTCTTCTTCCTG
AAGCAGCAGAGCCAGTTCCTGTGCTTGGCCGTGCAAGAAGGCAGCCTCGTGCTGTTGTATGACTTTGGGGCT
GGCCTGAAAAAGGCCGTCCCACTGCAGCCCCCACCGCCCCTGACCTCGGCCAGCAAGGCGATCCAGGTGTTC
CTGCTGGGGGGCAGCCGCAAGCGTGTGCTGGTGCGTGTGGAGCGGGCCACGGTGTACAGCGTGGAGCAGGAC
AATGATCTGGAGCTGGCCGACGCCTACTACCTGGGGGGCGTGCCGCCCGACCAGCTGCCCCCGAGCCTGCGA
TGGCTCTTCCCCACCGGAGGCTCAGTCCGTGGCTGCGTCAAAGGCATCAAGGCCCTGGGCAAGTATGTGGAC
CTCAAGCGGCTGAACACGACAGGCGTGAGCGCCGGCTGCACCGCCGACCTGCTGGTGGGGCGCGCCATGACT
TTCCATGGCCACGGCTTCCTTCGCCTGGCGCTCTCGAACGTGGCACCGCTCACTGGCAACGTCTACTCCGGC
TTCGGCTTCCACAGCGCCCAGGACAGTGCCCTGCTCTACTACCGGGCGTCCCCGGATGGGCTATGCCAGGTG
TCCCTGCAGCAGGGCCGTGTGAGCCTACAGCTCCTGAGGACTGAAGTGAAAACTCAAGCGGGCTTCGCCGAT
GGTGCCCCCCATTACGTCGCCTTCTACAGCAATGCCACGGGAGTCTGGCTGTATGTCGATGACCAGCTCCAG
CAGATGAAGCCCCACCGGGGACCACCCCCCGAGCTCCAGCCGCAGCCTGAGGGGCCCCCGAGGCTCCTCCTG
GGAGGCCTGCCTGAGTCTGGCACCATTTACAACTTCAGTGGCTGCATCAGCAACGTCTTCGTGCAGCGGCTC
CTGGGCCCACAGCGCGTATTTGATCTGCAGCAGAACCTGGGCAGCGTCAATGTGAGCACGGGCTGTGCACCC
GCCCTGCAAGCCCAGACCCCGGGCCTGGGGCCTAGAGGACTGCAGGCCACCGCCCGGAAGGCCTCCCGCCGC
AGCCGTCAGCCCGCCCGGCATCCTGCCTGCATGCTGCCCCCACACCTCAGGACCACCCGAGACTCCTACCAG
TTTGGGGGTTCCCTGTCCAGTCACCTGGAGTTTGTGGGCATCCTGGCCCGACATAGGAACTGGCCCAGTCTC
TCCATGCACGTCCTCCCGCGAAGCTCCCGAGGCCTCCTCCTCTTCACTGCCCGTCTGAGGCCCGGCAGCCCC
TCCCTGGCGCTCTTCCTGAGCAATGGCCACTTCGTTGCACAGATGGAAGGCCTCGGGACTCGGCTCCGCGCC
CAGAGCCGCCAGCGCTCCCGGCCTGGCCGCTGGCACAAGGTCTCCGTGCGCTGGGAGAAGAACCGGATCCTG
CTGGTGACGGACGGGGCCCGGGCCTGGAGCCAGGAGGGGCCGCACCGGCAGCACCAGGGGGCAGAGCACCCC
CAGCCCCACACCCTCTTTGTGGGCGGCCTCCCGGCCAGCAGCCACAGCTCCAAACTTCCGGTGACCGTCGGG
TTCAGCGGCTGTGTGAAGAGACTGAGGCTGCACGGGAGGCCCCTGGGGGCCCCCACACGGATGGCAGGGGTC
ACACCCTGCATCTTGGGCCCCCTGGAGGCGGGCCTGTTCTTCCCAGGCAGCGGGGGAGTTATCACTTTAGAC
CTCCCAGGAGCTACACTGCCTGATGTGGGCCTGGAACTGGAGGTGCGGCCCCTGGCAGTCACCGGACTGATC
TTCCACTTGGGCCAGGCCCGGACGCCCCCCTACTTGCAGTTGCAGGTCCTGCTGCGGGCGGATGACGGAGCA
GGGGAGTTCTCCACGTCAGTGACCCGCCCCTCAGTGCTGTGTGATGGCCAGTGGCACCGGCTAGCGGTGATG
AAAAGCGGGAATGTGCTCCGGCTGGAGGTGGACGCGCAGAGCAACCACACCGTGGGCCCCTTGCTGGCGGCT
GCAGCTGGTGCCCCAGCCCCTCTGTACCTCGGGGGCCTGCCTGAGCCCATGGCCGTGCAGCCCTGGCCCCCC
GCCTACTGCGGCTGCATGAGGAGGCTGGCGGTGAACCGGTCCCCCGTCGCCATGACTCGCTCTGTGGAGGTC
CACGGGCCAGTGGGGGCCAGTGGCTGCCCAGCCGCCTAGGACACAGCCAACCCCGGCCCCTGGTCAGGCCCC
TGCAGCTGCCTCACACCGCCCCTTGTGCTCGCCTCATAGGTGTCTATTTGGACTCTAAGCTCTACGGGTGAC
AGATCTTGTTTCTGAAGATGGTTTAAGTTATAGCTTCTTAAACGAAAGAATAAAATACTGCAAAATGTTTTT
ATATTTGGCCCTTCCACCCATTTTTAATTGTGAGAGATTTGTCACCAATCATCACTGGTTCCTCCTTAAAAA
TTAAAAAGTAACTTCTGTGTAAAAAAAAAA
[0055] In a search of public sequence databases, the NOV1b nucleic
acid sequence, located on chromsome 20 has 2495 of 2495 bases
(100%) identical to a gb:GENBANK-ID:HSLAMA5|acc:Z95636.1 mRNA from
Homo sapiens (H. sapiens mRNA for laminin alpha 5 chain) (E=0.0).
Public nucleotide databases include all GenBank databases and the
GeneSeq patent database.
[0056] The disclosed NOV1b polypeptide (SEQ ID NO:4) encoded by SEQ
ID NO:3 has 908 amino acid residues and is presented in Table 1B
using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV1b has no signal peptide and is
likely to be localized the microbody (peroxisome) with a certainty
of 0.5371. In other embodiments, NOV1b may also be localized to the
lysosome (lumen) with acertainty of 0.3191, the mitochondrial
matrix space with a certainty of 0.1000 or in the nucleus with a
certainty of 0.1000. TABLE-US-00005 TABLE 1D Encoded NOV1b protein
sequence (SEQ ID NO:4).
MYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSTDEDIGEQFAAVSLDRTLQFGHMSVTVERQMIQET
KGDTVAPGAEGLLNLRPDDFVPYVGGYPSTFTPPPLLRFPGYRGCIEMDTLNEEVVSLYNFERTFQLDTAVD
RPCARSKSTGDPWLTDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSGVLFFLKQQSQFLCLAVQEGSL
VLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVRVERATVYSVEQDNDLELADAYYLGGVPP
DQLPPSLRWLFPTGGSVRGCVKGIKALGKYVDLKRLNTTGVSAGCTADLLVGRAMTFHGHGFLRLALSNVAP
LTGNVYSGFGPHSAQDSALLYYRASPDGLCQVSLQQGRVSLQLLRTEVKTQAGFADGAPHYVAFYSNATGVW
LYVDDQLQQMKPHRGPPPELQPQPEGPPRLLLGGLPESGTIYNFSGCISNVFVQRLLGPQRVFDLQQNLGSV
NVSTGCAPALQAQTPGLGPRGLQATARKASRRSRQPARHPACMLPPHLRTTRDSYQFGGSLSSELEFVGILA
RHRNWPSLSMHVLPRSSRGLLLFTARLRPGSPSLALFLSNGHFVAQMEGLGTRLRAQSRQRSRPGRWHKVSV
RWEKNRILLVTDGARAWSQEGPHRQHQGAEHPQPHTLFVGGLPASSESSKLPVTVGFSGCVKRLRLHGRPLG
APTRMAGVTPCILGPLEAGLFFPGSGGVITLDLPGATLPDVGLELEVRPLAVTGLIFHLGQARTPPYLQLQV
LLRADDGAGEFSTSVTRPSVLCDGQWHRLAVMKSGNVLRLEVDAQSNHTVGPLLAAAAGAPAPLYLGGLPEP
MAVQPWPPAYCGCMRRLAVNRSPVAMTRSVEVHGAVGASGCPAA
[0057] A search of sequence databases reveals that the NOV1b amino
acid sequence has 908 of 913 amino acid residues (99%) identical
to, and 908 of 913 amino acid residues (99%) similar to, the 1645
amino acid residue ptnr:SWISSNEW-ACC:015230 protein from Homo
sapiens (Human) (Laminin Alpha-5 Chain) (E=0.0). Public amino acid
databases include the GenBank databases, SwissProt, PDB and
PIR.
[0058] NOV1b is expressed in at least the following tissues: brain,
Prostate, ovary, kidney, melanocyte, heart, uterus, breast, head
and neck, stomach, genitourinary tract, pancreas, lung, testis,
b-cell, dorsal root ganglia. Expression information was derived
from the tissue-sources of the sequences that were included in the
derivation of the sequence of CuraGen Acc. No. CG102167-01. The
sequence is predicted to be expressed in placenta because of the
expression pattern of (GENBANK-ID:
gb:GENBANK-ID:HSLAMA5|acc:Z95636.1) a closely related H. sapiens
mRNA for laminin alpha 5 chain.
[0059] NOV1c
[0060] A disclosed NOV1c nucleic acid of 10800 nucleotides (also
referred to as CG55974-02) encoding a novel human laminin alpha
5-like protein is shown in Table 1E. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
1-3 and ending with a TGA termination codon at nucleotides
10792-10794. The start and stop codons are in bold letters in Table
1E, and the 5' and 3' untranslated regions are underlined. Since
the start codon of NOV1c is not a traditional initiation codon, and
NOV1c has no termination codon, NOV1c could be a partial open
reading frame that could be extended in the 5' and/or 3'
direction(s). TABLE-US-00006 TABLE 1E NOV1c nucleotide sequence
(SEQ ID NO:5).
ATGGCGAAGCGGCTCTGCGCGGGGAGCGCACTGTGTGTTCGCGGCCCCCGGGGCCCCGCGCCGCTGCTGCTG
CACCCGCCCTACTTCAACCTGGCCGAGGGCGCCCGCATCGCCGCCTCCGCGACCTGCGGAGAGGAGGCCCCG
GCGCGCGGCTCCCCGCGCCCCACCGAGGACCTTTACTGCAAGCTGGTAGGGGGCCCCGTGGCCGGCGGCGAC
CCCAACCAGACCATCCAGGGCCAGTACTGTGACATCTGCACGGCTGCCAACAGCAACAAGGCACACCCCGCG
AGCAATGCCATCGATGGCACGGAGCGCTGGTGGCAGAGTCCACCGCTGTCCCGCGGCCTGGAGTACAACGAG
GTCAACGTCACCCTGGACCTGGGCCAGGTCTTCCACGTGGCCTACGTCCTCATCAAGTTTGCCAACTCACCC
CGGCCGGACCTCTGGGTGCTGGAGCGGTCCATGGACTTCGGCCGCACCTACCAGCCCTGGCAGTTCTTTGCC
GCCTCCAAGAGGGACTGTCTGGAGCGGTTCGGGCCACAGACGCTGGAGCGCATCACACGGGACGACGCGGCC
ATCTGCACCACCGAGTACTCACGCATCGTGCCCCTGGAGAACGGAGAGATCGTGGTGTCCCTGGTGAACGGA
CGTCCGGGCGCCATGAATTTCTCCTACTCGCCGCTGCTACGTGAGTTCACCAAGGCCACCAACGTCCGCCTG
CGCTTCCTGCGTACCAACACGCTGCTGGGCCATCTCATGGGGAAGGCGCTGCGGGACCCCACGGTCACCCGC
CCGTATTATTACAGCATCAAGGATATCAGCATCGGAGGCCGCTGTGTCTGCCACGGCCACGCGGATGCCTGC
GATGCCAAAGACCCCACGGACCCGTTCAGGCTGCAGTGCACCTGCCAGCACAACACCTGCGGGGGCACCTGC
GACCGCTGCTGCCCCGGCTTCAATCAGCAGCCGTGGAAGCCTGCGACTGCCAACAGTGCCAACGAGTGCCAG
TGTGAGTGCTACGGCCATGCCACCGACTGTTACTACGACCCTGAGGTGGACCGGCGCCGCGCCAGCCAGAGC
CTGGATGGCACCTATCAGGGTGGGGGTGTCTGTATCGACTGCCAGCACCACACCACCGGCGTCAACTGTGAG
CGCTGCCTGCCCGGCTTCTACCGCTCTCCCAACCACCCTCTCGACTCGCCCCACGTCTGCCGCGGCTGCAAC
TGCGAGTCCGACTTCACGGATGGCACCTGCGAGGACCTGACGGGTCGATGCTACTGCCGGCCCAACTTCTCT
GGGGAGCGGTGTGACGTGTGTGCCGAGGGCTTCACGGGCTTCCCAAGCTGCTACCGTGAGCACCTGCCAGGG
AATGACACCAGGGAGCAGGTGCTGCCAGCCGGCCAGATTGTGAGTTGTGACTGCAGCGCGGCAGGGACCCAG
GGCAACGCCTGCCGGAAGGACCCAAGGGTGGGACGCTGTCTGTGCAAACCCAACTTCCAAGGCACCCATTGT
GAGCTCTGCGCGCCAGGGTTCTACGGCCCCGGCTGCCCTGCCAGTGTTCCAGCCCTGGAGTGGCCGATGACC
GCTGTGACCCTGACACAGGCCAGTGCAGGTGCCGAGTGGGCTTCGAGGGGGCCACATGTGATCGCTGTGCCC
CCGGCTACTTTCACTTCCCTCTCTGCCAGTCACCCGCTCCGCTCTGCAGTGTGTGGCTGCAGCCCTGCAGGA
ACCTTGCCCGAGGGCTGCGATGAGGCCGGCCGCTGCCTATGCCAGCCTGAGTTTGCTGGACCTCATTGTGAC
CCGTGCCGCCCTGGCTACCATGGTTTCCCCAACTGCGCAGCATGCACCTGCGACCCTCGGGGAGCCCTGGAC
CAGCTCTGTGGGGCGGGAGGTTTGTGCCGCTGCCGCCCCGGCTACACAGGCACTGCCTGCCAGGAATGCAGC
CCCGGCTTTCACGGCTTCCCCAGCTGTCCTGCCACTGCTCTGCTGAAGGCTCCCTGCACGCAGCCTGTGACC
CCCGGAGTGGGCAGTGCAGCTGCCGGCCCCGTGCGGGGCTGCGGTGTGACACGTGTGTGCCCGGTGCCTACA
ACTTCCCCTACTGCGAAGCTGGCTCTTGCCACCCTGCCGGTCTGGCCCCCAGTGGATCCTGCCCTTCCTGAG
GCACAGGTTCCCTGTATGTGCCGGGCTCACGTGGAGGGGCCGAGCTGTGACCGCTGCAAACCTGGGTTCTGG
GGACTGAGCCCCAGCAACCCCGAAGGCTGTACCCGCTGCAGCTGCGACCTCAGGGGCACACTGGGTGGAGTT
GCTGAGTGCCAGCCGGGCACCGGCCAGTGCTTCTGCAAGCCCCACGTGTGCGGCCAGGCCTGCGCGTCCTGC
AAGGATGGCTTCTTTGGACTGGATCAGGCTGACTATTTTGGCTGCCGCAGCTGCCGGTGTGACATTGGCGGT
GCACTGGGCCAGAGCTGTGAACCGAGGACGGGCGTCTGCCGGTGCCGCCCCAACACCCAGGGCCCCACCTGC
AGCGAGCCTGCGAGGGACCACTACCTCCCGGACCTGCACCACCTGCGCCTGGAGCTGGAGGAGGCTGCCACA
CCTGAGGGTCACGCCGTGCGCTTTGGCTTCAACCCCCTCGAGTTCGAGAACTTCAGCTGGAGGGGCTACGCG
CAGATGGCACCTGTCCAGCCCAGGATCGTGGCCAGGCTGAACCTGACCTCCCCCGACCTTTTCTGGCTCGTC
TTCCGATACGTCAACCGGGGGGCCATGAGTGTGAGCGGGCGGGTCTCTGTGCGAGAGGAGGGCAGGTCGGCC
GCCTGTGCCAACTGCACAGCACAGAGTCAGCCCGTGGCCTTCCCACCCAGCACGGAGCCTGCCTTCATCACC
GTGCCCCAGAGGGGCTTCGGAGAGCCCTTTGTGCTGAACCCTGGCACCTGGGCCCTGCGTGTGGAGGCCGAA
GGGGTGCTCCTGGACTACGTGGTTCTGCTGCCTAGCGCATACTACGAGGCGGCGCTCCTGCAGCTGCGGGTG
ACTGAGGCCTGCACATACCGTCCCTCTGCCCAGCAGTCTCCCCCCAGCTGCCTCCTCTACACACACCTCCCC
CTGGATGGCTTCCCCTCGGCCGCCGGGCTGGAGGCCCTGTGTCGCCAGGACAACAGCCTGCCCCGGCCCTGC
CCCACGGAGCAGCTCAGCCCGTCGCACCCGCCACTGATCACCTGCACGGGCAGTGATGTGGACGTCCAGCTT
CAAGTGGCAGTGCCACAGCCAGGCCGCTATGCCCTAGTGGTGGAGTACGCCAATGAGGATGCCCGCCAGGAG
GTGGGCGTGGCCGTGCACACCCCACAGCGGGCCCCCCAGCAGGGGCTGCTCTCCCTGCACCCCTGCCTGTAC
AGCACCCTGTGCCGGGGCACTGCCCGGGATACCCAGGACCACCTGGCTGTCTTCCACCTGGACTCGGAGGCC
AGCGTGAGGCTCACAGCCGAACAGGCACGCTTCTTCCTGCACGGGGTCACTCTGGTGCCCATTGAGGAGTTC
AGCCCGGAGTTCGTGGAGCCCCGGGTCAGCTGCATCAGCAGCCACGGCGCCTTTGGCCCCAACAGTGCCGCC
TGTCTGCCCTCGCGCTTCCCAAAGCCGCCCCAGCCCATCATCCTCAGGGACTGCCAGGTGATCCCGCTGCCG
CCCGGCCTCCCGCTGACCCACGCGCAGGATCTCACTCCAGCCATGTCCCCAGCTGGACCCCGACCTCGGCCC
CCCACCGCTGTGGACCCTGATGCAGAGCCCACCCTGCTGCGTGAGCCCCAGGCCACCGTGGTCTTCACCACC
CATGTGCCCACGCTGGGCCGCTATGCCTTCCTGCTGCACGGCTACCAGCCAGCCCACCCCACCTTCCCCGTG
GAAGTCCTCATCAACGCCGGCCGCGTGTGGCAGGGTCACGCCAACGCCAGCTTCTGTCCACATGGCTACGGC
TGCCGCACCCTGGTGGTCTGTGAGGGCCAGGCCCTGCTGGACGTGACCCACAGCGAGCTCACTGTGACCGTG
CGTGTGCCCAAGGGCCGGTGGCTCTGGCTGGATTATGTACTCGTGGTCCCTGAGAACGTCTACAGCTTTGGC
TACCTCCGGGAGGAGCCCCTGGATAAATCCTATGACTTCATCAGCCACTGCGCAGCCCAGGGCTACCACATC
AGCCCCAGCAGCTCATCCCTGTTCTGCCGAAACGCTGCTGCTTCCCTCTCCCTCTTCTATAACAACGGAGCC
CGTCCATGTGGCTGCCACGAAGTAGGTGCTACAGGCCCCACGTGTGAGCCCTTCGGGGGCCAGTGTCCCTGC
CATGCCCATGTCATTGGCCGTGACTGCTCCCGCTGTGCCACCGGATACTGGGGCTTCCCCAACTGCAGGGCC
TGTGACTGCGGTGCCCGCCTCTGTGACGAGCTCACGGGCCAGTGCATCTGCCCGCCACGCACCATCCCGCCC
GACTGCCTGCTGTGCCAGCCCCAGACCTTTGGCTGCCACCCCCTGGTCGGCTGTGAGGAGTGTAACTGCTCA
GGGCCCGGCATCCAGGAGCTCACAGACCCTACCTGTGACACAGACAGCGGCCAGTGCAGGTGCAGACCCAAC
GTGACTGGGCGCCGCTGTGATACCTGCTCTCCGGGCTTCCATGGCTACCCCCGCTGCCGCCCCTGTGACTGT
CACGAGGCGGGCACTGCGCCTGGCGTGTGTGACCCCCTCACAGGGCAGTGCTACTGTAAGGAGAACGTGCAG
GGCCCCAAATGTGACCAGTGCAGCCTTGGGACCTTCTCACTGGATGCTGCCAACCCCAAAGGTTGCACCCGC
TGCTTCTGCTTTGGGGCCACGGAGCGCTGCCGGAGCTCGTCCTACACCCGCCAGGAGTTCGTGGATATGGAG
GGATGGGTGCTGCTGAGCACTGACCGGCAGGTGGTGCCCCACGAGCGGCAGCCAGGGACGGAGATGCTCCGT
GCAGACCTGCGGCACGTGCCTGAGGCTGTGCCCGAGGCTTTCCCCGAGCTGTACTGGCAGGCCCCACCCTCC
TACCTGGGGGACCGGGTAAGCTCCTACGGTGGGACCCTCCGTTATGAACTGCACTCAGAGACCCAGCGGGGA
GATGTCTTTGTCCCCATGGAGAGCAGGCCGGATGTGGTGCTGCAGGGCAACCAGATGAGCATCACATTCCTG
GAGCCGGCATACCCCACGCCTGGCCACGTTCACCGTGGGCAGCTGCAGCTGGTGGAGGGGAACTTCCGGCAT
ACGGAGACGCGCAACACTGTGTCCCGCGAGGAGCTCATGATGGTGCTGGCCAGCCTGGAGCAGCTGCAGATC
CGTGCCCTCTTCTCACAGATCTCCTCGGCTGTCTTCCTGCGCAGGGTGGCACTGGAGGTGGCCAGCCCAGCA
GGCCAGGGGGCCCTGGCCAGCAATGTGGAGCTGTGCCTGTGCCCCGCCAGCTACCGGGGGGACTCATGCCAG
GAATGTGCCCCCGGCTTCTATCGGGACGTCAAAGGTCTCTTCCTGGGCCGATGTGTGCCTTGTCAGTGCCAT
GGACACTCAGACCGCTGCCTCCCTGGCTCTGGCGTCTGTGTGTGCCAGCACAACACCGAAGGGGCCCACTGT
GAGCGCTGCCAGGCTGGCTTCGTGAGCAGCAGGGACGACCCCAGCGCCCCCTGTGTCAGCTGCCCCTGCCCC
CTCTCAGTGCCTTCCAACAGGTGTGCGCCCGGATTCTTTGGGAACCCAdTGGTGCTGGGCAGCTCCTGCCAG
CCATGCGACTGCAGCGGCAACGGTGACCCCAACTTGCTCTTCAGCGACTGCGACCCCCTGACGGGCGCCTGC
CGTGGCTGCCTGCGCCACACCACTGGGCCCCGCTGCGAGATCTGTGCCCCCGGCTTCTACGGCAACGCCCTG
CTGCCCGGCAACTGCACCCGTTGCGACTGTACCCCATGTGGGACAGAGGCCTGCGACCCCCACAGCGGGCAC
TGCCTGTGCAAGGCGGGCGTGACTGGGCGGCGCTGTGACCGCTGCCAGGAGGGACATTTTGGTTTCGATGGC
TGCGGGGGCTGCCGCCCGTCTGCTTGTGGACCGGCCGCCGAGGGCTCCGAGTGCCACCCCCAGAGCGGACAG
TGCCACTGCCGACCAGGGACCATGGGACCCCAGTGCCGCGAGTGTGCCCCTGGCTACTGGGGGCTCCCTGAG
CAGGGCTGCAGGCGTTGCCAGTGCCCTGGGGGCCGCTGTGACCCTCACACGGGCCGCTGCAACTGCCCCCCG
GGGCTCAGCGGGGAGCGCTGCGACACCTGCAGCCAGCAGCATCAGGTGCCTGTTCCAGGCGGGCCTGTGGGC
CACAGCATCCACTGTGAAGTGTGTGACCACTGTGTGGTCCTGCTCCTGGATGACCTGGAACGGGCCGGCGCC
CTCCTCCCCGCCATTCACGAGCAACTGCGTGGCATCAATGCCAGCTCCATGGCCTGGGCCCGTCTGCACAGG
CTGAACGCCTCCATCGCTGACCTGCAGGTACTGAGCGTCCTGGCCTTCCCTCCCCAACCCGGGCCAGTGCAG
GCCTTCACCTTTCGCCTCCCACAGAGCCAGCTCCGGAGCCCCCTGGGCCCCCGCCATGAGACGGCACAGCAG
CTGGAGGTGCTGGAGCAGCAGAGCACAAGCCTTCCTCCACAGGCCGTGGGGACCCGAGACCAGGCGAGCCAA
TTGCTGGCCGGCACCGAGGCCACACTGGGCCATGCGAAGACGCTGTTGGCGGCCATCCGGGCTGTGGACCGC
ACCCTGAGCGAGCTCATGTCCCAGACGGGCCACCTGGCGCTGGCCAATGCCTCGGCTCCATCAGGTGAGCAG
CTGCTCCGGACACTGGCCGAGGTGGAGCGGCTGCTCTGGGAGATGCGGGCCCGGGACCTGGGGGCCCCGCAG
GCAGCAGCTGAGGCTGAGTTGGCTGCAGCACAGAGAGTGCTGGCCCGGGTGCAGGAGCAGCTGAGCAGCCTC
TGGGAGGAGAACCAGGCACTGGCCACACAAACCCGCGACCGGCTGGCCCAGCACGAGGCCGGCCTCATGGAC
CTGCGAGAGGCTTTGAACCGGGCAGTGGACGCCACACGGGAGGCCCAGGAGCTCAACAGCCGCAACCAGGAG
CGCCTGGAGGAAGCCCTGCAAAGGAAGCAGGAGCTGTCCCGGGACAATGCCACCCTGCAGGCCACTCTGCAT
GCGGCTAGGGACACCCTGGCCAGCGTCTTCAGATTGCTGGAGGGGCTAAGTCCACTCAAATTCCAGGAGCTG
GAGCGCCTCGCCGCCAGCCTGGATGGGGCTCGGACCCCACTGCTGCAGAGGATGCAGACCTTCTCCCCGGCG
GGCAGCAAGCTGCGTCTAGTGGAGGCCGCCGAGGCCCACGCACAGCAGCTGGGCCAGCTGGCACTCAATCTG
TCCATCATCCTGGACGTCAACCAGGACCGCCTCACCCAGAGGGCCATCGAGGCCTCCAACGCCTACAGCCGC
ATCCTGCAGGCCGTGCAGGCTGCCGAGGATGCTGCTGGCCAGGCCCTGCAGCAGGCGGACCACACGTGGCAG
ACGGTGGTGCGGCAGGGCCTGGTGGACCGAGCCCAGCAGCTCCTGGCCAACAGCACTGCACTAGAAGAGGCC
ATGCTCCAGGAACAGCAGAGGCTGGGCCTTGGTGAGTGCTGGGCTCCGATGGGGGCCCTTAGGCCTGCTGGG
ACCCAGCTCCGAGATGTCCGGGCCAAGAAGCACCAGCTGGAGGCGCACATCCAGGCGGCGCAGGCCATGCTT
GCCATGGACACAGGTGAGACAAGCAAGAAGATCGCACATGCCAAGGCTGTGGCTGCTGAAGCCCAGGACACC
GCCACCCGTGTGCAGTCCCAGCTGCAGGCCATGCAGGAGAATGTGGAGCGGTGGCAGGGCCAGTACGAGGGC
CTGCGGGGCCAGGACCTGGGCCAGGCAGTGCTTGACGCAGGCTCTGCAGTGTCCACCCTGGAGAAGACGCTG
CCCCAGCTGCTGGCCAAGCTGAGCATCCTGGAGAACCGTGGGGTGCACAACGCCAGCCTGGCCCTGTCCGCC
AGCATTGGCCGCGTGCGAGAGCTCATTGCCCAGGCCCGGGGGGCTGCCAGTAAGGTGGTCAAGGTGCCCATG
AAGTTCAACGGGCGCTCAGGGGTGCAGCTGCGCACCCCACGGGATCTTGCCGACCTTCCTGCCTACACTGCC
CTCAAGTTCTACCTGCAGGGCCCAGAGCCTGAGCCTGGGCAGGGTACCGAGGATCGCTTTGTGATGTACATG
GGCAGCCGCCAGGCCACTGGGGACTACATGGGTGTGTCTCTGCGTGACAAGAAGGTGCACTGGGTGTATCAG
CTGGCTGAGGCGGGCCCTGCAGTCCTAAGCATCGATGAGGACATTGGGGAGCAGTTCGCAGCTGTCAGCCTG
GACAGGACTCTCCAGTTTGGCCACATGTCCGTCACAGTGGAGAGACAGATGATCCAGGAAACCAAGGGTGAC
ACGGTGGCCCCTGGGGCAGAGGGGCTGCTCAACCTGCGGCGAGACGACTTCGTCTTCTACGTCGGGGGGTAC
CCCAGTACCTTCACGCCCCCTCCCCTGCTTCGCTTCCCCGGCTACCGGGGCTGCATCGAGATGGACACGCTG
AATGAGGAGGTGGTCAGCCTCTACAACTTCGAGAGGACCTTCCAGCTGGACACGGCTGTGGACAGGCCTTGT
GCCCGGTCCAAGTCGACCGGGGACCCGTGGCTCACGGACGGCTCCTACCTGGACGGCACCGGCTTCGCCCGC
ATCAGCTTCGACAGTCAGATCAGCACCACCAAGCGCTTCGAGCAGGAGCTGCGGCTCGTGTCCTACAGCGGG
GTGCTCTTCTTCCTGAAGCAGCAGAGCCAGTTCCTGTGCTTGGCCGTGCAAGAAGGCAGCCTCGTGCTGTTG
TATGACTTTGGGCCTGGCCTGAAAAAGGCCGTCCCACTGCAGCCCCCACCGCCCCTGACCTCGGCCAGCAAG
GCGATCCAGGTGTTCCTGCTGGGGGGCAGCCGCAAGCGTGTGCTGGTGCGTGTGGAGCGGGCCACGGTGTAC
AGCGTGGAGCAGGACAATGATCTGGAGCTGGCCGACGCCTACTACCTGGGGGGCGTGCCGCCCGACCAGCTG
CCCAGCCTGCGACGGCTCTTCCCCACCGGAGGCTCAGTCCGTGGCTGCGTCAAAGGCATCAAGGCCCTGGGC
AAGTATGTGGACCTCAAGCGGCTGAACACGACAGGCGTGAGCGCCGGCTGCACCGCCGACCTGCTGGTGGGG
CGCGCCATGACTTTCCATGGCCACGGCTTCCTTCGCCTGGCGCTCTCGAACGTGGCACCGCTCACTGGCAAC
GTCTACTCCGGCTTCGGCTTCCACAGCGCCCAGGACAGTGCCCTGCTCTACTACCGGGCGTCCCCGGTGAGA
CCTCACCAGGTGTCCCTGCAGCAGGGCCGTGTGAGCCTACAGCTCCTGAGGACTGAAGTGAAAACTCAAGCG
GGCTTCGCCGATGGTGCCCCCCATTACGTCGCCTTCTACAGCAATGCCACGGGGGTCTGGCTGTATGTCGAT
GACCAGCTCCAGCAGATGAAGCCCCACCGGGGACCACCCCCCGAGCTCCAGCCGCAGCCTGAGGGGCCCCCG
AGGCTCCTCCTGGGAGGCCTGCCTGAGTCTGGCACCATTTACAACTTCAGTGGCTGCATCAGCAACGTCTTC
GTGCAGCGGCTCCTGGGCCCACAGCGCGTATTTGATCTGCAGCAGAACCTGGGCAGCGTCAATGTGAGCACG
GGCTGTGCACCCGCCCTGCAAGCCCAGACCCCGGGCCTGGGGCCTAGACAGGCCTCCCGCCGCAGCCGTCAG
CCCGCCCGGCATCCTGCCTGCATGCTGCCCCCACACCTCAGGACCACCCGAGACTCCTACCAGTTTGGGGGT
TCCCTGTCCAGTCACCTGGAGTTTGTGGGCATCCTGGCCCGACATAGGAACGTCTCCGTGCGCTGGGAGAAG
AACCGGATCCTGCTGGTGACGGACGGGGCCCGGGCCTGGAGCCAGGAGGGGCCGCACCGGCAGCACCAGGGG
GCAGAGCACCCCCAGCCCCACACCCTCTTTGTGGGCGGCCTCCCGGCCAGCAGCCACAGCTCCAAACTTCCG
GTGACCGTCGGGTTCAGCGGCTGTGTGAAGAGACTGAGGCTGCACGGGAGGCCCCTGGGGGCCCCCAGACGG
ATGGCAGGGGTCACACCCTGCATCTTGGGCCCCCTGGAGGCGGGCCTGTTCTTCCCAGGCAGCCGGGGAGTT
ATCACTTTAGGTCTGCCAGGAGCTACACTGCCTGATGTGGGCCTGGAACTGGAGGTGCGGCCCCTGGCAGTC
ACCGGACTGATCTTCCACTTGGGCCAGGCCCGGACGCCCCCCTACTTGCAGTTGCAGGTGCTACCCCGCCAG
GTCCTGCTGCGGGCGGATGACGGAGCACGGGAGTTCTCCACGTCAGTGACCCGCCCCTCAGTGCTGTGTGAT
GGCCAGTGGCACCGGCTAGCGGTGATGAAAAGCGGGAATGTGCTCCGGCTGGAGGTGGACGCGCAGAGCAAC
CACACCGTGGGCCCCTTGCTGGCGGCTGCAGCTGGTGCCCCAGCCCCTCTGTACCTCGGGGGCCTGCCTGAG
CCCATGGCCGTGCAGCCCTGGCCCCCCGCCTACTGCGGCTGCATGACGAGGCTGGCGGTGAACCGGTCCCCC
GTCGCCATGACTCGCTCTGTGGAGGTCCACGGGGCAGTGGGGGCCAGTGGCTGCCCAGCCGCCTAGAATAAA
[0061] In a search of public sequence databases, the NOV1c nucleic
acid sequence, located on chromsome 20 has 3800 of 4840 bases (78%)
identical to a gb:GENBANK-ID:MMU37501 |acc:U37501.1 mRNA from Mus
musculus (Mus musculus laminin alpha 5 chain (Lama5) mRNA, partial
cds) (E=0.0). Public nucleotide databases include all GenBank
databases and the GeneSeq patent database.
[0062] The disclosed NOV1c polypeptide (SEQ ID NO:6) encoded by SEQ
ID NO:5 has 3597 amino acid residues and is presented in Table 1F
using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV1c has a signal peptide and is
likely to be localized the mitochondrial matrix space with a
certainty of 0.4318. In other embodiments, NOV1c may also be
localized to the microbody (peroxisome) with acertainty of 0.3000,
the lysosome (lumen) with a certainty of 0.2055 or in the
mitochondrial inner membrane with a certainty of 0.1122. The most
likely cleavage site for NOV1c is between positions 14 and 15:
CVR-GP TABLE-US-00007 TABLE 1F Encoded NOV1c protein sequence (SEQ
ID NO:6).
MAKRLCAGSALCVRGPRGPAPLLLHPPYFNLAEGARIAASATCGEEAPARGSPRPTEDLYCKLVGGPVAGGD
PNQTIQGQYCDICTAANSNKAHPASNAIDGTERWWQSPPLSRGLEYNEVNVTLDLGQVFEVAYVLIKFANSP
RPDLWVLERSMDFGRTYQPWQFFAASKRDCLERFGPQTLERITRDDAAICTTEYSRIVPLENGEIVVSLVNG
RPGAMNFSYSPLLREFTKATNVRLRFLRTNTLLGHLMGKALRDPTVTRRYYYSIKDISIGGRCVCHGHADAC
DAKDPTDPFRLQCTCQHNTCGGTCDRCCPGFNQQPWKPATANSANECQCECYGEATDCYYDPEVDRRRASQS
LDGTYQGGGVCIDCQHHTTGVNCERCLPGFYRSPNHPLDSPHVCRGCNCESDFTDGTCEDLTGRCYCRPNFS
GERCDVCAEGFTGFPSCYREHLPGNDTREQVLPAGQTVSCDCSAAGTQGNACRKDPRVGRCLCKPNFQGTHC
ELCAPGFYGPGCPASVPALEWPMTAVTLTQASAGAEWASRGPHVIAVPPATFTSLSASEPLRSAVCGCSPAG
TLPEGCDEAGRCLCQPEFAGPHCDRCRPGYHGFPNCAACTCDPRGALDQLCGAGGLCRCRPGYTGTACQECS
PGFHGFPSCPATALLKAPCTQPVTPGVGSAAAGPVRGCGVTRVCPVPTTSPTAKLALATLPVWPPVDPALPE
AQVPCMCRAHVEGPSCDRCKPGFWGLSPSNPEGCTRCSCDLRGTLGGVAECQPGTGQCFCKPNVCGQACASC
KDGFFGLDQADYFGCRSCRCDIGGALGQSCEPRTGVCRCRPNTQGPTCSEPARDHYLPDLHHLRLELEEAAT
PEGHAVRFGFNPLEFENFSWRGYAQMAPVQPRIVARLNLTSPDLFWLVFRYVNRGAMSVSGRVSVREEGRSA
ACANCTAQSQPVAFPPSTEPAFITVPQRGFGEPFVLNPGTWALRVEAEGVLLDYVVLLPSAYYEAALLQLRV
TEACTYRPSAQQSPPSCLLYTHLPLDGFPSAAGLEALCRQDNSLPRPCPTEQLSPSHPPLITCTGSDVDVQL
QVAVPQPGRYALVVEYANEDARQEVGVAVHTPQRAPQQGLLSLHPCLYSTLCRGTARDTQDHLAVFELDSEA
SVRLTAEQARFELEGVTLVPTEEFSPEFVEPRVSCISSHGAFGPNSAACLPSRFPKPPQPTILRDCQVIPLP
PGLPLTHAQDLTPAMSPAGPRPRPPTAVDPDAEPTLLREPQATVVFTTHVPTLGRYAFLLHGYQPAHPTFPV
EVLINAGRVWQGHANASFCPHGYGCRTLVVCEGQALLDVTHSELTVTVRVPKGRWLWLDYVLVVPENVYSFG
YLREEPLDKSYDFISHCAAQCYHISPSSSSLFCRNAAASLSLFYNNGARPCGCHEVGATGPTCEPFGGQCPC
HAHVIGRDCSRCATGYWGFPNCRACDCGARLCDELTGQCICPPRTIPPDCLLCQPQTFGCEPLVGCEECNCS
GPGIQELTDPTCDTDSGQCRCRPNVTGRRCDTCSPGFHGYPRCRPCDCHEAGTAPGVCDPLTGQCYCKENVQ
GPKCDQCSLGTFSLDAANPKGCTRCFCFGATERCRSSSYTRQEFVDMEGWVLLSTDRQVVPHERQPGTEMLR
ADLRHVPEAVPEAFPELYWQAPPSYLGDRVSSYGGTLRYELHSETQRGDVFVPNESRPDVVLQGNQMSITFL
EPAYPTPGHVHRGQLQLVEGNFRHTETRNTVSREELNMVLASLEQLQIRALFSQISSAVPLRRVALEVASPA
GQGALASNVELCLCPASYRGDSCQECAPGFYRDVKGLFLGRCVPCQCHGHSDRCLPGSGVCVCQHNTEGAHC
ERCQAGFVSSRDDPSAPCVSCPCPLSVPSNRCAPGFFGNPLVLGSSCQPCDCSGNGDPNLLFSDCDPLTGAC
RGCLRETTGPRCEICAPGFYGNALLPGNCTRCDCTPCGTEACDPHSGECLCKAGVTGRRCDRCQEGNFGFDG
CGGCRPCACGPAAEGSECHPQSGQCECRPGTMGPQCRECAPGYWGLPEQGCRRCQCPGGRCDPETGRCNCPP
GLSGERCDTCSQQHQVPVPGGPVGHSIHCEVCDHCVVLLLDDLERAGALLPAIHEQLRGINASSMAWARLHR
LNASIADLQVLSVLAFPPQPGPVQAPTPRLPQSQLRSPLGPRHETAQQLEVLEQQSTSLPPQAVGTRDQASQ
LLAGTEATLGEAKTLLAAIRAVDRTLSELMSQTGHLGLANASAPSGEQLLRTLAEVERLLWEMRARDLCAPQ
AAAEAELAAAQRVLARVQEQLSSLWEENQALATQTRDRLAQHEACLMDLREALNRAVDATREAQELNSRNQE
RLEEALQRKQELSRDNATLQATLHAARDTLASVFRLLEGLSPLKFQELERLAASLDGARTPLLQEMQTFSPA
GSKLRLVEAAEAHAQQLGQLALNLSIILDVNQDRLTQRAIEASNAYSRILQAVQAAEDAAGQALQQADHTWQ
TVVRQGLVDRAQQLLANSTALEEAMLQEQQRLGLGECWAPMGALRPAGTQLRDVRAKKDQLEAHIQAAQAML
AMDTGETSKKIAHAKAVAAEAQDTATRVQSQLQAMQENVERWQGQYEGLRGQDLGQAVLDACSAVSTLEKTL
PQLLAKLSILENRGVHNASLALSASIGRVRELIAQARGAASKVVKVPMKFNGRSGVQLRTPRDLADLAAYTA
LKFYLQCPEPEPGQGTEDRFVMYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSIDEDIGEQFAAVSL
DRTLQFGHMSVTVERQMTQETKGDTVAPGAEGLLNLRPDDFVFYVGGYPSTFTPPPLLRFPGYRGCIEMDTL
NEEVVSLYNFERTFQLDTAVDRPCARSKSTCDPWLTDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSG
VLFFLKQQSQPLCLAVQEGSLVLLYDFGACLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVRVERATVY
SVEQDNDLELADAYYLGGVPPDQLPSLRRLFPTGGSVRGCVKGIKALGKYVDLKRLNTTGVSAGCTADLLVG
RAMTFHGHGFLRLALSNVAPLTCNVYSGFGFHSAQDSALLYYRASPVRPHQVSLQQGRVSLQLLRTEVKTQA
GFADGAPHYVAFYSNATGVWLYVDDQLQQMKPHRGPPPELQPQPEGPPRLLLGGLPESGTIYNFSGCISNVF
VQRLLGPQRVFDLQQNLGSVNVSTGCAPALQAQTPGLGPRQASRRSRQPARHPACMLPPHLRTTRDSYQFGG
SLSSHLEFVGILARHRNVSVRWEKNRILLVTDGARAWSQEGPHRQHQGAEHPQPHTLFVGGLPASSESSKLP
VTVGFSGCVKRLRLEGRPLGAPTEMACVTPCILGPLEAGLFFPGSGGVITLCLPGATLPDVGLELEVRPLAV
TGLIFHLGQARTPPYLQLQVLPRQVLLRADDGAGEFSTSVTRPSVLCDGQWHRLAVMKSGNVLRLEVDAQSN
HTVGPLLAAAAGAPAPLYLGGLPEPMAVQPWPPAYCGCMRRLAVNRSPVAMTRSVEVEGAVGASGCPAA
[0063] NOV1c is expressed in at least the following tissues:
Mammalian Tissue, Small Intestine, Bone Marrow, brain, Prostate,
ovary, kidney, melanocyte, heart, uterus, breast, head and neck,
stomach, genitourinary tract, pancreas, lung, testis, b-cell,
dorsal root ganglia. Expression information was derived from the
tissue sources of the sequences that were included in the
derivation of the sequence of CuraGen Acc. No. CG55974-02.
[0064] NOV1d
[0065] A disclosed NOV1d nucleic acid of 5204 nucleotides (also
referred to as 164875783) encoding a novel Human laminin alpha
5-like protein is shown in Table 1G. An open reading frame was
identified beginning with an TGT codon at nucleotides 3-5 and
ending with a TAG codon at nucleotides 49234925. The start and stop
codons are in bold letters and the 5' and 3' untranslated regions
are underlined in Table 1G. Because the start codon is not a
traditional initiation codon, NOV1d could be a partial reading
frame. NOV1d could extend further in the 5' direction.
TABLE-US-00008 TABLE 1G NOV1d nucleotide sequence (SEQ ID NO:7).
GCTGTGACCGCTGCCAGGAGGGACATTTTGGTTTCAATGGCTGCGGGGGCTGCCGCCCGTGTGCTTGTGGAC
CGGCCGCCGAGGGCTCCGAGTGCCACCCCCAGAGCGGACAGTGCCACTGCCGACCAGGGACCATGGGACCCC
AGTGCCGCGAGTGTGCCCCTGGCTACTGGGGGCTCCCTGAGCAGGGCTGCAGGCGCTGCCAGTGCCCTGGGG
GCCGCTGTGACCCTCACACGGGCCGCTGCAACTGCCCCCCGGGGCTCAGCGGGGAGCGCTGCGACACCTGCA
GCCAGCAGCATCAGGTGCCTGTTCCAGGCGGGCCTGTGGGCCACAGCATCCACTGTGAAGTGTGTGACCACT
GTGTGGTCCTGCTCCTGGATGACCTGGAACGGGCCGGCGCCCTCCTCCCCGCCATTCACGAGCAACTGCGTG
GCATCAATGCCAGCTCCATGGCCTGGGCCCGTCTGCACAGGCTGAACGCCTCCATCGCTGACCTGCAGAGCC
AGCTCCGGAGCCCCCTGGGCCCCCGCCATGAGACGGCACAGCAGCTGGAGGTGCTGGAGCAGCAGAGCACAA
GCCTCGGGCAGGACGCACGGCGGCTAGGCGGCCAGGCCGTGGGGACCCGAGACCAGGCGAGCCAATTGCTGG
CCGGCACCGAGGCCACACTGGGCCATGCGAAGACGCTGTTGGCGGCCATCCGGGCTGTGGACCGCACCCTGA
GCGAGCTCATGTCCCAGACGGGCCACCTGGGGCTGGCCAATGCCTCGGCTCCATCAGGTGAGCAGCTGCTCC
GGACACTGGCCGAGGTGGAGCGGCTGCTCTGGGAGATGCGGGCCCGGGACCTGGGGGCCCCGCAGGCAGCAG
CTGAGGCTGAGTTGGCTGCAGCACAGAGATTGCTGGCCCGGGTGCAGGAGCAGCTGAGCAGCCTCTGGGAGG
AGAACCAGGCACTGGCCACACAAACCCGCGACCGGCTGGCCCAGCACGAGGCCGGCCTCATGGACCTGCGAG
AGGCTTTGAACCGGGCAGTGGACGCCACACGGGAGGCCCAGGAGCTCAACAGCCGCAACCAGGAGCGCCTGG
AGGAAGCCCTGCAAAGGAAGCAGGAGCTGTCCCGGGACAATGCCACCCTGCAGGCCACTCTGCATGCGGCTA
GGGACACCCTGGCCAGCGTCTTCAGATTGCTGCACAGCCTGGACCAGGCTAAGGAGGAGCTGCAGCGCCTCG
CCGCCAGCCTGGACGGGGCTCGGACCCCACTGCTGCAGAGGATGCAGACCTTCTCCCCGGCGGGCAGCAAGC
TGCGTCTAGTGGAGGCCGCCGAGGCCCACGCACAGCAGCTGGGCCAGCTGGCACTCAATCTGTCCAGCATCA
TCCTGGACGTCAACCAGGACCGCCTCACCCAGAGGGCCATCGAGGCCTCCAACGCCTACAGCCGCATCCTGC
AGGCCGTGCAGGCTGCCGAGGATGCTGCTGGCCAGGCCCTGCAGCAGGCGGACCACACGTGGCCGACGGTGG
TGCGGCAGGGCCTGGTGGACCGAGCCCAGCAGCTCCTGGCCAACAGCACTGCACTAGAAGAGGCCATGCTCC
AGGAACAGCAGAGGCTGGGCCTTGTGTGGGCTGCCCTCCAGGGTGCCAGGACCCAGCTCCGAGATGTCCGGG
CCAAGAAGGACCAGCTGGAGGCGCACATCCAGGCGGCGCAGGCCATGCTTGCCATGGACACAGACGAGACAA
GCAAGAAGATCGCACATGCCAAGGCTGTGGCTGCTGAAGCCCAGGACACCGCCACCCGTGTGCAGTCCCAGC
TGCAGGCCATGCAGGAGAATGTGGAGCGGTGGCAGGGCCAGTACGAGGGCCTGCGGGGCCAGGACCTGGGCC
AGGCAGTGCTTGACGCAGGCCACTCAGTGTCCACCCTGGAGAAGACGCTGCCCCAGCTGCTGGCCAAGCTGA
GCATCCTGGAGAACCGTGGGGTGCACAACGCCAGCCTGGCCCTGTCCGCCAGCATTGGCCGCGTGCGAGAGC
TCATTGCCCAGGCCCGGGGGGCTGCCAGTAAGGTCAAGGTGCCCATGAAGTTCAACGGGCGCTCAGGGGTGC
AGCTGCGCACCCCACGGGATCTTGCCGACCTTGCTGCCTACACTGCCCTCAAGTTCTACCTGCAGGGCCCAG
AGCCTGAGCCTGGGCAGGGTACCGAGGATCGCTTTGTGATGTACATGGGCAGCCGCCAGGCCACTGGGGACT
ACATGGGTGTGTCTCTGCGTGACAAGAAGGTGCACTGGGTGTATCAGCTGGGTGAGGCGGGCCCTGCAGTCC
TAAGCATCGATGAGGACATTGGGGAGCAGTTCGCAGCTGTCAGCCTGGACAGGACTCTCCAGTTTGGCCACA
TGTCCGTCACAGTGGAGAGACAGATGATCCAGGAAACCAAGGGTGACACGGTGGCCCCTGGGGCAGAGGGGC
TGCTCAACCTGCGGCCAGACGACTTCGTCTTCTACGTCGGGGGGTACCCCAGTACCTTCACGCCCCCTCCCC
TGCTTCGCTTCCCCGGCTACCGGGGCTGCATCGAGATGGACACGCTGAATGAGGAGGTGGTCAGCCTCTACA
ACTTCGAGAGGACCTTCCAGCTGGACACGGCTGTGGACAGGCCTTGTGCCCGCTCCAAGTCGACCGGGGACC
CGTGGCTCACGGACGGCTCCTACCTGGACGGCACCGGCTTCGCCCGCATCAGCTTCGACAGTCAGATCAGCA
CCACCAAGCGCTTCGAGCAGGAGCTGCGGCTCGTGTCCTACAGCGGGGTGCTCTTCTTCCTGAAGCAGCAGA
GCCAGTTCCTGTGCTTGGCCGTGCAAGAAGGCAGCCTCGTGCTGTTGTATGACTTTGGGGCTGGCCTGAAAA
AGGCCGTCCCACTGCAGCCCCCACCGCCCCTGACCTCGGCCAGCAAGCCGATCCAGGTGTTCCTGCTGGGGG
GCAGCCGCAAGCGTGTGCTGGTGCGTGTGGAGCGGGCCACGGTGTACAGCGTGGAGCAGGACAATGATCTGG
AGCTGGCCGACGCCTACTACCTGGGGGGCGTGCCGCCCGACCAGCTGCCCCCGAGCCTGCGATGGCTCTTCC
CCACCGGAGGCTCAGTCCGTGGCTGCGTCAAAGGCATCAAGGCCCTGGGCAAGTATGTGGACCTCAAGCGGC
TGAACACGACAGGCGTGAGCGCCGGCTGCACCGCCGACCTGCTGGTGGGGCGCGCCATGACTTTCCATGGCC
ACGGCTTCCTTCGCCTGGCGCTCTCGAACGTGGCACCGCTCACTGGCAACGTCTACTCCGGCTTCGGCTTCC
ACAGCGCCCAGGACAGTGCCCTGCTCTACTACCGGGCGTCCCCGGATGGGCTATGCCAGGTGTCCCTGCAGC
AGGGCCGTGTGAGCCTACAGCTCCTGAGGACTGAAGTGAAAACTCAAGCGGGCTTCGCCGATGGTGCCCCCC
ATTACGTCGCCTTCTACAGCAATGCCACGGGAGTCTGGCTGTATGTCGATGACCAGCTCCAGCAGATGAAGC
CCCACCGGGGACCACCCCCCGAGCTCCAGCCGCAGCCTGAGGGGCCCCCGAGGCTCCTCCTGCGAGGCCTGC
CTGAGTCTGGCACCATTTACAACTTCAGTGGCTGCATCAGCAACGTCTTCGTGCAGCGGCTCCTGGGCCCAC
AGCGCGTATTTGATCTGCAGCAGAACCTGGGCAGCGTCAATGTGAGCACGGGCTGTGCACCCGCCCTGCAAG
CCCAGACCCCGGGCCTGGGGCCTAGAGCACTGCAGGCCACCGCCCGGAAGGCCTCCCGCCGCAGCCGTCAGC
CCGCCCGGCATCCTGCCTGCATGCTGCCCCCACACCTCAGGACCACCCGAGACTCCTACCAGTTTGGGGGTT
CCCAGACCCCGGGCCTGGGGCCTAGAGGACTGCAGGCCACCGCCCGGAAGGCCTCCCGCCGCAGCCGTCAGC
TCCTCCCGCGAAGCTCCCGAGGCCTCCTCCTCTTCACTGCCCGTCTGAGGCCCGGCAGCCCCTCCCTGGCGC
TCTTCCTGAGCAATGGCCACTTCGTTGCACAGATGGAAGGCCTCGGGACTCGCCTCCGCGCCCAGAGCCGCC
AGCGCTCCCGGCCTGGCCGCTGGCACAAGGTCTCCGTGCGCTGGGAGAAGAACCGGATCCTGCTGGTGACGG
ACGGGGCCCGGGCCTGGAGCCAGGAGGGGCCGCACCGGCAGCACCAGGGGGCAGAGCACCCCCAGCCCCACA
CCCTCTTTGTGGGCGGCCTCCCGGCCAGCAGCCACAGCTCCAAACTTCCGGTGACCGTCGGGTTCAGCGGCT
GTGTGAAGAGACTGAGGCTGCACGGGAGGCCCCTGGGGGCCCCCACACGGATGGCAGGGGTCACACCCTGCA
TCTTGGGCCCCCTGGAGGCGGGCCTGTTCTTCCCAGGCAGCGGGGGAGTTATCACTTTAGACCTCCCAGGAG
CTACACTGCCTGATGTGGGCCTGGAACTGGAGGTGCGGCCCCTGGCAGTCACCGGACTGATCTTCCACTTGG
GCCAGGCCCGGACGCCCCCCTACTTGCAGTTGCAGGTCCTGCTGCGGGCGGATGACGGAGCAGGGGAGTTCT
CCACGTCAGTGACCCGCCCCTCAGTGCTGTGTGATGGCCAGTGGCACCGGCTAGCGGTGATGAAAAGCGGGA
ATGTGCTCCGGCTGGAGGTGGACGCGCAGAGCAACCACACCGTGGGCCCCTTGCTGGCGCCTGCAGCTGGTG
CCCCAGCCCCTCTGTACCTCGGGGGCCTGCCTGAGCCCATGGCCGTGCAGCCCTGGCCCCCCGCCTACTGCG
GCTGCATGAGGAGGCTGGCGGTGAACCGGTCCCCCGTCGCCATGACTCGCTCTGTGGAGGTCCACGGGGCAG
TGGGGGCCAGTGGCTGCCCAGCCGCCTAGGACACAGCCAACCCCGGCCCCTGGTCAGGCCCCTGCAGCTGCC
TCACACCGCCCCTTGTGCTCGCCTCATAGGTGTCTATTTGGACTCTAAGCTCTACGGGTGACAGATCTTGTT
TCTGAAGATGGTTTAAGTTATAGCTTCTTAAACGAAAGAATAAAATACTGCAAAATGTTTTTATATTTGGCC
CTTCCACCCATTTTTAATTGTGAGAGATTTGTCACCAATCATCACTGGTTCCTCCTTAAAAATTAAAAAGTA
ACTTCTGTGTAAAAAAAAAA
[0066] In a search of public sequence databases, the NOV1d nucleic
acid sequence, located on chromsome 20 has 4573 of 4573 bases
(100%) identical to a gb:GENBANK-ID:AB011105|acc:AB011105.1 mRNA
from Homo sapiens (Homo sapiens mRNA for KIAA0533 protein, partial
cds) (E=0.0). Public nucleotide databases include all GenBank
databases and the GeneSeq patent database.
[0067] The disclosed NOV1d polypeptide (SEQ ID NO:8) encoded by SEQ
ID NO:7 has 1640 amino acid residues and is presented in Table 1H
using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV1d has no signal peptide and is
likely to be localized the cytoplasm with a certainty of 0.5050. In
other embodiments, NOV1b may also be localized to the microbody
(peroxisome) with acertainty of 0.3000, the lysosome (lumen) with a
certainty of 0.2741 or in the mitochondrial matrix space with a
certainty of 0.1000. TABLE-US-00009 TABLE 1H Encoded NOV1d protein
sequence (SEQ ID NO:8).
CDRCQEGHFGFNGCGGCRPCACGPAAEGSECHPQSGQCHCRPGTMGPQCRECAPGYWGLPEQGCRRCQCPGG
RCDPHTGRCNCPPGLSGERCDTCSQQHQVPVPGGPVGHSIHCEVCDHCVVLLLDDLERAGALLPAIHEQLRG
INASSMAWARLHRLNASTADLQSQLRSPLGPRHETAQQLEVLEQQSTSLGQDARRLGGQAVGTRDQASQLLA
GTEATLGHAKTLLAAIRAVDRTLSELMSQTGHLGLANASAPSGEQLLRTLAEVERLLWEMRARDLGAPQAAA
EAELAAAQRLLARVQEQLSSLWEENQALATQTRDRLAQHEAGLMDLREALNRAVDATREAQELNSRNQERIE
EALQRKQELSRDNATLQATLHAARDTLASVFRLLHSLDQAKEELERLAASLDGARTPLLQRMQTFSPAGSKL
RLVEAAEAHAQQLGQLALNLSSIILDVNQDRLTQRAIEASNAYSRILQAVQAAEDAAGQALQQADHTWATVV
RQGLVDRAQQLLANSTALEEAMLQEQQRLGLVWAALQGARTQLRDVRAKKDQLEAHIQAAQAMLAMDTDETS
KKIAHAKAVAAEAQDTATRVQSQLQAMQENVERWQGQYEGLRGQDLGQAVLDAGHSVSTLEKTLPQLLAKLS
ILENRGVHNASLALSASIGRVRELIAQARGAASKVKVPMKFNGRSGVQLRTPRDLADLAAYTALKFYLQGPE
PEPGQGTEDRFVMYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSIDEDIGEQFAAVSLDRTLQFGHM
SVTVERQMIQETKGDTVAPGAEGLLNLRPDDFVFYVGGYPSTFTPPPLLRFPGYRGCIEMDTLNEEVVSLYN
FERTFQLDTAVDRPCARSKSTGDPWLTDGSYLDGTGFARISFDSQISTTKREEQELRLVSYSGVLFFLKQQS
QFLCLAVQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVRVERATVYSVEQDNDLE
LADAYYLGGVPPDQLPPSLRWLFPTGGSVRGCVKGIKALGKYVDLKRLNTTGVSAGCTADLLVGRAMTFHGH
GFLRLALSNVAPLTGNVYSGFGFHSAQDSALLYYRASPDGLCQVSLQQGRVSLQLLRTEVKTQAGFADGAPH
YVAPYSNATGVWLYVDDQLQQMKPHRGPPPELQPQPEGPPRLLLGGLPESGTIYNFSGCISNVFVQRLLGPQ
RVFDLQQNLGSVNVSTGCAPALQAQTPGLGPRGLQATARKASRRSRQPARHPACMLPPHLRTTRDSYQFGGS
LSSHLEFVGILARHRNWPSLSMHVLPRSSRGLLLFTARLRPGSPSLALFLSNGHFVAQMEGLGTRLRAQSRQ
RSRPGRWHKVSVRWEKNRILLVTDGARAWSQEGPHRQHQGAEHPQPHTLFVGGLPASSHSSKLPVTVGFSGC
VKRLRLHGRPLGAPTRMAGVTPCILGPLEAGLFFPGSGGVITLDLPGATLPDVGLELEVRPLAVTGLIFHLG
QARTPPYLQLQVLLRADDGAGEFSTSVTRPSVLCDGQWHRLAVMKSGNVLRLEVDAQSNHTVGPLLAAAAGA
PAPLYLGGLPEPMAVQPWPPAYCGCMRRLAVNRSPVAMTRSVEVHGAVGASGCPAA
[0068] A search of sequence databases reveals that the NOV1d amino
acid sequence has 908 of 913 amino acid residues (99%) identical
to, and 1640 of 1645 amino acid residues (99%) identical to, and
1640 of 1645 amino acid residues (99%) similar to, the 1645 amino
acid residue ptnr:SWISSNEW-ACC:015230 protein from Homo sapiens
(Human) (Laminin Alpha-5 Chain) (E=0.0). Public amino acid
databases include the GenBank databases, SwissProt, PDB and
PIR.
[0069] Homologies to any of the above NOV1 proteins will be shared
by the other NOV1 proteins insofar as they are homologous to each
other as shown below. Any reference to NOV1 is assumed to refer to
all four of the NOV1 proteins in general, unless otherwise
noted.
[0070] The disclosed NOV1a polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 1I.
TABLE-US-00010 TABLE 1I BLAST results for NOV1a Gene Index/ Length
Identity Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi|7459688|pir||T10053 laminin alpha 5 3635 2441/3319 2666/3319 0.0
chain - mouse (73%) (79%) (fragment) gi|2497593|sp|Q00174| Laminin
alpha 3712 793/2179 1106/2179 0.0 LMA_DROME chain precursor (36%)
(50%) gi|14786772|ref|XP.sub.-- laminin, alpha 5 1634 1060/1213
1063/1213 0.0 037217.1| [Homo sapiens] (87%) (87%) (XM_037217)
gi|2281044|emb|CAB09137.1| laminin alpha 5 953 571/620 571/620 0.0
(Z95636) chain [Homo (92%) (92%) sapiens] gi|17136292|ref|NP.sub.--
LanA-P1; 3712 790/2179 1105/2179 0.0 476617.1| headline; (36%)
(50%) (NM_057269) laminin; laminin A; laminin alpha; laminin
alpha3,5; laminin alpha- chain [Drosophila melanogaster]
[0071] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 1J. In the
ClustalW alignment of the NOV1 proteins, as well as all other
ClustalW analyses herein, the black outlined amino acid residues
indicate regions of conserved sequence (i.e., regions that may be
required to preserve structural or functional properties), whereas
non-highlighted amino acid residues are less conserved and can
potentially be altered to a much broader extent without altering
protein structure or function.
[0072] The presence of identifiable domains in NOV1, as well as all
other NOVX proteins, was determined by searches using software
algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and
Prints, and then determining the Interpro number by crossing the
domain match (or numbers) using the Interpro website
(http:www.ebi.ac.uk/interpro). DOMAIN results for NOV1 as disclosed
in Tables 1K-1L, were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST analyses. This BLAST
analysis software samples domains found in the Smart and Pfam
collections. For Table 1K and all successive DOMAIN sequence
alignments, fully conserved single residues are indicated by black
shading or by the sign (|) and "strong" semi-conserved residues are
indicated by grey shading or by the sign (+). The "strong" group of
conserved amino acid residues may be any one of the following
groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MELV, MILF, HY,
FYW.
[0073] Tables 1K-S list the domain descriptions from DOMAIN
analysis results against NOV1a. This indicates that the NOV1a
sequence has properties similar to those of other proteins known to
contain this domain. Below are representative domain results. There
are additional areas on NOV1 a that also have homology to these
Domains. TABLE-US-00011 TABLE 1K Domain Analysis of NOV1a
gnl|Smart|smart00136, LamNT, Laminin N-terminal domain (domain VI);
N- terminal domain of laminins and laminin-related protein such as
Unc-6/ netrins. (SEQ ID NO:52) CD-Length = 239 residues, 96.7%
aligned Score = 271 bits (692), Expect = 6e-73 Query: 26
PPYFNLAEGARIAASATCGEEAPARGSPRPTEDLYCKLVGGPVAGGDPNQTIQGQYCDIC 85 | +
||| | + ||+||||+ | | |||||| | ||+ || | Sbjct: 9
PEFVNLAFGRPVTASSTCGEQGPER---------YCKLVGR---------TEQGKKCDYC 50
Query: 86
TAANSNKAHPASNAIDGTE----RWWQSPPLSRGLEYNEVNVTLDLGQVFHVAYVLIKFA 141 |
+ ++||| | || |||| ||| | + ||+|||||+ ||+ ||++|| Sbjct: 51
DARDPRRSHPAENLTDGNNPGNPTWWQSEPLSNGPQ--NVNLTLDLGKEFHLTYVILKFC 108
Query: 142
NSPRPDLWVLERSMDFGRTYQPWQFFAASKRDCLERFGPQTLERITR--DDAAICTTEYS 199
|||| | +|||| |||+|+||+|+|++ || || ||+ + +||+||| Sbjct: 109
-SPRPSLAILERS-DFGKTWQPYQYFSS---DCRRTFGRPPRGPITKGNEQEVLCTSEYS 163
Query: 200
RIVPLENGEIVVSLVNGRPGAMNFSYSPLLREFTKATNVRLRFLRTNTLLGHLMGKALRD 259
||||| ||| | + ||| | +| ||+|+|+ |||+|+| | ||| || | || Sbjct: 164
DIVPLEGGEIAFSTLEGRPSATDFDNSPVLQEWVTATNIRVRLTRLNTLGDDLMDK--RD 221
Query: 260 PTVTRRYYYSIKDISIGG 277 | ||| |||+| ||++|| Sbjct: 222
PEVTRSYYYAISDIAVGG 239
[0074] TABLE-US-00012 TABLE 1L Domain Analysis of NOV1a
gnl|Pfam|pfam00055, laminin_Nterm, Laminin N-terminal (Domain VI).
(SEQ ID ID:53) CD-Length = 237 residues, 100.0% aligned Score = 219
bits (559), Expect = 2e-57 Query: 24
LHPPYFNLAEGARIAASATCGEEAPARGSPRPTEDLYCKLVGGPVAGGDPNQTIQGQYCD 83 +|
||| | ++|++||| +| + || | + | | Sbjct: 1
CYPATGNLAIGRALSATSTCGLHSP---------EPYCILSH--LQPRDKK-------CF 42
Query: 84
ICTAANSNKA--HPASNAIDGTER----WWQSPPLSRGLEYNEVNVTLDLGQVFHVAYVL 137 +|
+ + | || | | |||| + |++| | +|||| || ||+ Sbjct: 43
LCDSNSPNPRNSHPISFLTDTFNPQSPTWWQSETMQNGVQYPNVTITLDLEAEFHFTYVI 102
Query: 138
IKFANSPRPDLWVLERSMDFGRTYQPWQFFAASKRDCLERFG--PQTLERITRDDA-AIC 194 |
| + || + ||| ||| |+ |+|++| || + |+ | | + +| Sbjct: 103
ITFK-TFRPAAMIYERSSDFG-TWIPYQYYAY---DCEATYPGIPRRPIRTGRAEDDVLC 157
Query: 195
TTEYSRIVPLENGEIVVSLVNGRPGAMNFSYSPLLREFTKATNVRLRFLRTNTLLGHLMG 254 |+
|| | || ||++ | + ||| | || || |+|+ ||||+|+ | +|| +|+ Sbjct: 158
TSRYSDIEPLTEGEVIFSTLEGRPSADNFDPSPRLQEWLKATNIRITLTRLHTLGDNLLD 217
Query: 255 KALRDPTVTRRYYYSIKDISICG 277 || | +|||+| || +|| Sbjct:
218 ---SDPEVLEKYYYAISDIVVGG 237
[0075] TABLE-US-00013 TABLE 1M Domain Analysis of NOV1a
gnl|Smart|smart00281, LamB, Laminin B domain (SEQ ID NO:54)
CD-Length = 127 residues, 98.4% aligned Score = 152 bits (385),
Expect = 2e-37 Query: 1674
PELYWQAPPSYLGDRVSSYGGTLRYELHSETQRGDVFVPMESRPDVVLQGNQMSITFLEP 1733
+|| || +|||+|+|||| ||| | + + | + | |||+|+|| + ++ Sbjct: 3
EPVYWVAPEQFLGDKVTSYGGKLRYTLSFDGREGGTTL---SAPDVILEGNGLRLSHPAQ 59
Query: 1734
AYPTPGHVHRGQLQLVEGNFRHTETRNTVSREELMMVLASLEQLQIRALFSQISSAVFLR 1793 |
| +++ | |+++ | |+||+||||||+| + ||| +|+ | | Sbjct: 60
GPPLPDEETTNEVRFREENWQYFGGR-PVTREDLMMVLANLTAILIRATYSEQQLASRLS 118
Query: 1794 RVALEVASP 1802 |+|||| | Sbjct: 119 DVSLEVAVP 127
[0076] TABLE-US-00014 TABLE 1N Domain Analysis of NOV1a
gnl|Pfam|pfam00052, laminin_B, Laminin B (Domain IV). (SEQ ID
NO:55) CD-Length = 135 residues, 100.0% aligned Score = 92.4 bits
(228), Expect = 4e-19 Query: 1677
YWQAPPSYLGDRVSSYGGTLRYELHSETQRGDVFVFMESRPDVVLQGNQMSITFLEPAYP 1736
||+ | +|||+|+|||| |+| + | | |||+|+|| + ++ | Sbjct:
YWRLPERFLGDQVTSYGGKLKYSV-----AFDGVGTSNSEPDVILKGNGLRLSVPYMAQG 55
Query: 1737
TP---GHVERGQLQLVEGNFRETETRNTVSREELMMVLASLEQLQIRALFSQISSAVFLR 1793 +
++| | | +++ |+||+ + |||+| + ||| +| + | Sbjct: 56
NSYPSEVRVKYTVRLHE-TFWDFQSQPAVTREDFLSVLANLTAILIRATYSAGQAQSRLD 114
Query: 1794 RVALEVASPAGQGA-LASNVE 1813 |+||+| | | |+ || Sbjct: 115
DVSLEIARPGAAGPVPATWVE 135
[0077] TABLE-US-00015 TABLE 10 Domain Analysis of NOV1a
gnl|Smart|smart00282, LamG, Laminin G domain (SEQ ID NO:56)
CD-Length = 135 residues, 88.1% aligned Score = 76.6 bits (187),
Expect = 2e-14 Query: 2759
FVMYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSIDEDI--GEQFAAVSLDRTLQ 2816
++| ||+ ||++ + ||| ++ | || +||| |+ | |+ ||++| + Sbjct: 17
LLLYAGSKG-GGDFLALELRDGRLVLRYDLG-SGPARLTSDPTPLNDGQWHRVSVERNGR 74
Query: 2817
FGHMSVTVERQMIQETKGDTVAPGAEGLLNLRPDDFVFYVGGYPSTFTPPPLLRFPGYRG 2876
+|| ++ |+ || +|+| | |+|| | | | ||+|| Sbjct: 75
RVTLSVDGGNRVSGES------PGGSTILDL---DGPLYLGGLPEDLKLPGLPVTPGFRG 125
Query: 2877 CIEMDTLNEE 2886 || +| + Sbjct: 126 CIRNLKVNGK 135
[0078] TABLE-US-00016 TABLE 1P Domain Analysis of NOV1a
gnl|Pfam|pfam00053, laminin_EGF, Laminin EGF-like (Domains III and
V). This family is like pfam00008 but has 8 conserved cysteines
instead of 6. (SEQ ID NO:57) CD-Length = 49 residues, 100.0%
aligned Score = 59.3 bits (142), Expect = 4e-09 Query: 1561
CDCHEAGTAPGVCDPLTGQCYCKENVQGPKCDQCSLGTFSLDAANPKGC 1609 |||+ |+ |||
|||| || | | +||+| | + | + +|| Sbjct: 1
CDCNPHGSLSDTCDPETCQCLCKPGVTGRRCDRCKPGYYGLPSDPGQGC 49
[0079] TABLE-US-00017 TABLE 1Q Domain Analysis of NOV1a
gnl|Smart|smart00180, EGF_Lam, Laminin-type epidermal growth
factor-like domai (SEQ ID NO:58) CD-Length = 47 residues, 87.2%
aligned Score = 55.8 bits (133), Expect = 4e-08 Query: 1979
CDCTPCGT-EACDPHSGHCLCKAGVTGRRCDRCQEGHFGFD 2018 ||| || +|| |+ | ||
||||||||| |++| Sbjct: 1 CDCDPGGSASTCDPETGQCECKPNTTGRRCDRCAPGYYGLP
41
[0080] TABLE-US-00018 TABLE 1R Domain Analysis of NOV1a
gnl|Pfam|pfam01576, Myosin_tail, Myosin tail. The myosin molecule
is a multi-subunit complex made up of two heavy chains and four
light chains it is a fundamental contractile protein found in all
eukaryote cell types. This family consists of the coiled-coil
myosin heavy chain tail region. The coiled-coil is composed of the
tail from two molecules of myosin. These can then assemble into the
macromolecular thick filament. The coiled-coil region provides the
structural backbone the thick filament. (SEQ ID NO:59) CD-Length =
860 residues, 60.6% aligned Score = 53.1 bits (126), Expect = 3e-07
Query: 2205
RHETAQQLEVLEQQSTSLPPQAVGTRDQASQLLAGTEATLGHAKTLLA-AIRAVDRTLSE 2263 |
+ +++|| | ++ +| | +| || | + | | + |+ Sbjct: 69
RADLSRELEELSERLE----EAGGATAAQIELNKKREAELAKLRKDLEEANLQHEEALAT 124
Query: 2264
LMSQTGHLGLANASAPSGEQL-----LRTLAEVERLLWEMRARDLGAPQAAAEAELAAAQ 2318 |
+ | +| ||+ + || |+ + || | + |+ Sbjct: 125
LRKK--H---QDAINELSEQIEQLQKQKAKAEKEKSQLQAEVDDLLAQLDSITKAKLNAE 179
Query: 2319
RVLARVQEQLSSLW----EENQALA--TQTRDRLAQHEAGLM-DLREALNRAVDATREAQ 2371 +
+++ ||| | | + | | + || + | | || + + ++ Sbjct: 180
KKAKQLESQLSELQVKLDELQRQLNDLTSQKSRLQSENSDLTRQLEEAEAQVSNLSKLKS 239
Query: 2372
ELNSRNQERLEEALQRKQELSRDNATLQATLHAARDTLASVFRLLEGLSPLK---FQELE 2428
+| |+ |||| + +| ||+ | ||| | | |+ || | | ++| Sbjct: 240
QLESQ----LEEAKRSLEEESRERANLQAQLRQLEHDLDSLREQLEEESEAKAELERQLS 295
Query: 2429
RLAASLDGARTPL----LQRMQTFSPAGSKL--------RLVEAAEAHAQQLGQLALNLS 2476 +
| + |+ | + || ||| | | + | Sbjct: 296
KANAEIQQWRSKFESEGALRAEELEELKKKLNQKISELEEAAEAANAKCDSLEKTKSRLQ 355
Query: 2477
IILDVNQDRLTQRAIEASNAYSRILQAVQAAEDAAGQALQQADHTWQTVVR--QGLVDRA 2534
|+ | +| +|| + |+ | | |+ | | +| | Sbjct: 356
SELEDL-----QIELERANAAASELEKKQKNFDKILAE-------WKRKVDELQAELDTA 403
Query: 2535
QQ--------LLANSTALEEAMLQ------EQQRLGLGECWAPMGALRPAGTQLRDVRAK 2580
|+ | ||| | | + | | | | + ++ Sbjct: 404
QREARNLSTELFRLKNELEELKDQVEALRRENKNLQD-EIHDLTDQLGEGGRNVHELEKA 462
Query: 2581
KDQLEAHIQAAQAMLAMDTGETSKKIAHAKAVAAEAQDTATRVQSQLQAMQENVER---- 2636 +
+||| || |++ | + + | | +| ++ +|| Sbjct: 463
RRRLEAEKDELQA--ALEEAEAA---------LELEESKVLRAQVELSQIRSEIERRLAE 511
Query: 2637
WQGQYEGLRGQ-----DLGQAVLDA----GSAVSTLEKTLPQLLAKLSI-LE--NRGVHN 2684 +
++| | + || |+| + | |+| | + +| | |+ |+ Sbjct: 512
KEEEFENTRKNEQRAIESLQATLEAETKGKAEASRLKKKLEGDINELEIALDHANKANAE 571
Query: 2685 ASLALSASIGRVRELIAQ 2702 | + +|+|| | Sbjct: 572
AQKNVKKYQQQVKELQTQ 589
[0081] TABLE-US-00019 TABLE 1S Domain Analysis of NOV1a
gnl|Pfam|pfam00054, laminin_G, Laminin G domain. (SEQ ID NO:60)
CD-Length = 134 residues, 89.6% aligned Score = 47.8 bits (112),
Expect = 1e-05 Query: 2760
VMYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSIDEDIGE-QFAAVSLDRTLQFG 2818
++| |+ |++ + ||| ++ | || +||||+ + + + ++ | |+| + | Sbjct: 10
LLYGGT-NTDRDFLALELRDGRLEVSYDLG-SGPAVVRSGDRLNDGKWHRVELERNGRKG 67
Query: 2819
HMSVTVERQMIQETKGDTVAPGAEGLLNLRPDDFVFYVGGYPSTFTPPPLLRF-PGYRGC 2877
+|| | + |+ | |+| | |||| | | ++|| Sbjct: 68
TLSVDGEESVDGESPSGPDVPEE--NLDL---DTPLYVGGLPELSVKRLLAAISTSFKGC 122
Query: 2878 IEMDTLN 2884 | +| Sbjct: 123 IRDVIVN 129
[0082] Laminins are the major noncollagenous components of basement
membranes that mediate cell adhesion, growth, migration, and
differentiation. They are composed of distinct but related alpha,
beta and gamma chains. The three chains form a cross-shaped
molecule that consist of a long arm and three short globular arms.
The long arm consist of a coiled coil structure contributed by all
three chains and cross-linked by interchain disulfide bonds. Beside
different types of globular domains each subunit contains, in its
first half, consecutive repeats of about 60 amino acids in length
that include eight conserved cysteines. The tertiary structure of
this domain is remotely similar in its N-terminal to that of the
EGF-like module. It is known as a `LE` or `laminin-type EGF-like`
domain. The number of copies of the LE domain in the different
forms of laminins is highly variable; from 3 up to 22 copies have
been found (1).
[0083] Miner et al (1) identified a fifth member of the alpha
subfamily of vertebrate laminin chains. Consistent with the
trimeric structure of laminin, all basal laminae characterized to
that time contained at least 1 beta and at least 1 gamma chain. For
the alpha chains, on the other hand, the situation was less clear.
Using PCR, Miner et al. identified a novel murine alpha chain
called alpha-5. Sequence analysis revealed a close relationship to
the only known Drosophila alpha chain, suggesting that the
ancestral alpha laminin gene is more similar to laminin alpha-5
than it is to laminin alpha-1, alpha-2, alpha-3, or alpha-4.
Analysis of RNA expression showed that alpha-5 is widely expressed
in adult tissues, with highest levels in lung, heart, and kidney.
It is speculated that the Laminin alpha 5 may be a major laminin
chain of adult basal laminae. Dirkin et al (2) mapped the LAMA5
gene to 20q13.2-q13.3; the mouse gene (Lama5) was mapped by linkage
analysis to a syntenic region of distal chromosome 2.
[0084] The novel sequence described here is a human homolog of
mouse laminin alpha 5. Because of its expression pattern (3-4), it
is suggested that laminin laminin alpha5 plays role in the
development of the human lung and skin morphogenesis. In addition,
there is distinct temporal and spatial expression of these chains
during proliferative and differentiation stages, possibly
reflecting different functions (4).
[0085] The disclosed NOV1 nucleic acid of the invention encoding a
Human laminin alpha 5-like protein includes the nucleic acid whose
sequence is provided in Table 1A, 1C, 1E, 1G, or a fragment
thereof. The invention also includes a mutant or variant nucleic
acid any of whose bases may be changed from the corresponding base
shown in Table 1A, 1C, 1E, or 1G while still encoding a protein
that maintains its Human laminin alpha 5-like activities and
physiological functions, or a fragment of such a nucleic acid. The
invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, by way
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 31% percent of
the bases may be so changed.
[0086] The disclosed NOV1 protein of the invention includes the
Human laminin alpha 5-like protein whose sequence is provided in
Table 1B, 1D, 1F, or 1H. The invention also includes a mutant or
variant protein any of whose residues may be changed from the
corresponding residue shown in Table 1B, 1D, 1F, or 1H while still
encoding a protein that maintains its Human laminin alpha 5-like
activities and physiological functions, or a functional fragment
thereof. In the mutant or variant protein, up to about 54% percent
of the residues may be so changed.
[0087] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0088] The above defined information for this invention suggests
that this Human laminin alpha 5-like protein (NOV1) may function as
a member of a "Human laminin alpha 5 family". Therefore, the NOV1
nucleic acids and proteins identified here may be useful in
potential therapeutic applications implicated in (but not limited
to) various pathologies and disorders as indicated below. The
potential therapeutic applications for this invention include, but
are not limited to: protein therapeutic, small molecule drug
target, antibody target (therapeutic, diagnostic, drug
targeting/cytotoxic antibody), diagnostic and/or prognostic marker,
gene therapy (gene delivery/gene ablation), research tools, tissue
regeneration in vivo and in vitro of all tissues and cell types
composing (but not limited to) those defined here.
[0089] The NOV1 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in cancer
including but not limited to various pathologies and disorders as
indicated below. For example, a cDNA encoding the Human laminin
alpha 5-like protein (NOV1) may be useful in gene therapy, and the
Human laminin alpha 5-like protein (NOV1) may be useful when
administered to a subject in need thereof. By way of nonlimiting
example, the compositions of the present invention will have
efficacy for treatment of patients suffering from Von Hippel-Lindau
(VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis,
hypercalceimia, Parkinson's disease, Huntington's disease, cerebral
palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders,
addiction, anxiety, pain, neurodegeneration; Cholesteryl ester
storage disease; Corneal dystrophy, Thiel-Behnke type;
Dubin-Johnson syndrome; Leukemia, T-cell acute lymphocytic; Retinol
binding protein, deficiency of; SEMD, Pakistani type;
Spinocerebellar ataxia, infantile-onset, with sensory neuropathy;
Split hand/foot malformation, type 3; Tolbutamide poor metabolizer;
Urofacial syndrome; Warfarin sensitivity; Wolman disease,
neuroprotection, fertility, diabetes, autoimmune disease, renal
artery stenosis, interstitial nephritis, glomerulonephritis,
polycystic kidney disease, systemic lupus erythematosus, renal
tubular acidosis, IgA nephropathy, cardiomyopathy, atherosclerosis,
hypertension, congenital heart defects, aortic stenosis, atrial
septal defect (ASD), atrioventricular (A-V) canal defect, ductus
arteriosus, pulmonary stenosis, subaortic stenosis, ventricular
septal defect (VSD), valve diseases, tuberous sclerosis,
sclerodema, obesity, transplantation, ulcers, systemic lupus
erythematosus, autoimmune disease, asthma, emphysema, scleroderma,
allergy, ARDS, or other pathologies or conditions. The NOV1 nucleic
acid encoding the Human laminin alpha 5-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.
[0090] NOV1 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV1 substances for use in therapeutic or diagnostic methods.
These antibodies may be generated according to methods known in the
art, using prediction from hydrophobicity charts, as described in
the "Anti-NOVX Antibodies" section below. The disclosed NOV1
proteins have multiple hydrophilic regions, each of which can be
used as an immunogen. These novel proteins can be used in assay
systems for functional analysis of various human disorders, which
will help in understanding of pathology of the disease and
development of new drug targets for various disorders.
[0091] NOV2
[0092] NOV2 includes three novel Human Hurpin/PI 13-like proteins
disclosed below. The disclosed sequences have been named NOV2a,
NOV2b, and NOV2c.
[0093] NOV2a
[0094] A disclosed NOV2a nucleic acid of 3105 nucleotides (also
referred to as CG55999-01) encoding a novel Human Hurpin/PI 13-like
protein is shown in Table 2A. An open reading frame was identified
beginning with an ATG initiation codon at nucleotides 38-40 and
ending with a TAA codon at nucleotides 1238-1240. A The start and
stop codons are in bold letters in Table 2A. TABLE-US-00020 TABLE
2A NOV2a nucleotide sequence (SEQ ID NO:9).
TGTTGTTCTTGCTATTCTAGGTCTCGCTAAAATCATCATGGATTCACTTGGCGCCGTCAGCACTCGACTTGG
GTTTGATCTTTTCAAAGAGCTGAAGAAAACAAATGATGGCAACATCTTCTTTTCCCCTGTGGGCATCTTAAC
TGCAATTGGCATGGTCCTCCTGGGGACCCGAGGAGCCACCGCTTCCCAGTTGGAGGAGGTGTTTCACTCTGA
AAAAGAGACGAAGAGCTCAAGAATAAAGGCTGAAGAAAAAGAGGTGGTAAGAATAAAGGCTGAAGGAAAAGA
GATTGAGAACACAGAAGCAGTACATCAACAATTCCAAAAGTTTTTGACTGAAATAAGCAAACTCACTAATGA
TTATGAACTGAACATAACCAACAGGCTGTTTGGAGAAAAAACATACCTCTTCCTTCAAAAATACTTAGATTA
TGTTGAAAAATATTATCATGCATCTCTGGAACCTGTTGATTTTGTAAATGCAGCCGATGAAAGTCGAAAGAA
GATTAATTCCTGGGTTGAAAGCAAAACAAATGAAAAAATCAAGGACTTGTTCCCAGATGGCTCTATTAGTAG
CTCTACCAAGCTGGTGCTGGTGAACATGGTTTATTTTAAAGGGCAATGGGACAGGGAGTTTAAGAAAGAAAA
TACTAAGGAAGAGAAATTTTGGATGAATAAGAGCACAAGTAAATCTGTACAGATGATGACACAGAGCCATTC
CTTTAGCTTCACTTTCCTGGAGGACTTGCAGGCCAAAATTCTAGGGATTCCATATAAAAACAACGACCTAAG
CATGTTTGTGCTTCTGCCCAACGACATCGATGGCCTGGAGAAGATAATAGATAAAATAAGTCCTGAGAAATT
GGTAGAGTGGACTAGTCCAGGGCATATGGAAGAAAGAAAGGTGAATCTGCACTTGCCCCGGTTTGAGGTGGA
GGACGGTTACGATCTAGAGGCGGTCCTGGCTGCCATGGGGATGGGCGATGCCTTCAGTGAGCACAAAGCCGA
CTACTCGGGAATGTCGTCAGGCTCCGGGTTGTACGCCCAGAAGTTCCTGCACAGTTCCTTTGTGGCAGTAAC
TGAGGAAGGCACCGAGGCTGCAGCTGCCACCGGCATAGGCTTTACTGTCACATCCGCCCCAGGTCATGAAAA
TGTTCACTGCAATCATCCCTTCCTGTTCTTCATCAGGCACAATGAATCCAACAGCATCCTCTTCTTCGGCAG
ATTTTCTTCTCCTTAAGATGATCGTTGCCATGGCATTGCTGCTTTTAGCAAAAAACAACTACCAGTGTTACT
CATATGATTATGAAAATCGTCCATTCTTTTAAATGTTGTCTCACTTGCATTTCCAGTCTTGGCCATCAAATC
AATGATTTAATGACTCCAATAATGTGTGTGTTTATAACCATCCTCGAAAGTGAAATGTCCTTTTTTTTGTGC
CATGCGTAAGGTGAGTCAAACCAAACCTCATTGATAATCTCCCCTTTGGTTTCCTTTGAAAGTAAATTGGTA
TCTTGTAGTTTTGTGCACACGAAAGGAGAGAAAGTTTCTCCAGTAAAGAGTACGAACTAGTAATTTTGGGGG
GTCTCTCTAATTCTGGTATTTTGACATGTTATAATACGCAAGTAAAATAAAACAATAGTTTACTCAGCTCAT
GTTACTATTCCCCAACAGATATTGTGGCAAATCACACATAGGAAAGAGGATTTGGGAATACAGTAGCAAAAC
ATAAATTAAAACTCAAATGCCCAGGACAAAATAAAACAATATACCAGATGGAGAGGATGCCCGTATTTTCAT
CTTCCATTCTAACATTATCCATTGTTAGATGCATAAGCATTTTGATATTGTGTAATAAATGTGGTATTTGAG
AAGATAAATGATGTAGTTGATCAGTAATCCTCCTCTATCACCTTTTTAGACTTTGTAAGGTAAATATTTGGA
CTAACTTTTAGAAAAGTTTCCCTTTTTTTCTCCATTTACATTTTTCTGGTTTTTTTTTTTTTTTTTGAGTGA
GGTACGAGTATTACCAAATGATATTTTCTGAAGATGCTTTTTGGAAAGCTCTGAATCTATACCTAATGCTCT
TAATTATTGGCTTGTTTCATTTTTTTCCTCCAGTTTTTAACAAGATCACATAACTGGCTTATTTTTAACAGC
TTTGTCAAACTACAATTTACATGCCGTAAAATGTACACACTGTAATTTTATAATTCATTGACTTTTAGTAAA
TTTTCTAGCGTTATGCATCGCCACAATCCAGTTTTAGAATATTTCCATGACCCTAAGAAGTTTCCTCATGTC
TATTAATATTCCCAATCCTAGGCACCACTGAGTTGTTTTCTGTCTTTATAAGTTTTTCTTTCTACATCTTAT
ATAAATGGAATCATAATACATGTAGTATTTTGTGTCTGGCGTCTTGCACTTAGCATGGTGTTCTTGAGGTTC
ATCTGTTGTAGTATGTATTGATACTTAGGATTTTTTTATTGCCGAATACTATTCCATTGCATGGAAAAGACC
TATTTTATTTCTAGGTTCACCAGTTGAGGGACATTTGGATTGTTCCCACTTCTTGGGCTGTTAGGAATAATG
TTGCTCTGAACATGTAAATAAAGATCTTTGTGTTCACATATGTTTTTCATTTTCTGTTGGGGAGATTCCCTA
GGCTAGAAATTGCTGGGCCATATGAAAAATCAATAGTTAGCTTTGTAAGAAACAGTCAAACCGTTTTCCCAA
CGTGACATTTTATATTCCCACCAGGAATGTTTAAAACTAGTGTCTTCAAATCCTCACCAACATCCAGGATTG
TGTCTTTATGATTATAGCCATTTTTGTAGGTACAAAGTGGCATCTCATGGTGGTTTTAATTTGCATTTCCAT
AATATCTAATTAGGTTGAGCTTTTTTTATGTGCTTATTGGCCATTTGTTTGACTTTGTTTGGTGAAATGTAT
ACAAATCATTTGCTCATTTTTAATTTGGGTTGTCTGTCTTGTCTTCTCATTTTATTGAGTTAAATGAGTTCT
TAATAATCTCTGGCTTACAAGTCCTTAATTTATCAAATATATGATACGTGGACATTTCCTCATAAAAAAAAA
AAAAAAAAA
[0095] The disclosed NOV2a nucleic acid sequence, localized to the
q21.3-22 region of chromsome 18, has 2854 of 2866 bases (99%)
identical to a gb:GENBANK-ID:HSPI13711|acc:AJ001696.2 mRNA from
Homo sapiens (Homo sapiens mRNA for hurpin, clone R7-1.1)
(E=0.0).
[0096] A NOV2a polypeptide (SEQ ID NO:10) encoded by SEQ ID NO:9
has 400 amino acid residues and is presented using the one-letter
code in Table 2B. Signal P, Psort and/or Hydropathy results predict
that NOV2a contains a signal peptide and is likely to be localized
to the plasma membrane with a certainty of 0.7900. In other
embodiments, NOV2a may also be localized to the microbody
(peroxisome) with a certainty of 0.7106, the Golgi body with a
certainty of 0.3000, or the endoplasmic reticulum (membrane) with a
certainty of 0.2000. The most likely cleavage site for NOV2a is
between positions 50 and 51: ATA-SQ. TABLE-US-00021 TABLE 2B
Encoded NOV2a protein sequence (SEQ ID NO:10).
MDSLGAVSTRLGFDLFKELKKTNDGNIFFSPVGILTAIGMVLLGTRGATASQLEEVFHSEKETKSSRIKAEE
KEVVRIKAEGKEIENTEAVHQQFQKFLTEISKLTNDYELNITNRLFGEKTYLFLQKYLDYVEKYYHASLEPV
DFVNAADESRKKINSWVESKTNEKIKDLFPDGSISSSTKLVLVNMVYFKGQWDREFKKENTKEEKEWMWKST
SKSVQMMTQSHSFSFTFLEDLQAKILGIPYKNNDLSMFVLLPNDIDGLEKIIDKISPEKLVEWTSPGHMEER
KVNLHLPRFEVEDGYDLEAVLAAMGMGDAFSEHKADYSGMSSGSGLYAQKFLHSSFVAVTEEGTEAAAATGI
GFTVTSAPGHENVHCNHPFLFFIRHNESNSILFFGRFSSP
[0097] The disclosed NOV2a amino acid sequence has 391 of 400 amino
acid residues (97%) identical to, and 391 of 400 amino acid
residues (97%) similar to, the 391 amino acid residue
ptnr:SWISSNEW-ACC:Q9UIV8 protein from Homo sapiens (Human) (Hurpin
(HACAT UV-Repressible Serpin) (Protease Inhibitor 13) (Headpin)
(E=5.8e.sup.-206).
[0098] NOV2a is expressed in at least the following tissues:
adrenal gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. This information was derived by determining the tissue
sources of the sequences that were included in the invention
including but not limited to SeqCalling sources, Public EST
sources, and/or RACE sources.
[0099] In addition, the sequence is predicted to be expressed in
keratinocytes because of the expression pattern of (GENBANK-ID:
gb:GENBANK-ID:HSPI13711|acc:AJ001696.2) a closely related Homo
sapiens mRNA for hurpin, clone R7-1.1 homolog.
[0100] NOV2b
[0101] In the present invention, the target sequence identified
previously, NOV2a, was subjected to the exon linking process to
confirm the sequence. PCR primers were designed by starting at the
most upstream sequence available, for the forward primer, and at
the most downstream sequence available for the reverse primer. In
each case, the sequence was examined, walking inward from the
respective termini toward the coding sequence, until a suitable
sequence that is either unique or highly selective was encountered,
or, in the case of the reverse primer, until the stop codon was
reached. Such primers were designed based on in silico predictions
for the full length cDNA, part (one or more exons) of the DNA or
protein sequence of the target sequence, or by translated homology
of the predicted exons to closely related human sequences sequences
from other species. These primers were then employed in PCR
amplification based on the following pool of human cDNAs: adrenal
gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. Usually the resulting amplicons were gel purified, cloned
and sequenced to high redundancy. The resulting sequences from all
clones were assembled with themselves, with other fragments in
CuraGen Corporation's database and with public ESTs. Fragments and
ESTs were included as components for an assembly when the extent of
their identity with another component of the assembly was at least
95% over 50 bp. In addition, sequence traces were evaluated
manually and edited for corrections if appropriate. These
procedures provide the sequence reported below, which is designated
NOV2b. This differs from the previously identified sequence (NOV2a)
in having 2 different aminoacids.
[0102] A disclosed NOV2b nucleic acid of 1238 nucleotides (also
referred to as CG55999-O.sub.2) encoding a novel Human Hurpin/PI
13-like protein is shown in Table 2C. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
24-26 and ending with a TAA codon at nucleotides 1224-1226. A The
start and stop codons are in bold letters in Table 2C and the 5'
and 3' untranslated regions are underlined. TABLE-US-00022 TABLE 2C
NOV2b nucleotide sequence (SEQ ID NO:11).
TTCTAGGTCTCGCTAAAATCATCATGGATTCACTTGGCGCCGTCAGCACTCGACTTGGGTTTGATCTTTTCA
AAGAGCTGAAGAAAACAAATGATGGCAACATCTTCTTTTCCCCTGTGGGCATCTTGACTGCAATTGGCATGG
TCCTCCTGGGGACCCGAGGAGCCACCGCTTCCCAGTTGGAGGAGGTGTTTCACTCTGAAAAAGAGACGAAGA
GCTCAAGAATAAAGGCTGAAGAAAAAGAGGTGGTAAGAATAAAGGCTGAAGGAAAAGAGATTGAGAACACAG
AAGCAGTACATCAACAATTCCAAAAGTTTTTGACTGAAATAAGCAAACTCACTAATGATTATGAACTGAACA
TAACCAACAGGCTGTTTGGAGAAAAAACATACCTCTTCCTTCAAAAATACTTAGATTATGTTGAAAAATATT
ATCATGCATCTCTGGAACCTGTTGATTTTGTAAATGCAGCCGATGAAAGTCGAAAGAAGATTAATTCCTGGG
TTGAAAGCAAAACAAATGAAAAAATCAAGGACTTGTTCCCAGATGGCTCTATTAGTAGCTCTACCAAGCTGG
TGCTGGTGAACATGGTTTATTTTAAAGGGCAATGGGACAGGGGGTTTAAGAAAGAAAATACTAAGGAAGAGA
AATTTTGGATGAATAAGAGCACAAGTAAATCTGTACAGATGATGACACAGAGCCATTCCTTTAGCTTCACTT
TCCTGGAGGACTTGCAGGCCAAAATTCTAGGGATTCCATATAAAAACAACGACCTAAGCATGTTTGTGCTTC
TGCCCAACGACATCGATGGCCTGGAGAAGATAATAGATAAAATAAGTCCTGAGAAATTGGTAGAGTGGACTA
GTCCAGGGCATATGGAAGAAAGAAAGGTGAATCTGCACTTGCCCCGGTTTGAGGTGGAGGACAGTTACGATC
TAGAGGCGGTCCTGGCTGCCATGGGGATGGGCGATGCCTTCAGTGAGCACAAAGCCGACTACTCGGGAATGT
CGTCAGGCTCCGGGTTGTACGCCCAGAAGTTCCTGCACAGTTCCTTTGTGGCAGTAACTGAGGAAGGCACCG
AGGCTGCAGCTGCCACTGGCATAGGCTTTACTGTCACATCCGCCCCAGGTCATGAAAATGTTCACTGCAATC
ATCCCTTCCTGTTCTTCATCAGGCACAATGAATCCAACAGCATCCTCTTCTTCGGCAGATTTTCTTCTCCTT
AAGATGATCGTTGC
[0103] The disclosed NOV2b nucleic acid sequence, localized to the
q21.3 region of chromsome 18, has 999 of 1013 bases (98%) identical
to a gb:GENBANK-ID:AF169949|acc:AF169949.1 mRNA from Homo sapiens
(Homo sapiens headpin mRNA, complete cds) (E=1.4e.sup.-215).
[0104] A NOV2b polypeptide (SEQ ID NO: 12) encoded by SEQ ID NO: 11
has 400 amino acid residues and is presented using the one-letter
code in Table 2D. Signal P, Psort and/or Hydropathy results predict
that NOV2b contains a signal peptide and is likely to be localized
to the plasma membrane with a certainty of 0.7900. In other
embodiments, NOV2b may also be localized to the microbody
(peroxisome) with a certainty of 0.7147, the Golgi body with a
certainty of 0.3000, or the endoplasmic reticulum (membrane) with a
certainty of 0.2000. The most likely cleavage site for NOV2b is
between positions 50 and 51: ATA-SQ. TABLE-US-00023 TABLE 2D
Encoded NOV2b protein sequence (SEQ ID NO:12).
MDSLGAVSTRLGFDLFKELKKTNDGNIFFSPVGILTAIGMVLLGTRGATASQLEEVFHSEKETKSSRIKAEE
KEVVRIKAEGKEIENTEAVHQQFQKFLTEISKLTNDYELNITNRLFGEKTYLFLQKYLDYVEKYYHASLEPV
DFVNAADESRKKINSWVESKTNEKIKDLFPDGSISSSTKLVLVNMVYFKGQWDRGFKKENTKEEKFWMNKST
SKSVQMMTQSHSFSFTFLEDLQAKILGTPYKNNDLSMFVLLPNDIDGLEKIIDKISPEKLVEWTSPGHMEER
KVNLELPRFEVEDSYDLEAVLAAMGMGDAFSEHKADYSGMSSGSGLYAQKFLHSSFVAVTEEGTEAAAATGI
GFTVPSAPGEENVHCNEPFLFFIHHNESNSILFFGRFSSP
[0105] The disclosed NOV2b amino acid sequence has 390 of 400 amino
acid residues (97%) identical to, and 390 of 400 amino acid
residues (97%) similar to, the 391 amino acid residue
ptnr:SPTREMBL-ACC:Q9UKG0 protein from Homo sapiens (Human)
(HEADPIN) (E=5.3e.sup.-205).
[0106] NOV2b is expressed in at least the following tissues:
adrenal gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea
and uterus, Buccal mucosa, Cervix, Coronary Artery, Skin,
Vulva.
[0107] Expression information was derived from the tissue sources
of the sequences that were included in the derivation of the
sequence of CuraGen Acc. No. CG55999-02.
[0108] The sequence is predicted to be expressed in keratinocytes
because of the expression pattern of (GENBANK-ID:
gb:GENBANK-ID:AF169949|acc:AF169949.1) a closely related Homo
sapiens headpin mRNA, complete cds homolog in species Homo
sapiens.
[0109] NOV2c
[0110] A disclosed NOV2c nucleic acid of 1559 nucleotides (also
referred to as CG55999-05) encoding a novel Human Hurpin/PI 13-like
protein is shown in Table 2E. An open reading frame was identified
beginning with an ATG initiation codon at nucleotides 353-355 and
ending with a TAA codon at nucleotides 1553-1555. A The start and
stop codons are in bold letters in Table 2E and the 5' and 3'
untranslated regions are underlined. TABLE-US-00024 TABLE 2E NOV2c
nucleotide sequence (SEQ ID NO:13).
AAGCTTCCATGAAGAGGAGGCTGTGGAGAGGCAGAGACAGGCAGGGTCAGACAGAGAGCAAGAGAATAAAGC
CATTAAAACATTAACCCTGCTCCGCGGGGAAATAAGAACTGAGCACCACCGGATGACGGAAGACTCCAGTAG
ATTGATGGATGTCTCCCAGCAAGAGAAGGCCAAGAGAGGACGTGAGAAGCAGGCAGCAGCGACCTTTCACCA
AAAGGGTGGAAATCCCTGTATTCCGGATCGATGCAAGAAGAGGAATAGAAGCAGAAAGGATTCCCCTGACAC
AGAGTAATTCAAATGTTCAGTTTTGATTGTTGTTCTTGCTATTCTAGGTCTCGCTAAAATCATCATGGATTC
ACTTGGCGCCGTCAGCACTCGACTTGGGTTTGATCTTTTCAAAGAGCTGAAGAAAACAAATGATGGCAACAT
CTTCTTTTCCCCTGTGGGCATCTTGACTGCAATTGGCATGGTCCTCCTGGGGACCCGAGGAGCCACCGCTTC
CCAGTTGGAGGAGGTGTTTCACTCTGAAAAAGAGACGAAGAGCTCAAGAATAAAGGCTGAAGAAAAAGAGGT
GGTAAGAATAAAGGCTGAAGGAAAAGAGATTGAGAACACAGAAGCAGTACATCAACAATTCCAAAAGTTTTT
GACTGAAATAAGCAAACTCACTAATGATTATGAACTGAACATAACCAACAGGCTGTTTGGAGAAAAAACATA
CCTCTTCCTTCAAAAATACTTAGATTATGTTGAAAAATATTATCATGCATCTCTGGAACCTGTTGATTTTGT
AAATGCAGCCGATGAAAGTCGAAAGAAGATTAATTCCTGGGTTGAAAGCAAAACAAATGAAAAAATCAAGGA
CTTGTTCCCAGATGGCTCTATTAGTAGCTCTACCAAGCTGGTGCTGGTGAACATGGTTTATTTTAAAGGGCA
ATGGGACAGCGAGTTTAAGAAAGAAAATACTAAGGAAGAGAAATTTTGGATGAATAAGAGCACAAGTAAATC
TGTACAGATGATGACACAGAGCCATTCCTTTAGCTTCACTTTCCTGGAGGACTTGCAGGCCAAAATTCTAGG
GATTCCATATAAAAACAACGACCTAAGCATGTTTGTGCTTCTGCCCAACGACATCGATGGCCTGGAGAAGAT
AATAGATAAAATAAGTCCTGAGAAATTGGTAGAGTGGACTAGTCCAGGGCATATGGAAGAAAGAAAGGTGAA
TCTGCACTTGCCCCGGTTTGAGGTGGAGGACAGTTACGATCTAGAGGCGGTCCTGGCTGCCATGGGGATGGG
CGATGCCTTCAGTGAGCACAAAGCCGACTACTCGGGAATGTCGTCAGGCTCCGGGTTGTACGCCCAGAAGTT
CCTGCACAGTTCCTTTGTGGCAGTAACTGAGGAAGGCACCGAGGCTGCAGCTGCCACTGGCATAGGCTTTAC
TGTCACATCCGCCCCAGGTCATGAAAATGTTCACTGCAATCATCCCTTCCTGTTCTTCATCAGGCACAATGA
ATCCAACAGCATCCTCTTCTTCGGCAGATTTTCTTCTCCTTAAGATG
[0111] The disclosed NOV2c nucleic acid sequence, localized to the
q21.3-22 region of chromsome 18, has 519 of 519 bases (100%)
identical to a gb:GENBANK-ID:AF216854|acc:AF216854.1 mRNA from Homo
sapiens (Homo sapiens headpin gene, complete cds)
(E=2.3e.sup.-303).
[0112] A NOV2c polypeptide (SEQ ID NO:14) encoded by SEQ ID NO:13
has 400 amino acid residues and is presented using the one-letter
code in Table 2F. Signal P, Psort and/or Hydropathy results predict
that NOV2c contains a signal peptide and is likely to be localized
to the plasma membrane with a certainty of 0.7900. In other
embodiments, NOV2c may also be localized to the microbody
(peroxisome) with a certainty of 0.7024, the Golgi body with a
certainty of 0.3000, or the endoplasmic reticulum (membrane) with a
certainty of 0.2000. The most likely cleavage site for NOV2c is
between positions 50 and 51: ATA-SQ. TABLE-US-00025 TABLE 2F
Encoded NOV2c protein sequence (SEQ ID NO:14).
MDSLGAVSTRLGFDLFKELKKTNDGNIFFSPVGILTAIGMVLLGTRGATASQLEEVFHSEKETKSSRIKAEE
KEVVRIKAEGKEIENTEAVHQQFQKFLTEISKLTNDYELNITNRLFGEKTYLFLQKYLDYVEKYYHASLEPV
DFVNAADESRKKINSWVESKTNEKIKDLFPDGSISSSTKLVLVNMVYFKGQWDREFKKENTKEEKFWMNKST
SKSVQNMTQSHSFSFTFLEDLQAKILGIPYKNNDLSMFVLLPNDIDGLEKIIDKTSPEKLVEWTSPGHMEER
KVNLHLPRFEVEDSYDLEAVLAAMGMGDAFSEHKADYSGMSSGSGLYAQKFLHSSFVAVTEEGTEAAAATGI
GFTVTSAPGHENVHCNHPFLFFIRHNESNSILFFGRFSSP
[0113] The disclosed NOV2c amino acid sequence has 391 of 400 amino
acid residues (97%) identical to, and 391 of 400 amino acid
residues (97%) similar to, the 391 amino acid residue
ptnr:SPTREMBL-ACC:Q9UKG0 protein from Homo sapiens (Human)
(Headpin) (E=1.1e.sup.-205)
[0114] NOV2d
[0115] A disclosed NOV2d nucleic acid of 818 nucleotides (also
referred to as CG55999-06) encoding a novel Human Hurpin/PI 13-like
protein is shown in Table 2G. An open reading frame was identified
beginning with an ATG initiation codon at nucleotides 40-42 and
ending with a TAA codon at nucleotides 565-567. A The start and
stop codons are in bold letters in Table 2G and the 5' and 3'
untranslated regions are underlined. TABLE-US-00026 TABLE 2G NOV2d
nucleotide sequence (SEQ ID NO:15).
ACCGTCTCTCCAAAAACCCGAGGTCTCGCTAAAATCATCATGGATTCACTTGGCGCCGTCAGCACTCGACTT
GGGTTTGATCTTTTCAAAGAGCTGAAGAAAACAAATGATGGCAACATCTTCTTTTCCCCTGTGGGCATCTTG
ACTGCAATTGGCATGGTCCTCCTGGGGACCCGAGGAGCCACCGCTTCCCAGTTGGAGGAGGTGTTTCACTCT
GAAAAAGAGACGAAGAGCTCAAGAATAAAGGCTGAAGAAAAAGAGGTGGTAAGAATAAAGGCTGAAGGAAAA
GAGATTGAGAACACAGAAGCAGTACATCAACAATTCCAAAAGTTTTTGACTGAAATAAGCAAACTCACTAAT
GATTATGAACTGAACATAACCAACAGGCTGTTTGGAGAAAAAACATACCTCTTCCTTCAAAAATACTTAGAT
TATGTTGAAAAATATTATCATGCATCTCTGGAACCTGTTGATTTTGTAAATGCAGCCGATGAAAGTCGAAAG
AAGATTAATTCCTGGGTTGAAAGCAAAACAAATGATGTGCAAACTGAGGCACAGAGAGTTTAAATAACTTGC
CCAAGATTCCTCAGCTGATAAGAGGCAAACTGGATGCTAACAGAGGCATCTGACCCCAGAGTCTGGACTCTT
AACCATGAACCTTAATTTATCCACTGGGATAAATAGGCGATGGGCAAAATGAGAACCTCCCCGTCGATTCTG
CCAGCAAACCCTTTGTCAGCAAGGCCCTCAGAAAAAATCAAGGACTTGTTCCCAGATGGCTCTATTAGTAGC
TCTACCAAGCTGGTGCTGGTGACATG
[0116] The disclosed NOV2d nucleic acid sequence, localized to the
q21.3-22 region of chromsome 18, has 214 of 214 bases (100%)
identical to a gb:GENBANK-ID:AF216854|acc:AF216854.1 mRNA from Homo
sapiens (Homo sapiens headpin gene, complete cds)
(E=1.3e.sup.-134).
[0117] A NOV2d polypeptide (SEQ ID NO:16) encoded by SEQ ID NO:15
has 175 amino acid residues and is presented using the one-letter
code in Table 2H. Signal P, Psort and/or Hydropathy results predict
that NOV2d contains a signal peptide and is likely to be localized
to the plasma membrane with a certainty of 0.7900. In other
embodiments, NOV2d may also be localized to the microbody
(peroxisome) with a certainty of 0.3878, the Golgi body with a
certainty of 0.3000, or the endoplasmic reticulum (membrane) with a
certainty of 0.2000. The most likely cleavage site for NOV2d is
between positions 50 and 51: ATA-SQ. TABLE-US-00027 TABLE 2H
Encoded NOV2d protein sequence (SEQ ID NO:16).
MDSLGAVSTRLGFDLFKELKKTNDGNIFFSPVGILTAIGMVLLGTRGATASQLEEVFHSEKETKSSRIKAEE
KEVVRIKAEGKETENTEAVEQQFQKFLTEISKLTNDYELNITNRLFGEKTYLFLQKYLDYVEKYYHASLEPV
DFVNAADESRKKINSWVESKTNDVETEAQRV
[0118] The disclosed NOV2d amino acid sequence has 157 of 167 amino
acid residues (94%) identical to, and 158 of 167 amino acid
residues (94%) similar to, the 391 amino acid residue
ptnr:SWISSNEW-ACC:Q9UIV8 protein from Homo sapiens (Human) (HURPIN
(HACAT UV-Repressible Serpin) (Protease Inhibitor 13) (Headpin))
(E=2.2e.sup.-76).
[0119] NOV2d is expressed in at least the following tissues:
Mammalian Tissue, Coronary Artery, Buccal mucosa, Pituitary Gland,
Cervix, Uterus, Vulva, Skin. Expression information was derived
from the tissue sources of the sequences that were included in the
derivation of the sequence of CuraGen Acc. No. CG55999-06.
[0120] NOV2e
[0121] A disclosed NOV2e nucleic acid of 1062 nucleotides (also
referred to as 166485357) encoding a novel Human Hurpin/PI 13-like
protein is shown in Table 2I. An open reading frame was identified
beginning with an GGA initiation codon at nucleotides 1-3 and
ending with a GAG codon at nucleotides 1060-1062. A The start and
stop codons are in bold letters in Table 21. Since the start and
stop codons are not traditional initiation and termination codons,
NOV2e may be a partial reading frame that extends further in the 5'
and 3' directions. TABLE-US-00028 TABLE 2I NOV2e nucleotide
sequence (SEQ ID NO:197).
GGATCCTCCCAGTTGGAGGAGGTGTTTCACTCTGAAAAAGAGACGAAGAGCTCAAGAATAAAGGCTGAAGAA
AAAGAGGTGGTAAGAATAAAGGCTGAAGGAAAAGAGATTGAGAACACAGAAGCAGTACATCAACAATTCCAA
AAGTTTTTGACTGAAATAAGCAAACTCACTAATGATTATGAACTGAACATAACCAACAGGCTGTTTGGAGAA
AAAACATACCTCTTCCTTCAAAAATACTTAGATTATGTTGAAAAATATTATCATGCATCTCTGGAACCTGTT
GATTTTGTAAATGCAGCCGATGAAAGTCGAAAGAAGATTAATTCCTGCGTTGAAAGCAAAACAAATGAAAAA
ATCAAGGACTTGTTCCCAGATGGCTCTATTAGTAGCTCTACCAAGCTGGTGCTGGTGAACATGGTTTATTTT
AAAGGGCAATGGGACAGGGAGTTTAAGAAAGAAAATACTAAGGAACAGAAATTTTGGATGAATAAGAGCACA
AGTAAATCTGTACAGATGATGACACAGAGCCATTCCTTTAGCTTCACTTTCCTGGAGGACTTGCAGGCCAAA
ATTCTAGGGATTCCATATAAAAACAACGACCTAAGCATGTTTGTGCTTCTGCCCAACGACATCGATGGCCTG
GAGAAGATAATAGATAAAATAAGTCCTGAGAAATTGGTAGAGTGGACTAGTCCAGGGCATATGGAAGAAAGA
AAGGTGAATCTGCACTTGCCCCGGTTTGAGGTGGAGGACAGTTACGATCTAGAGGCGGTCCTGGCTGCCATG
GGGATGGGCGATGCCTTCAGTGAGCACAAAGCCGACTACTCGGGAATGTCGTCAGGCTCCGGGTTGTACGCC
CAGAAGTTCCTGCACAGTTCCTTTGTGGCAGTAACTGAGGAAGGCACCGAGGCTGCAGCTGCCACTGGCATA
GGCTTTACTGTCACATCCGCCCCAGGTCATGAAAATGTTCACTGCAATCATCCCTTCCTGTTCTTCATCAGG
CACAATGAATCCAACAGCATCCTCTTCTTCGGCAGATTTTCTTCTCCTCTCGAG
[0122] A NOV2e polypeptide (SEQ ID NO:14) encoded by SEQ ID NO:13
has 354 amino acid residues and is presented using the one-letter
code in Table 2J. TABLE-US-00029 TABLE 2J Encoded NOV2e protein
sequence (SEQ ID NO:198).
GSSQLEEVFHSEKETKSSRIKAEEKEVVRIKAEGKEIENTEAVHQQFQKFLTEISKLTNDYELNITNRLFGE
KTYLFLQKYLDYVEKYYEASLEPVDFVNAADESRKKINSWVESKTNEKIKDLFPDGSISSSTKLVLVNMVYF
KGQWDREFKKENTKEEKFWMNKSTSKSVQMMTQSHSFSFTFLEDLQAKILGIPYKNNDLSMFVLLPNDIDGL
EKIIDKISPEKLVEWTSPGHMEERKVNLHLPRFEVEDSYDLEAVLAAMGMGDAFSEHKADYSGMSSGSGLYA
QKFLHSSFVAVTEEGTEAAAATGIGFTVTSAPGHENVHCNHPFLFFIRHNESNSILFFGRFSSPLE
[0123] NOV2a also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 2I. TABLE-US-00030 TABLE 2K BLAST
results for NOV2 Gene Index/ Protein/ Length Identity Positives
Identifier Organism (aa) (%) (%) Expect gi|8393956|ref| serine (or
391 364/400 364/400 0.0 NP_036529.1| cysteine) (91%) (91%)
(NM_012397) proteinase inhibitor, clade B gi|6018510|emb| hurpin
[Homo 390 362/400 363/400 0.0 CAA04937.1| sapiens] (90%) (90%)
(AJ001698) gi|7522623|pir|| headpin 391 363/400 363/400 0.0 JC7118
serine (90%) (90%) proteinase inhibitor- human gi|9801227|emb|
hurpin [Homo 339 311/400 312/400 e-162 CAC03569.1| sapiens] (77%)
(77%) (AJ278717) gi|266995|sp|P29508| SQUANOUS 390 209/401 274/401
e-107 SCC1_HUMAN CELL (52%) (68%) CARCINOMA ANTIGEN 1 (SCCA-1)
(PROTEIN T4- A)
[0124] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 2L.
[0125] Tables 2M-N lists the domain description from DOMAIN
analysis results against NOV2a. This indicates that the NOV2a
sequence has properties similar to those of other proteins known to
contain this domain. TABLE-US-00031 TABLE 2M Domain Analysis of
NOV2 gnl|Pfam|pfam00079, serpin, Serpin (S protease inhibitor).
Structure is a multi-domain fold containing a bundle of helices and
a beta sandwich. (SEQ ID ID:66) CD-Length = 377 residues, 98.4%
aligned Score = 318 bits (816), Expect = 3e-88 Query: 3
SLGAVSTRLGFDLFKEL-KKTNDGNIFFSPVGILTAIGMVLLGTRGATASQLEEVFHSEK 61 | +
+ | |+||| ++ | ||||||| | +|+ |+ || +| ||+|+ || Sbjct: 7
KLASANADFAFSLYKELVEQNPDKNIFFSPVSISSALAMLSLGAKGNTATQILEVLGFNL 66
Query: 62
ETKSSRIKAEEKEVVRIKAEGKEIENTEAVHQQFQKFLTEISKLTNDYELNITNRLFGEK 121 |
+|| || | |+++ +| | || +| Sbjct: 67
TETSE----------------------AEIHQGFQELLQELNRPDTGLQLTTGNALFVDK 104
Query: 122
TYLFLQKYLDYVEKYYHASLEPVDFVNAADESRKKINSWVESKTNEKIKDLFPDGSISSS 181 +
| ++|+ ++ | + + ||| + +|++|+|| ||| || ||||| | + | Sbjct: 105
SLKLLDEFLEDSKRLYQSEVFSVDF-SDPEEAKKQINDWVEKKTQGKIKDLLKD--LDSD 161
Query: 182
TKLVLVNMVYFKGQWDREFKKENTKEEKFWMNKSTSKSVQMMTQSHSFSFTFLEDLQAKI 241 |
||||| +||||+| + | | |+|| | ++| |+ | || | +| + |+| |+ Sbjct: 162
TVLVLVNYIYFKGKWKKPFDPELTEEEDFHVDKKTTVKVPMMNQLGTFYYFRDEELNCKV 221
Query: 242
LGIPYKNNDLSMFVLLPNDIDGLEKIIDKISPERLVEWTSPGHMEERKVNLHLPRFEVED 301 |
+||| | || +||+++ ||++ +||| | +| +|| |+| |+||+| +| Sbjct: 222
LELPYKGNATSMLFILPDEVGKLEQVEAALSPETLRKWLE--NMEPREVELYLPKFSIEG 279
Query: 302
GYDLEAVLAAMGMGDAFSEHRADYSGMSSGSGLYAQKFLHSSFVAVTEEGTEAAAATGIG 361
|||+ ||| +|+ | || +|| ||+| | | +| + + | ||||||||||| Sbjct: 280
TYDLKDVLAKLGITDLFSN-QADLSGISEDEDLKVSKAVHKAVLEVDEEGTEAAAATGAI 338
Query: 362 FTVTSAPGHENVHCNHPFLFFIRHNESNSILFFGRFSSP 400 | | + |||| |
+ + |||| |+ +| Sbjct: 339 IVPRSLPPELEFTADRPFLFLIYDDPTGSILFMGKVVNP
377
[0126] TABLE-US-00032 TABLE 2N Domain Analysis of NOV2
gnl|Smart|smart00093, SERPIN, SERine Proteinase INhibitors (SEQ ID
NO:67) CD-Length = 360 residues, 100.0% aligned Score = 312 bits
(800), Expect = 2e-86 Query: 13
FDLFKELKKTN-DGNIFFSPVGILTAIGMVLLGTRGATASQLEEVFHSEKETKSSRIKAE 71
|||+||| | + | ||||||| | +|+ |+ || +|+||+|+ || | Sbjct: 1
FDLYKELAKESPDKNIFFSPVSISSALAMLSLGAKGSTATQILEVLGFNLTETSE----- 55
Query: 72
EKEVVRIKAEGKEIENTEAVHQQFQKFLTEISKLTNDYELNITNRLFGEKTYLFLQKYLD 131
+|| || | +++ | +| | || +|+ | +|+ Sbjct: 56
-----------------ADIHQGFQHLLHLLNRPDNKLQLRTANALFVDKSLKLLDSFLE 98
Query: 132
YVEKYYHASLEPVDFVNAADESRKKINSWVESKTNEKIKDLFPDGSISSSTKLVLVNMVY 191
|+| | | ++ ||| + |+|++|+|| ||+ || ||||| | + |+||||| +| Sbjct: 99
DVKKLYGAEVQSVDFSDPAEEAKKQINDWVKKKTQGKIKDLLSD--LDPDTRLVLVNAIY 156
Query: 192
FKGQWDREFKKENTKEEKFWMNKSTSKSVQMMTQ-SHSFSFTFLEDLQAKILGIPYKNND 250
|||+| | |||+|| |+++++|+ | ||+| +| + |+| ++| +||| | Sbjct: 157
FKGKWKTPFDPENTREEDFYVDETTTVKVPMMSQTGRTFRYGRDEELNCQVLELPYKGN- 215
Query: 251
LSMFVLLPNDIDGLEKIIDKISPEKLVEWTSPGHMEERKVNLHLPRFEVEDGYDLEAVLA 310 ||
++||++ ||| + ++|| | +|| + +| | |+||+|++| |||+ || Sbjct: 216
ASMLIILPDEG-GLETVEKALTPETLKKWTK--SLTKRSVELYLPKFKLEISYDLKDVLE 272
Query: 311
AMGMGDAFSEHKADYSGMSSGSGLYAQKFLHSSFVAVTEEGTEAAAATGIGFTVTSAPGH 370
+|+ | || ||| ||+| | | +| +|+ | |||||||||||+ | | Sbjct: 273
KLGITDLFSN-KADLSGISEDKDLKVSKVVHKAFLEVNEEGTEAAAATGVIIVPRSLP-P 330
Query: 371 ENVHCNHPFLFFIRHNESNSILFFGRFSSP 400 | |||| || | + |||| |+
+| Sbjct: 331 PEFKANRPFLFLIRDNPTGSILFMGKVVNP 360
[0127] Proteolysis is the key feature of programmed cell death.
Extracellular proteinases can activate cell surface receptors which
trigger apoptosis, and the effector machinery requires the
activation and activity of numerous intracellular proteinases
(primarily caspases). Effective control of proteolysis is essential
for homeostasis and can occur at two levels: regulation of
proteinase activation, and regulation of the activated proteinase.
The serpins, a family of proteins that inhibit chymotrypsin-like
serine proteinases, control activated proteinases and several have
been implicated in the regulation of cell death. Hurpin is a novel
serine proteinase inhibitor recently cloned by Abts H F et al.
(1999, J. Mol. Biol., Vol. 293:29-39). It has-nearly 59% amino acid
identity with the squamous cell carinoma antigen1 (SCCA1) and
squamous cell carcinoma antigen 2 (SCCA2). Expression of hurpin
appears to be related to the activation or proliferation state of
keratinocytes.
[0128] The disclosed NOV2 nucleic acid of the invention encoding a
Human Hurpin/Pi 13-like protein includes the nucleic acid whose
sequence is provided in Tables 2A, 2C, 2E, 2G or a fragment
thereof. The invention also includes a mutant or variant nucleic
acid any of whose bases may be changed from the corresponding base
shown in Tables 2A, 2C, 2E, or, 2G while still encoding a protein
that maintains its Human Hurpin/Pi 13-like activities and
physiological functions, or a fragment of such a nucleic acid. The
invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, byway
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 3 percent of the
bases may be so changed.
[0129] The disclosed NOV2 protein of the invention includes the
Human Hurpin/PI 13-like protein whose sequence is provided in
Tables 2B, 2D, 2F, or 2H. The invention also includes a mutant or
variant protein any of whose residues may be changed from the
corresponding residue shown in Table 2B, 2D, 2F, or 2H while still
encoding a protein that maintains its Human Hurpin/PI 13-like
activities and physiological functions, or a functional fragment
thereof. In the mutant or variant protein, up to about 48 percent
of the residues may be so changed.
[0130] The NOV2 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in
Colorectal cancer; Combined factor V and VIII deficiency; Cone-rod
retinal dystrophy-1; Leukemia/lymphoma, B-cell, 2;
Lymphoma/leukemia, B-cell, variant; Protoporphyria, erythropoietic;
Protoporphyria, erythropoietic, recessive, with liver failure;
Obesity, autosomal dominant; Osteosarcoma; cancer, skin psoriasis,
and/or other pathologies and disorders.
[0131] NOV2 nucleic acids and polypeptides 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. These antibodies may be generated according to
methods known in the art, using prediction from hydrophobicity
charts, as described in the "Anti-NOVX Antibodies" section below.
The disclosed NOV2 protein has multiple hydrophilic regions, each
of which can be used as an immunogen. These novel proteins can be
used in assay systems for functional analysis of various human
disorders, which are useful in understanding of pathology of the
disease and development of new drug targets for various
disorders.
[0132] NOV3
[0133] NOV3 includes three novel Set Binding Factor (SBF1)-like
proteins disclosed below. The disclosed sequences have been named
NOV3a and NOV3b.
[0134] NOV3a
[0135] A disclosed NOV3a nucleic acid of 5316 nucleotides (also
referred to as CG56019-01) encoding a novel Set Binding Factor
(SBF1)-like protein is shown in Table 3A. An open reading frame was
identified beginning with a ATT initiation codon at nucleotides 3-5
and ending with a TGA codon at nucleotides 5172-5174. The start and
stop codons are in bold letters, and the 5' and 3' untranslated
regions are underlined. Because the start codon is not a
traditional initiation codon, NOV3 could be a partial open reading
frame that extends further in the 5' direction. TABLE-US-00033
TABLE 3A NOV3a Nucleotide Sequence (SEQ ID NO:17)
GAATTCGGCACGAGGTCTTCCTGTCCCGGAGCTACCAGCGGCTCGCCGATGCCTGTAGGGGCCTCCTGGCA
CTGCTGTTTCCTCTCAGATACAGCTTCACCTATGTGCCCATCCTGCCGGCTCAGCTGCTGGAGGTCCTCAG
CACACCCACGCCCTTCATCATTGGGGTCAACGCGGCCTTCCAGGCAGAGACCCAGGAGCTGCTCGATGTGA
TTGTTGCTGATCTGGATGGAGGGACGGTCACCATTCCTGAGTGTGTGCACATTCCACCCTTGCCAGAGCCA
CTGCAGAGTCAGACGCACAGTGTGCTGAGCATGGTCCTGGACCCGGAGCTGGAGTTGGCTGACCTCGCCTT
CCCTCCGCCCACGACATCCACCTCCTCCCTGAAGATGCAGGACAAGGAGCTGCGCGCGGTCTTCCTGCGGC
TGTTCGCTCAGCTGCTGCAGGGCTATCGCTGGTGCCTGCACGTCGTGCGCATCCACCCGGAGCCTGTCATC
CGCTTCCATAAGGCAGCCTTCCTGGGGCAGCGTGGGCTGGTAGAGGACGATTTCCTGATGAAGGTGCTGGA
GGGCATGGCCTTTGCTGGCTTTGTGTCAGAGCGTGGGGTCCCATACCGCCCTACGGACCTGTTCGATGAGC
TGGTGGCCCACGAGGTGGCAAGGATGCGGGCGGATGAGAACCACCCCCAGCGTGTCCTGCGTCACGTCCAG
GAACTGGCAGAGCAGCTCTACAAGAACGAGAACCCGTACCCAGCCGTGGCGATGCACAAGGTACAGAGGCC
TCGTGGACCAGGCTGCAGCCAAGATGCAGGGTGCACCCCCAGCTGTGAAGGCCGAGAGGAGGACCACCGTG
CCCTCAGGGCCCCCCATGACTGCCATACTGGAGCGGTGCAGTGGGCTGCATGTCAACAGCGCCCGGCGGCT
GGAGGTTGTGCGCAACTGCATCTCCTACGTGTTTGAGGGGAAAATGCTTGAGGCCAAGAAGCTGCTCCCAG
CCGTGTTGAGGGCCCTGAAGGGGCGAGTTGCCCGCCGCTGCCTCGCCCAGGAGCTGCACCTGCATGTGCAG
CAGAACCGTGCGGTCCTGGACCACCAGCAGTTTGACTTTGTCGTCCGTATGATGAACTGCTGCCTGCAGGA
CTGCACTTCTCTGGACGAGCATGGCATTGCGGCGGCTCTGCTGCCTCTGGTCACAGCCTTCTGCCGGAAGC
TGAGCCCGGGGGTGACGCAGTTTGCATACAGCTGTGTGCAGGAGCACGTGGTGTGGAGCACGCCACAGTTC
TGGGAGGCCATGTTCTATGGGGATGTGCAGACTCACATCCGGGCCCTCTACCTGGAGCCCACGGAGGACCT
GGCCCCCGCCCAGGAGGTTGGGGAGGCACCTTCCCAGGAGGACGAGCGCTCTGCCCTAGACGTGGCTTCTG
AGCAGCGGCGCTTGTGGCCAACTCTGAGTCGTGAGAAGCAGCAGGAGCTGGTGCAGAAGGAGGAGAGCACG
GTGTTCAGCCAGGCCATCCACTATGCCAACCGCATGAGCTACCTCCTCCTGCCCCTGGACAGCAGCAAGAG
CCGCCTACTTCGGGAGCGTGCCGGGCTGGGCGACCTGGAGAGCGCCAGCAACAGCCTGGTCACCAACAGCA
TGGCTGGCAGTGTGGCCGAGAGCTATGACACGGAGAGCGGCTTCGAGGATGCAGAGACCTGCGACGTAGCT
GGGGCTGTGGTCCGCTTCATCAACCGCTTTGTGGACAAGGTCTGCACGGAGAGTGGGGTCACCAGCGACCA
CCTCAAGGGGCTGCATGTCATGGTGCCAGACATTGTCCAGATGCACATCGAGACCCTGGAGGCCGTGCAGC
GGGAGAGCCGGAGGCTGCCGCCCATCCAGAAGCCCAAGCTGCTGCGGCCGCGCCTGCTGCCGGGTGAGGAG
TGTGTGCTGGACGGCCTGCGCGTCTACCTGCTGCCGGATGGGCGTGAGGAGGGCGCGGGGGGCAGTGCTGG
GGGACCAGCATTGCTCCCAGCTGAGGGCGCCGTCTTCCTCACCACGTACCGGGTCATCTTCACGGGGATGC
CCACGGACCCCCTGGTTGGGGAGCAGGTGGTGGTCCGCTCCTTCCCGGTGGCTGCGCTGACCAAGGAGAAG
CGCATCAGCGTCCAGACCCCTGTGGACCAGCTCCTGCAGGACGGGCTCCAGCTGCGCTCCTGCACATTCCA
GCTGCTGAAAATGGCCTTTGACGAGGAGGTGGGGTCTGACAGCGCCGAGCTCTTCCGTAAGCAGCTGCATA
AGCTGCGGTACCCGCCGGACATCAGGGCCACCTTTGCGTTCACCTTGGGCTCTGCCCACACACCTGGCCGG
CCACCGCGAGTCACCAAGGACAAGGGTCCTTCCCTCAGAACCCTGTCCCGGAACCTGGTCAAGAACGCCAA
GAAGACCATCGGGCGGCAGCATGTCACTCGCAAGAAGTACAACCCCCCCAGCTGGGAGCACCGGGGCCAGC
CGCCCCCTGAGGACCAGGAGGACGAGATCTCAGTGTCGGAGGAGCTGGAGCCCAGCACGCTGACCCCGTCC
TCAGCCCTGAAGCCCTCCGACCGCATGACCATGAGCAGCCTGGTGGAAAGGGCTTGCTGTCGCGACTACCA
GCGCCTCGGTCTGGGCACCCTGAGCAGCAGCCTGAGCCGGGCCAAGTCTGAGCCCTTCCGCATTTCTCCGG
TCAACCGCATGTATGCCATCTGCCGCAGCTACCCAGGGCTGCTGATCGTGCCCCAGAGTGTCCAGGACAAC
GCCCTGCAGCGCGTGTCCCGCTGCTATCGCCAGAACCGCTTCCCCGTGGTCTGCTGGCGCAGCGAGCGGTC
CAAGGCGGTGCTGCTGCGCTCTGGAGGCCTGCATGGCAAAGGTGTCGTCGGCCTCTTCAAGGCCCAGAACG
CACCTTCTCCAGGCCAGTCCCAGGCGGACTCGAGTAGCCTGGAGCAGGAGAAGTACCTGCAGGCTGTGGTC
AGCTCCATGCCCCGCTACGCCGACGCGTCGGGACGCAACACGCTTAGCGGCTTCTCCTCAGCCCACATGGG
CAGTCACGTTCCCAGCCCCAGAGCCAGGGTCACCACGCTGTCCAACCCCATGGCGGCCTCGGCCTCCAGAC
GGACCGCACCCCGAGGTAAGTGGGGCAGTGTCCGGACCAGTGGACGCAGCAGTGGCCTTGGCACCGATGTG
GGCTCCCCGGCTAGCTGGCAGAGACGCGCTGGCCCCACCCCAGGCCAACGGGGGCCCTCCCGACCCGGCTT
CCTGCGTCCGCAGCGAGCAGCCCTCTATATCCTTGGGGACAAAGCCCAGCTCAAGGGTGTGCGGTCAGACC
CCCTGCAGCAGTGGGAGCTGGTGCCCATTGAGGTATTCGAGGCACGGCAGGTGAAGGCTAGCTTCAAGAGG
CTGCTGAAAGCATGTGTCCCAGGCTGCCCCGCTGCTGAGCCCAGCCCAGCCTCCTTCCTGCGCTCACTGGA
GGACTCAGAGTGGCTGATCCAGATCCACAAGCTGCTGCAGGTGTCTGTGCTGGTGGTGGAGCTCCTGGATT
CAGGCTCCTCCGTGCTGGTGGGCCTGGAGGATGGCTGGGACATCACCACCCAGGTGGTATCCTTGGTGCAG
CTGCTCTCAGACCCCTTCTACCGCACGCTGGAGGGCTTTCGCCTGCTGGTGGAGAAGGAGTGGCTGTCCTT
CGGCCATCGCTTCAGCCACCGTGGAGCTCACACCCTGGCCGGGCAGAGCAGCGGCTTCACACCCGTCTTCC
TGCAGTTCCTGGACTGCGTACACCAGGTCCACCTGCAGTTCCCCATGGAGTTTGAGTTCAGCCAGTTCTAC
CTCAAGTTCCTCGGCTACCACCATGTGTCCCGCCGTTTCCGGACCTTCCTGCTCGACTCTGACTATGAGCG
CATTGAGCTGGGGCTGCTGTATGAGGAGAAGGGGGAACGCAGGGGCCAGGTGCCGTGCAGGTCTGTGTGGG
AGTATGTGGACCGGCTGAGCAAGAGGACGCCTGTGTTCCACAATTACATGTATGCGCCCGAGGACGCAGAG
GTCCTGCGGCCCTACAGCAACGTGTCCAACCTGAAGGTGTGGGACTTCTACACTGAGGAGACGCTGGCCGA
GGCCCTCCCTATGACTGGGAACTGGCCCAGGGGCCCCCTGAACCCCCAGAGGAAGAACGGTCTGATGGAGG
CGTCCCCAGAGCAGCGCCGCGTGGTGTGGCCCTGTTACGACAGCTGCCCGCGGGCCCAGCCTGACGCCATC
TCACGCCTGCTGGAGGAGCTGCAGAGGCTGGAGACAGAGTTGGGCCAACCCGCTGAGCGCTGGAAGGACAC
CTGGGACCGGGTGAAGGCTGCACAGCGCCTCGAGGGCCGGCCAGACGGCCGTGGCACCCCTAGCTCCCTCC
TTGTGTCCACCGCACCCCACCACCGTCGCTCGCTGGGTGTGTACCTGCAGGAGGGGCCCGTGGGCTCCACC
CTGAGCCTCAGCCTGGACAGCGACCAGAGTAGTGGCTCAACCACATCCGGCTCCCGTCAGGCTGCCCGCCG
CAGCACCAGCACCCTGTACAGCCAGTTCCAGACAGCAGAGAGTGAGAACAGGTCCTACGAGGGCACTCTGT
ACAAGAAGGGGGCCTTCATGAAGCCTTGGAAGGCCCGCTGGTTCGTGCTGGACAAGACCAAGCACCAGCTG
CGCTACTACGACCACCGTGTGGACACAGAGTGCAAGGGTGTCATCGACTTGGCGGAGGTGGAGGCTGTGGC
ACCTGGCACGCCCACTATGGGTGCCCCTAAGACTGTGGACGAGAAGGCCTTCTTTGACGTGAAGACAACGC
GTCGCGTTTACAACTTCTGTGCCCAGGACGTGCCCTCGGCCCAGCAGTGGGTGGACCGGATCCAGAGCTGC
TGTCGGACGCCTGAGCCTCCCAGCCCTGCCCGGCTGCTCTGCTCTCGTTACCGACCACTAGGGGTGGCAGG
GCCGCCCCGGCCATGTTTACAGCCCCGGCCCTCGACAGTACTGAGCCCCGAGCCCCCAGCACTTGTGTGTA
CAGCCCCCGTCCCCGCCCCGCCCCGCCCGGCCGGCCCTAACTTATTTTGGCGTCACAGCTGAGCACCGTGC
CGGGAGGTGGCCAAGGTACAGCCCGCAATGGGCCTGTAAATAGTCCGGCCCCGTCAGCGTGTGCTGGTCCA
CGGGCTCAGGCGAGTTTCTAGAAAGAGTCTATATAAAGAGAGAACTAACGCCAAAAAAAA
[0136] The disclosed NOV3a nucleic acid sequence maps to the q13.3
region of chromosome 22 and has 3553 of 3902 bases (91%) identical
to a gb:GENBANK-ID:HSU93181|acc:U93181.1 mRNA from Homo sapiens
(Homo sapiens nuclear dual-specificity phosphatase (SBF1) mRNA,
partial cds) (E=0.0).
[0137] A disclosed NOV3a protein (SEQ ID NO:18) encoded by SEQ ID
NO:17 has 1723 amino acid residues, and is presented using the
one-letter code in Table 3B. Signal P, Psort and/or Hydropathy
results predict that NOV3a does have a signal peptide, and is
likely to be localized to the mitochondrial membrane space with a
certainty of 0.5000. In other embodiments NOV3a is also likely to
be localized to the microbody (peroxisome) with a certainty of
0.3000, to mitochondrial inner membrane with a certainty of 0.2187,
or to the mitochondrial intermembrane space with a certainty of
0.2187. The most likely cleavage site for NOV3a is between
positions 33 and 34, (SFT-YV). TABLE-US-00034 TABLE 3B Encoded
NOV3a protein sequence (SEQ ID NO:18).
IRHEVFLSRSYQRLADACRGLLALLFPLRYSFTYVPILPAQLLEVLSTPTPFIIGVNAAFQAETQELLDVI
VADLDGGTVTIPECVHIPPLPEPLQSQTHSVLSMVLDPELELADLAFPPPTTSTSSLKMQDKELRAVFLRL
FAQLLQGYRWCLHVVRIEPEPVIRFHKAAFLGQRGLVEDDFLMKVLEGMAFAGFVSERGVPYRPTDLFDEL
VAHEVARMRADENHPQRVLRHVQELAEQLYKNENPYPAVAMHKVQRPGESSHLRRVPRPFPRLDEGTVQWI
VDQAAAKMQGAPPAVKAERRTTVPSGPPMTAILERCSGLHVNSARRLEVVRNCISYVFEGKMLEAKKLLPA
VLRALKGRVARRCLAQELHLHVQQNRAVLDHQQFDFVVRMMNCCLQDCTSLDEHGIAAALLPLVTAFCRKL
SPGVTQFAYSCVQEHVVWSTPQFWEAMFYGDVQTHIRALYLEPTEDLAPAQEVGEAPSQEDERSALDVASE
QRRLWPTLSREKQQELVQKEESTVFSQAIHYANRMSYLLLPLDSSKSRLLRERAGLGDLESASNSLVTNSM
AGSVAESYDTESGFEDAETCDVAGAVVRFINRFVDKVCTESGVTSDHLKGLHVMVPDIVQMHIETLEAVQR
ESRRLPPIQKPKLLRPRLLPGEECVLDGLRVYLLPDGREEGAGGSAGGPALLPAEGAVFLTTYRVIFTGMP
TDPLVGEQVVVRSFPVAALTKEKRISVQTPVDQLLQDGLQLRSCTFQLLKMAFDEEVGSDSAELFRKQLHK
LRYPPDIRATFAFTLGSAHTPGRPPRVTKDKGPSLRTLSRNLVKNAKKTIGRQHVTRKKYNPPSWEHRGQP
PPEDQEDEISVSEELEPSTLTPSSALKPSDRMTMSSLVERACCRDYQRLGLGTLSSSLSRAKSEPFRISPV
NRMYAICRSYPGLLIVPQSVQDNALQRVSRCYRQNRFPVVCWRSERSKAVLLRSGGLEGKGVVGLFKAQNA
PSPGQSQADSSSLEQEKYLQAVVSSMPRYADASGRNTLSGFSSAHMGSHVPSPRARVTTLSNPMAASASRR
TAPRGKWGSVRTSGRSSGLGTDVGSRLAGRDALAPPQANGGPPDPGFLRPQRAALYILGDKAQLKGVRSDP
LQQWELVPIEVFEARQVKASFKKLLKACVPGCPAAEPSPASFLRSLEDSEWLIQIHKLLQVSVLVVELLDS
GSSVLVGLEDGWDITTQVVSLVQLLSDPFYRTLEGFRLLVEKEWLSFGHRPSHRGAHTLAGQSSGFTPVFL
QFLDCVHQVHLQFPMEFEFSQFYLKFLGYHHVSRRPRTELLDSDYERIELGLLYEEKGERRGQVPCRSVWE
YVDRLSKRTPVFENYMYAPEDAEVLRPYSNVSNLKVWDFYTEETLAEALPMTGNWPRGPLNPQRKNGLMEA
SPEQRRVVWPCYDSCPRAQPDAISRLLEELQRLETELGQPAERWKDTWDRVKAAQRLEGRPDGRGTPSSLL
VSTAPHHRRSLGVYLQEGPVGSTLSLSLDSDQSSGSTTSGSRQAARRSTSTLYSQFQTAESENRSYEGTLY
KKGAFMKPWKARWFVLDKTKHQLRYYDHRVDTECKGVIDLAEVEAVAPGTPTMGAPKTVDEKAFFDVKTTR
RVYNFCAQDVPSAQQWVDRIQSCCRTPEPPSPARLLCSRYRPLGVAGPPRPCLQPRPSTVLSPEPPALVCT
APVPAPPRPAGPNLFWRHS
[0138] The disclosed NOV3a amino acid has 1047 of 1079 amino acid
residues (97%) identical to, and 1052 of 1079 amino acid residues
(97%) similar to, the 1697 amino acid residue
ptnr:SPTREMBL-ACC:060228 protein from Homo sapiens (Human) (Nuclear
Dual-Specificity Phosphatase) (E=0.0).
[0139] NOV3 is expressed in at least the following tissues: Adrenal
gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. This information was derived by determining the tissue
sources of the sequences that were included in the invention
including but not limited to SeqCalling sources, Public EST
sources, and/or RACE sources.
[0140] NOV3b
[0141] In the present invention, the target sequence identified
previously, NOV3a, was subjected to the exon linking process to
confirm the sequence. PCR primers were designed by starting at the
most upstream sequence available, for the forward primer, and at
the most downstream sequence available for the reverse primer. In
each case, the sequence was examined, walking inward from the
respective termini toward the coding sequence, until a suitable
sequence that is either unique or highly selective was encountered,
or, in the case of the reverse primer, until the stop codon was
reached. Such primers were designed based on in silico predictions
for the full length cDNA, part (one or more exons) of the DNA or
protein sequence of the target sequence, or by translated homology
of the predicted exons to closely related human sequences sequences
from other species. These primers were then employed in PCR
amplification based on the following pool of human cDNAs: adrenal
gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. Usually the resulting amplicons were gel purified, cloned
and sequenced to high redundancy. The resulting sequences from all
clones were assembled with themselves, with other fragments in
CuraGen Corporation's database and with public ESTs. Fragments and
ESTs were included as components for an assembly when the extent of
their identity with another component of the assembly was at least
95% over 50 bp. In addition, sequence traces were evaluated
manually and edited for corrections if appropriate. These
procedures provide the sequence reported below, which is designated
NOV3b. This differs from the previously identified sequence (NOV3a)
by coding for 50 additional bases at 5' end that includes a signal
peptide.
[0142] A disclosed NOV3b nucleic acid of 5740 nucleotides (also
referred to as Curagen Accession No. CG56019-O.sub.2) encoding a
novel Set Binding Factor 1-like protein is shown in Table 3C. An
open reading frame was identified beginning with an ATG initiation
codon at nucleotides 396-398 and ending with a TGA codon at
nucleotides 2439-2441. A putative untranslated region downstream
from the termination codon are underlined in Table 3C. The start
and stop codons are in bold letters. TABLE-US-00035 TABLE 3C NOV3b
nucleotide sequence (SEQ ID NO:19).
ACGCGTGTGGAGGATGCCACAGAGAGGGAGGAAGAGGGGGATGAGGGAGGCCAGACCCACCTGTCTCCCACA
GCACCTGCCCCATCTGCCCAGCTGTTTGCACCGAAGACGCTGGTACTGGTGTCGCGACTCGACCACACGGGG
GGGTTCAGGACCAGCCTTGGCCTCATCTATGCCATCCACGTGGAGGGCCTGAATGTGTGCCTGGAGAACGTG
ATTGGGAACCTGCTGACGTGCACTGTGCCCCTGGCTGGGGGCTCGCAGAGGACGATCTCTTTGGGGGCTGGT
GACCGGCAGGTCATCCAGACTCCACTGGCCGACTCGCTGCCCGTCAGCCGCTGCAGCGTGGCCCTGCTCTTC
CGCCAGCTAGGTGAGCCTGTCTGTCCCACCCCCGCATGGCTTCCCCTCGGGGACTGGTAGTTAGGGATGTGG
GTTCTCACTCTGCCTGCGTGGGGGCCCAGGGTCCTCTCCAAGCTTCTCTTCTTCCTTTAGGCATCACCAACG
TGCTGTCTTTGTTCTGTGCCGCCCTCACGGAGCACAAGGTTCTCTTCCTGTCCCGGAGCTACCAGCGGCTCG
CCGATGCCTGTAGGGGCCTCCTGGCACTGCTGTTTCCTCTCAGATACAGCTTCACCTATGTGCCCATCCTGC
CGGCTCAGCTGCTGGAGGTCCTCAGCACACCCACGCCCTTCATCATTGGGGTCAACGCGGCCTTCCAGGCAG
AGACCCAGGAGCTGCTCGATGTGATTGTTGCTGATCTGGATGGAGGGACGGTCACCATTCCTGAGTGTGTGC
ACATTCCACCCTTGCCAGAGCCACTGCAGAGTCAGACGCACAGTGTGCTGAGCATGGTCCTGGACCCGGAGC
TGGAGTTGGCTGACCTCGCCTTCCCTCCGCCCACGACATCCACCTCCTCCCTGAAGATGCAGGACAAGGAGC
TGCGCGCGGTCTTCCTGCGGCTGTTCGCTCAGCTGCTGCAGGGCTATCGCTGGTGCCTGCACGTCGTGCGCA
TCCACCCGGAGCCTGTCATCCGCTTCCATAAGGCAGCCTTCCTGGGCCAGCGTGGGCTGGTAGAGGACGATT
TCCTGATGAAGGTGCTGGAGGGCATGGCCTTTGCTGGCTTTGTGTCAGAGCGTGGGGTCCCATACCGCCCTA
CGGACCTGTTCGATGAGCTGGTGGCCCACGAGGTGGCAAGGATGCGGGCGGATGAGAACCACCCCCAGCGTG
TCCTGCGTCACGTCCAGGAACTGGCAGAGCAGCTCTACAAGAACGAGAACCCGTACCCAGCCGTGGCGATGC
ACAAGGTACAGAGGCCCGGTGAGAGCAGCCACCTGCGACGGGTGCCCCGACCCTTCCCCCGGCTGGATGAGG
GCACCGTGCAGTGGATCGTGGACCAGGCTGCAGCCAAGATGCAGGGTGCACCCCCAGCTGTGAAGGCCGAGA
GGAGGACCACCGTGCCCTCAGGGCCCCCCATGACTGCCATACTGGAGCGGTGCAGTGGGCTGCATGTCAACA
GCGCCCGGCGGCTGGAGGTTGTGCGCAACTGCATCTCCTACGTGTTTGAGGGGAAAATGCTTGAGGCCAAGA
AGCTGCTCCCAGCCGTGTTGAGGGCCCTGAAGGGGCGAGCTGCCCGCCGCTGCCTCGCCCAGGAGCTGCACC
TGCATGTGCAGCAGAACCGTGCGGTCCTGGACCACCAGCAGTTTGACTTTGTCGTCCGTATGATGAACTGCT
GCCTGCAGGACTGCACTTCTCTGGACGAGCATGGCATTGCGGCGGCTCTGCTGCCTCTGGTCACAGCCTTCT
GCCGGAAGCTGAGCCCGGGGGTGACGCAGTTTGCATACAGCTGTGTGCAGGAGCACGTGGTGTGGAGCACGC
CACAGTTCTGGGAGGCCATGTTCTATGGGGATGTGCAGACTCACATCCGGGCCCTCTACCTGGAGCCCACGG
AGGACCTGGCCCCCGCCCAGGAGGTTGGGGAGGCACCTTCCCAGGAGGACGAGCGCTCTGCCCTAGACGTGG
CTTCTGAGCAGCGGCGCTTGTGGCCAACTCTGAGTCGTGAGAAGCAGCAGGAGCTGGTGCAGAAGGAGGAGA
GCACGGTGTTCAGCCAGGCCATCCACTATGCCAACCGCATGAGCTACCTCCTCCTGCCCCTGGACAGCAGCA
AGAGCCGCCTACTTCGGGAGCGTGCCGGGCTGGGCGACCTGGAGAGCGCCAGCAACAGCCTGGTCACCAACA
GCATGGCTGGCAGTGTGGCCGAGAGCTATGACACGGAGAGCGGCTTCGAGGATGCAGAGACCTGCGACGTAG
CTGGGGCTGTGGTCCGCTTCATCAACCGCTTTGTGGACAAGGTCTGCACGGAGAGTGGGGTCACCAGCGACC
ACCTCAAGGGGCTGCATGTCATGGTGCCAGACATTGTCCAGATGCACATCGAGACCCTGGAGCCCGTGCAGC
GGGAGAGCCGGAGGCTGCCGCCCATCCAGAAGCCCAAGCTGCTGCGGCCGCGCCTGCTGCCGGGTGAGGAGT
GTGTGCTGGACGGCCTGCGCGTCTACCTGCTGCCGGATGGGCGTGAGGAGGGCGCGGGGGGCAGTGCTGGGG
GACCAGCATTGCTCCCAGCTGAGGGCGCCGTCTTCCTCACCACGTACCGGGTCATCTTCACGGGGATGCCCA
CGGACCCCCTGGTTGGGGAGCAGGTGGTGGTCCGCTCCTTCCCGGTGGCTGCGCTGACCAAGGAGAAGCGCA
TCAGCGTCCAGACCCCTGTGGACCAGCTCCTGCAGGACGGGCTCCAGCTGCGCTCCTGCACATTCCAGCTGC
TGAAAATGGCCTTTGACGAGGAGGTGGGGTCTGACAGCGCCGAGCTCTTCCGTAAGCAGCTGCATAAGCTGC
GGTACCCGCCGGACATCAGGGCCACCTTGCGTTCACCTTGGGCTCTGCCCACACACCTGGCCCGGCCACCGC
GAGTCACCAAGGACAAGGGTCCTTCCCTCAGAACCCTGTCCCGGAACCTGGTCAAGAACGCCAAGAAGACCA
TCGGGCGGCAGCATGTCACTCGCAAGAAGTACAACCCCCCCAGCTGGGAGCACCGGGGCCAGCCGCCCCCTG
AGGACCAGGAGGACCAGATCTCAGTGTCGGAGGAGCTGGAGCCCAGCACGCTGACCCCGTCCTCAGCCCTGA
AGCCCTCCGACCGCATGACCATGAGCAGCCTGGTGGAAAGGGCTTGCTGTCGCGACTACCAGCGCCTCGGTC
TGGGCACCCTGAGCAGCAGCCTGAGCCGGGCCAAGTCTGAGCCCTTCCGCATTTCTCCGGTCAACCGCATGT
ATGCCATCTGCCGCAGCTACCCAGGGCTGCTGATCGTGCCCCAGAGTGTCCAGGACAACGCCCTGCAGCGCG
TGTCCCGCTGCTACCGCCAGAACCGCTTCCCCGTGGTCTGCTGGCGCAGCGGGCGGTCCAAGGCGGTGCTGC
TGCGCTCTGGAGGCCTGCATGGCAAAGGTGTCGTCGGCCTCTTCAAGGCCCAGAACGCACCTTCTCCAGGCC
AGTCCCAGGCGGACTCGAGTAGCCTGGAGCAGGAGAAGTACCTGCAGGCTGTGGTCAGCTCCATGCCCCGCT
ACGCCGACGCGTCGGGACGCAACACGCTTAGCGGCTTCTCCTCAGCCCACATGGGCAGTCACGGTAAGTGGG
GCAGTGTCCGGACCAGTGGACGCAGCAGTGGCCTTGGCACCGATGTGGGCTCCCGGCTAGCTGGCAGAGACG
CGCTGGCCCCACCCCAGGCCAACGGGGGCCCTCCCGACCCGGGCTTCCTGCGTCCGCAGCGAGCAGCCCTCT
ATATCCTTGGGGACAAAGCCCAGCTCAAGGGTGTGCGGTCAGACCCCCTCCAGCAGTGGGAGCTGGTGCCCA
TTGAGGTATTCGAGGCACGGCAGGTGAAGGCTAGCTTCAAGAAGCTGCTGAAAGCATGTGTCCCAGGCTGCC
CCGCTGCTGAGCCCAGCCCAGCCTCCTTCCTGCGCTCACTGGAGGACTCAGAGTGGCTGATCCAGATCCACA
AGCTGCTGCAGGTGTCTGTGCTGGTGGTGGAGCTCCTGGATTCAGGCTCCTCCGTGCTGGTGGGCCTGGAGG
ATGGCTGGGACATCACCACCCAGGTGGTATCCTTGGTGCAGCTGCTCTCAGACCCCTTCTACCGCACGCTGG
AGGGCTTTCGCCTGCTGGTGGAGAAGGAGTGGCTGTCCTTCGGCCATCGCTTCAGCCACCGTGGAGCTCACA
CCCTGGCCGGGCAGAGCAGCGGCTTCACACCCGTCTTCCTGCAGTTCCTGGACTGCGTACACCAGGTCCACC
TGCAGTTCCCCATGGAGTTTGAGTTCAGCCAGTTCTACCTCAAGTTCCTCGGCTACCACCATGTGTCCCGCC
GTTTCCGGACCTTCCTGCTCGACTCTGACTATGAGCGCATTGAGCTGGGGCTGCTGTATGAGGAGAAGGGGG
AACGCAGGGGCCAGGTGCCGTGCAGGTCTGTGTGGGAGTATGTGGACCGGCTGAGCAAGAGGACGCCTGTGT
TCCACAATTACATGTATGCGCCCGAGGACGCAGAGGTCCTGCGGCCCTACAGCAACGTGTCCAACCTGAAGG
TGTGGGACTTCTACACTGAGGAGACGCTGGCCGAGGGCCCTCCCTATGACTGGGAACTGGCCCAGGGGCCCC
CTGAACCCCCAGAGGAAGAACGGTCTGATGGAGGCGCTCCCCAGAGCAGGCGCCGCGTGGTGTGGCCCTGTT
ACGACAGCTGCCCGCGGGCCCAGCCTGACGCCATCTCACGCCTGCTGGAGGAGCTGCAGAGGCTGGAGACAG
AGTTGGGCCAACCCGCTGAGCGCTGGAAGGACACCTGGGACCGGGTGAAGGCTGCACAGCGCCTCGAGGGCC
GGCCAGACGGCCGTGGCACCCCTAGCTCCCTCCTTGTGTCCACCGCACCCCACCACCGTCGCTCGCTGGGTG
TGTACCTGCAGGAGGGGCCCGTGGGCTCCACCCTGAGCCTCAGCCTGGACAGCGACCAGAGTAGTGGCTCAA
CCACATCCGGCTCCCGTCAGGCTGCCCGCCGCAGCACCAGCACCCTGTACAGCCAGTTCCAGACAGCAGAGA
GTGAGAACAGGTCCTACGAGGGCACTCTGTACAAGAAGGGGGCCTTCATGAAGCCTTGGAAGGCCCGCTGGT
TCGTGCTGGACAAGACCAAGCACCAGCTGCGCTACTACGACCACCGTGTGGACACAGAGTGCAAGGGTGTCA
TCGACTTGGCGGAGGTGGAGGCTGTGGCACCTGGCACGCCCACTATGGGTGCCCCTAAGACCGTGGACGAGA
AGGCCTTCTTTGACGTGAAGACAACGCGTCGCGTTTACAACTTCTGTGCCCAGGACGTGCCCTCGGCCCAGC
AGTGGGTGGACCGGATCCAGAGCTGCCTGTCGGACGCCTGAGCCTCCCAGCCCTGCCCGGCTGCTCTGCTTC
CGGTCGTTACCGACCACTAGGGGTGGTGTTGGGACACCTGGGCGAGATGTGAGGGTGGGCTCACTTGAGCCA
CTGAAACCAGCCAGGTCTTCCCTCAGGCCGGACAGATGGCGCCTGACCAAAGTTCCTGGCACCTGGAAAACC
CACAGCAGGGCACGAGTGACCTGAGAGGCCCACTCAGGCAGAAGAGACGCAAGCTGGGCCGTCCAACTGGTT
TCAACTGCCAGCTTTACCAATGCAGCATTTATTTTAAAATTAAATTAAATTA
[0143] In a search of public sequence databases, the NOV3b nucleic
acid sequence, located on chromosome 11, has 4947 of 4963 bases
(99%) identical to a gb:GENBANK-ID:HSU93181|acc:U93181.1 mRNA from
Homo sapiens (Homo sapiens nuclear dual-specificity phosphatase
(SBF1) mRNA, partial cds) (E=0.0). Public nucleotide databases
include all GenBank databases and the GeneSeq patent database.
[0144] The disclosed NOV3b polypeptide (SEQ ID NO:20) encoded by
SEQ ID NO:19 has 1681 amino acid residues and is presented in Table
3B using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV3b has a signal peptide and is
likely to be localized to the Golgi body with a certainty of
0.9000. In other embodiments, NOV3b may also be localized to the
plasma membrane with a certainty of 0.7900, in the microbody
(peroxisome) with a certainty of 0.3525, or in the endoplasmic
reticulum (membrane) with a certainty of 0.2000. The most likely
cleavage site for NOV3b is between positions 46 and 47, ALT-EH.
TABLE-US-00036 TABLE 3D Encoded NOV3b protein sequence (SEQ ID
NO:20).
MASPRGLVVRDVGSHSAWVGAQGPLQASLLPLGITNVLSLFCAALTEHKVLFLSRSYQRLADACRGLLALLF
PLRYSFTYVPILPAQLLEVLSTPTPFIIGVNAAFQAETQELLDVIVADLDGGTVTIPECVHIPPLPEPLQSQ
THSVLSMVLDPELELADLAFPPPPTSTSSLKMQDKELRAVELRLFAQLLQGYRWCLEVVRIHPEPVIRFHKA
APLGQRGLVEDDFLMKVLEGMAFAGFVSERGVPYRPTDLFDELVAEEVARMRADENHPQRVLRHVQELAEQL
YKNENPYPAVAMHKVQRPGESSHLRRVPRPFPRLDEGTVQWIVDQAAAKMQGAPPAVKAERRTTVPSGPPMT
AILERCSGLHVNSARRLEVVRNCISYVFEGKMLEAKKLLPAVLRALKGRAARRCLAQELHLHVQQNRAVLDH
QQFDPVVRMMNCCLQDCTSLDEHGIAAALLPLVTAFCRKLSPGVTQFAYSCVQEHVVWSTPQFWEAMFYGDV
QTHIRALYLEPTEDLAPAQEVGEAPSQEDERSALDVASEQRRLWPTLSREKQQELVQKEESTVFSQAIHYAN
RMSYLLLPLDSSKSRLLRERAGLGDLESASNSLVTNSMAGSVAESYDTESGFEDAETCDVAGAVVRFINRFV
DKVCTESGVTSDHLKGLHVMVPDIVQMHIETLEAVQRESRRLPPIQKPKLLRPRLLPGEECVLDGLRVYLLP
DGREEGAGGSAGGPALLPAEGAVFLTTYRVIFTGMPTDPLVGEQVVVRSFPVAALTKEKRISVQTPVDQLLQ
DGLQLRSCTFQLLKMAFDEEVGSDSAELFRKQLEKLRYPPDIRATFAFTLGSAHTPGRPPRVTKDKGPSLRT
LSRNLVKNAKKTIGRQHVTRKKYNPPSWEHRGQPPPEDQEDEISVSEELEPSTLTPSSALKPSDRMTMSSLV
ERACCRDYQRLGLGTLSSSLSRAKSEPFRISPVNRMYAICRSYPGLLIVPQSVQDNALQRVSRCYRQNRFPV
VCWRSGRSKAVLLRSGGLHGKGVVGLFKAQNAPSPGQSQADSSSLEQEKYLQAVVSSMPRYADASGRNTLSG
FSSAHMGSHGKWGSVRTSGRSSGLGTDVGSRLAGRDALAPPQANGGPPDPGFLRPQRAALYILGDKAQLKGV
RSDPLQQWELVPIEVFEARQVKASFKKLLKACVPGCPAAEPSPASFLRSLEDSEWLIQIHKLLQVSVLVVEL
LDSGSSVLVGLEDGWDITTQVVSLVQLLSDPFYRTLEGFRLLVEKEWLSFGHRFSHRGAHTLAGQSSGFTPV
FLQFLDCVHQVHLQFPNEFEFSQFYLKFLGYEHVSRRFRTFLLDSDYERIELGLLYEEKGERRGQVPCRSVW
EYVDRLSKRTPVFHNYMYAPEDAEVLRPYSNVSNLKVWDFYTEETLAEGPPYDWELAQGPPEPPEEERSDGG
APQSRRRVVWPCYDSCPRAQPDAISRLLEELQRLETELGQPAERWKDTWDRVKAAQRLEGRPDGRGTPSSLL
VSTAPHHRRSLGVYLQEGPVGSTLSLSLDSDQSSGSTTSGSRQAARRSTSTLYSQFQTAESENRSYEGTLYK
KGAFMKPWKARWFVLDKTKHQLRYYDHRVDTECKGVIDLAEVEAVAPGTPTMGAPKTVDEKAFFDVKTTRRV
YNFCAQDVPSAQQWVDRIQSCLSDA
[0145] A search of sequence databases reveals that the NOV3b amino
acid sequence has 1631 of 1631 amino acid residues (100%) identical
to, and 1631 of 1631 amino acid residues (100%) similar to, the
1631 amino acid residue ptnr:SPTREMBL-ACC:Q9UGB8 protein from Homo
sapiens (Human) (DJ579N16.2 (Set Binding Factor 1)) (E=0.0). Public
amino acid databases include the GenBank databases, SwissProt, PDB
and PIR.
[0146] NOV3b is expressed in at least the following tissues:
Adrenal gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. Expression information was derived from the tissue sources
of the sequences that were included in the derivation of the
sequence of CuraGen Acc. No. CG56019-02. The sequence is predicted
to be expressed with a similar pattern to (GENBANK-ID:
gb:GENBANK-ID:HSU93181|acc:U93181.1) a closely related Homo sapiens
nuclear dual-specificity phosphatase (SBF1) mRNA, partial cds
homolog.
[0147] NOV3a also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 3E. TABLE-US-00037 TABLE 3E BLAST
results for NOV3a Gene Index/ Protein/ Length Identity Positives
Identifier Organism (aa) (%) (%) Expect gi|3015538|gb|AAC39675.1|
nuclear 1697 1578/1723 1578/1723 0.0 (U93181) dual- (91%) (91%)
specificity phosphatase [Homo sapiens] gi|6572379|emb|CAB63063.1|
dJ579N16.2 1631 1495/1653 1501/1653 0.0 (AL096767) (SET binding
(90%) (90%) factor 1) [Homo sapiens] gi|17485528|ref|XP.sub.-- SET
binding 1327 1015/1066 1016/1066 0.0 037447.2| factor 1 (95%) (95%)
(XM_037447) [Homo sapiens] gi|12698077|dbj|BAB21857.1| KIAA1766
1123 544/934 683/934 0.0 (AB051553) protein (58%) (72%) [Homo
sapiens] gi|15292603|gb|AAK93570.1| SD10541p 1728 596/1574 859/1574
0.0 (AY052146) [Drosophila (37%) (53%) melanogaster]
[0148] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 3F.
[0149] Tables 3G-3I list the domain descriptions from DOMAIN
analysis results against NOV3a. This indicates that the NOV3a
sequence has properties similar to those of other proteins known to
contain this domain. TABLE-US-00038 TABLE 3G Domain Analysis of
NOV3a gnl|Pfam|pfam02141, DENN, DENN (AEX-3) domain. (SEQ ID NO:73)
CD-Length = 146 residues, 51.4% aligned Score = 69.3 bits (168),
Expect = 2e-12 Query: 5
VFLSRSYQRLADACRGLLALLFPLRYSFTYVPILPAQLLEVLSTPTPFIIGVNAAFQAET 64 +|
|| |+ | ++|||+| + |+|+||| | +|| |||++||| ++| Sbjct: 70
LFHSRKLSTLSSCCEAVVALLYPFEWQCPYIPLLPASLADVLLAPTPTYLIVPSSFFDNK 129
Query: 65 QELL---DVIVADLD 76 | ||| ||| Sbjct: 130 LLELPPSDVICVDLD
144
[0150] TABLE-US-00039 TABLE 3H Domain Analysis of NOV3a
gnl|Smart|smart00233, PH, Pleckstrin homology domain.; Domain
commonly found in eukaryotic signalling proteins. The domain family
possesses multiple functions including the abilities to bind
inositol phosphates, and various proteins. PH domains have been
found to possess inserted domains (such as in PLC gamma,
syntrophins) and to be inserted within other domains. Mutations in
Brutons tyrosine kinase (Btk) within its PH domain cause X-linked
agammaglobulinaemia (XLA) in patients. Point mutations cluster into
the positively charged end of the molecule around the predicted
binding site for phosphatidylinositol lipids. (SEQ ID NO:74)
CD-Length = 104 residues, 96.2% aligned Score = 63.5 bits (153),
Expect = 9e-11 Query: 1557
YEGTLYKKGA-FMKPWKARWFVLDKTKHQLRYYDHR---VDTECKGVIDLAEVEAVAPGT 1612
|| | || + | || |+||| | || + ++ || | |+ Sbjct: 3
KEGWLLKKSSGGKKSWKKRYFVL--FNGVLLYYKSKKKKSSSKPKGSIPLSGCTVREAPD 60
Query: 1613 PTMGAPKTVDEKAFFDVKT-TRRVYNFCAQDVPSAQQWVDRIQSCCR 1658 +
+| |++ | |+ |+ ++||+ ++ Sbjct: 61
S-----DSDKKKNCFEIVTPDRKTLLLQAESEEERKEWVEALRKAIA 102
[0151] TABLE-US-00040 TABLE 3I Domain Analysis of NOV3a
gnl|Pfam|pfam00169, PH, PH domain. PH stands for pleckatrin
homology. (SEQ ID NO:75) CD-Length = 100 residues, 97.0% aligned
Score = 53.1 bits (126), Expect = 1e-07 Query: 1558
EGTLYKKGAFMKP-WKARWFVLDKTKHQLRYYDHRV-DTECKGVIDLAEVEAVAPGTPTM 1615
|| | || | || |+| | | || + | || | |+ Sbjct: 4
EGWLLKKSTVKKKRWKKRYFFL--FNDVLIYYKDKKKSYEPKGSIPLSGCSVEDVPDSEF 61
Query: 1616 GAPKTVDEKAFFDVKTTRR--VYNFCAQDVPSAQQWVDRIQSCCR 1658 | |
+++ + |+ | |+ ||| | Sbjct: 62
KRPNC------FQLRSRDGKETFILQAESEEERQDWIKAIQSAIR 100
[0152] Mammalian SET domain-containing proteins define a
distinctive class of chromatin-associated factors that are targets
for growth control signals and oncogenic activation. By yeast
two-hybrid screening, Cui X et. al. (1998, Nat. Genet. Vol. 18:
331-337) identified Sbf1 (also known as nuclear dual-specificity
phosphatase) as a protein interacting with the SET domain in the
protooncoprotein homolog of Drosophila trithorax, Hrx. Sbf1, shares
extensive sequence similarity with myotubularin, a dual specificity
phosphatase (dsPTPase) that is mutated in a subset of patients with
inherited myopathies. Both Sbf1 and myotubularin interact with the
SET domains of Hrx and other epigenetic regulatory proteins, but
Sbf1 lacks phosphatase activity due to several evolutionarily
conserved amino acid changes in its structurally preserved
catalytic pocket. Sbf1 has shown to prevent myoblast
differentiation in vitro and induce oncogenic changes in NIH 3T3
fibroblasts. Furthermore, it also functions as SET domain-dependent
positive regulator of growth-inducing kinase signaling pathways
(Immaculata De Vivo et al., 1998, Proc. Natl. Acad. Sci. USA, vol
95: 9471-9476).
[0153] The disclosed NOV3 nucleic acid of the invention encoding a
Set Binding Factor (SBF1)-like protein includes the nucleic acid
whose sequence is provided in Table 3A, 3C, or a fragment thereof.
The invention also includes a mutant or variant nucleic acid any of
whose bases may be changed from the corresponding base shown in
Table 3A, or 3C while still encoding a protein that maintains its
Set Binding Factor (SBF1)-like activities and physiological
functions, or a fragment of such a nucleic acid. The invention
further includes nucleic acids whose sequences are complementary to
those just described, including nucleic acid fragments that are
complementary to any of the nucleic acids just described. The
invention additionally includes nucleic acids or nucleic acid
fragments, or complements thereto, whose structures include
chemical modifications. Such modifications include, by way of
nonlimiting example, modified bases, and nucleic acids whose sugar
phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 9 percent of the
bases may be so changed.
[0154] The disclosed NOV3 protein of the invention includes the Set
Binding Factor (SBF1)-like protein whose sequence is provided in
Table 3B, or 3D. The invention also includes a mutant or variant
protein any of whose residues may be changed from the corresponding
residue shown in Table 3B, or 3D while still encoding a protein
that maintains its Set Binding Factor (SBF1)-like activities and
physiological functions, or a functional fragment thereof. In the
mutant or variant-protein, up to about 63 percent of the residues
may be so changed.
[0155] The protein similarity information, expression pattern, and
map location for the Set Binding Factor (SBF1)-like protein and
nucleic acid (NOV3) disclosed herein suggest that NOV3 may have
important structural and/or physiological functions characteristic
of the citron kinase-like family. Therefore, the NOV3 nucleic acids
and proteins of the invention are useful in potential diagnostic
and therapeutic applications. 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, as well as 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), and (v) a composition promoting tissue regeneration in
vitro and in vivo.
[0156] The NOV3 nucleic acids and proteins of the invention are
useful in potential diagnostic and therapeutic applications
implicated in various diseases and disorders described below. For
example, the compositions of the present invention will have
efficacy for treatment of patients suffering from Von Hippel-Lindau
(VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis,
hypercalceimia, Parkinson's disease, Huntington's disease, cerebral
palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders,
addiction, anxiety, pain, neurodegeneration; Cholesteryl ester
storage disease; Corneal dystrophy, Thiel-Behnke type;
Dubin-Johnson syndrome; Leukemia, T-cell acute lymphocytic; Retinol
binding protein, deficiency of; SEMD, Pakistani type;
Spinocerebellar ataxia, infantile-onset, with sensory neuropathy;
Split hand/foot malformation, type 3; Tolbutamide poor metabolizer;
Urofacial syndrome; Warfarin sensitivity; Wolman disease, and/or
other pathologies.
[0157] NOV3 nucleic acids and polypeptides 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. These antibodies may be generated according to
methods known in the art, using prediction from hydrophobicity
charts, as described in the "Anti-NOVX Antibodies" section below.
For example the disclosed NOV3 protein have multiple hydrophilic
regions, each of which can be used as an immunogen. This novel
protein also has value in development of powerful assay system for
functional analysis of various human disorders, which will help in
understanding of pathology of the disease and development of new
drug targets for various disorders.
[0158] NOV4
[0159] A disclosed NOV4 nucleic acid of 762 nucleotides (designated
CuraGen Acc. No. CG55692-01) encoding a novel TSPAN-1-like protein
is shown in Table 4A. An open reading frame was identified
beginning with an ATG initiation codon at nucleotides 9-11 and
ending with a TAA codon at nucleotides 732-734. A putative
untranslated region downstream from the termination codon is
underlined in Table 4A, and the start and stop codons are in bold
letters. TABLE-US-00041 TABLE 4A NOV4 Nucleotide Sequence (SEQ ID
NO:21)
GACACACCATGCAGTGCTTCAAATTCATTAAGGTCATGATGTTCCTCTTCAATCAACTCATCTTTCTCTG
TGGTGCAGCCCTGTTGGCTGTGGGAATATGGGTAACCGTCGATGGGACATCTTTCCTGAAGGTCTTCGGA
TCACTATCATCCAGTGCCATGCAGTTTGTCAACGTGGGCTACTTCCTCATCGCCGCTGGTGCTGTGCTCT
TCATTTTTGGTTTCCTGGGCTGCTATGGTGCTCCCTCTGAGAAACAAGTGTGTGCTCTGGTGATGTTCTT
TTCCATCCTCCTCATCATCTTCATCGCTGAGATTGCAGGTGCTGTGGTTGCTTTGGTGTACACCACATTG
GCTGAACAATTCCTGACACTCCTGGTGGTGCCTGCTATCGAAAAAGACTATGGTTACCAGACTGATTTCA
CCCAAGTATGGAACACTACAATGGAAGAGTTGCATTGCTGTGGCTTTAACAACTACACAGATTTTAATGC
CTCACGTTTCGTCAAAGAGAATAAAGTCTTCCCCCCACCCTGTTGTGCCAACCCTGGCAACCATACAGTT
GAACCATGCACCGAGGAGAAGGCCAAAAGTATGAAAGTACAGCGTTGTTTCAAAGAGATTCTGCATAGAA
TCAGAAACAATGCAGTCACTGTGGGTGGTGTGGCAGTTGGAGTTGCGGCCCTAGAGCTGGCTGCCATGGT
TGTATCCATGTATCTATACTGCAATCTGAAATAAGACTACTACTTCCTCCTGACTTGCTGCC
[0160] The nucleic acid sequence, localized to chromosome 12, has
616 of 765 bases (80%) identical to a
gb:GENBANK-ID:AF065388|acc:AF065388.1 mRNA from Homo sapiens (Homo
sapiens tetraspan NET-1 mRNA, complete cds) (E=1.8e.sup.-100).
[0161] A NOV4 polypeptide (SEQ ID NO:22) encoded by SEQ ID NO:21 is
241 amino acid residues and is presented using the one letter code
in Table 4B. Signal P, Psort and/or Hydropathy results predict that
NOV4 has no signal peptide and is likely to be localized at the
plasma membrane with a certainty of 0.6400. In other embodiments,
NOV4 may also be localized to the Golgi body with a certainty of
0.4600, the endoplasmic reticulum (membrane) with a certainty of
0.3700, or the endoplasmic reticulum (lumen) with a certainty of
0.1000. The most likely cleavage site for NOV4 is between positions
36 and 37: VDG-TS. TABLE-US-00042 TABLE 4B NOV4 protein sequence
(SEQ ID NO:22)
MQCFKFIKVMMFLFNQLIFLCGAALLAVGIWVTVDGTSFLKVFGSLSSSAMQFVNVGYFLIAAGAVLEIFGFLG
CYGAPSEKQVCALVMFFSILLIIFIAEIAGAVVALVYTTLAEQFLTLLVVPAIEKDYGYQTDFTQVWNTTMEEL
HCCGFNNYTDFNASRFVKENKVFPPPCCANPGNHTVEPCTEEKAKSMKVQGCFKEILHRIRNNAVTVGGVAVGV
AALELAAMVVSMYLYCNLK
[0162] The full amino acid sequence of the protein of the invention
was found to have 177 of 241 amino acid residues (73%) identical
to, and 197 of 241 amino acid residues (81%) similar to, the 241
amino acid residue ptnr:SPTREMBL-ACC:060635 protein from Homo
sapiens (Human) (TSPAN-1) (E=6.1e.sup.-92).
[0163] NOV4 is expressed in at least Colon, Testis, prostate,
melanocyte, heart, uterus, kidney, stomach because of the
expression pattern of (GENBANK-ID:AF065388|acc:AF065388.1) a
closely related Homo sapiens tetraspan NET-1 mRNA, complete cds
homolog in species Homo sapiens:
[0164] NOV4 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 4C. TABLE-US-00043 TABLE 4C BLAST
results for NOV4 Gene Index/ Length Identity Positives Identifier
Protein/Organism (aa) (%) (%) Expect gi|13097420|gb| Similar to 240
210/241 213/241 e-104 AAH03448.1|AAH03448 tetraspan 1 [Mus (87%)
(88%) (BC003448) musculus] gi|5032197|ref| tetraspan 1 [Homo 241
161/242 179/242 3e-67 NP_005718.1| sapiens] (66%) (73%) (NM_005727)
gi|12643622|sp| TETRASPANIN 1 241 160/242 179/242 7e-67
O60635|TSN1_HUMAN (TSPAN-1) (66%) (73%) (TETRASPAN NET-1)
(TETRASPANIN TM4- C) gi|6601561|gb| neuronal 247 76/217 111/217
2e-17 AAF19031.1|AF206661_1 tetraspanin (35%) (51%) (AF206661)
[Gallus gellus] gi|17570135|ref| tetraspanin/ 282 52/165 83/165
1e-16 NP_510445.1| [Caenorhabditis (31%) (49%) (NM_078044)
elegans]
[0165] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 4D.
[0166] Table 4E lists the domain description from DOMAIN analysis
results against NOV4. This indicates that the NOV4 sequence has
properties similar to those of other proteins known to contain this
domain. TABLE-US-00044 TABLE 4E Domain Analysis of NOV4
gnl|pfam|pfam00335, transmembrane4, Tetraspanin family. (SEQ ID
NO:81) CD-Length = 222 residues, 100.0% aligned Score = 126 bits
(316), Expect = 2e-30 Query: 8
KVMMFLFNQLIFLCGAALLAVGIWVTVDGTSFLKVFGSLSSSAMQFVNVGYFLIAAGAVL 67 |
++|| | | +||| |||||||+ || +|| ++ ||||| | ||| ||+| Sbjct: 1
KYLLFLLNLLFWLCGILLLAVGIWLLVDLSSFSELLGSLSSLV-----AAYVLIAVGAIL 55
Query: 68
FIFGFLGCYGAPSEKQVCALVMFFSILLIIFIAEIAGAVVALVYTTLAEQFLTLLVVPAI 127 |+
||||| || | + | | ++| ||+||| |+| ++| |+ | | + || Sbjct: 56
FLVGFLGCCGAIRESR-CLLGLYFVFLLLIFILEVAAGILAFVFRDKLESSLNESLKNAI 114
Query: 128
EKDYGYQTDFTQVWNTTMEELHCCGFNNYTDFNASRFVKENKVFPPPCCANPGNHTVEPC 187 +
| | |+ |+ ||| | |||+ |++ + | || + Sbjct: 115
KNYYDTDPDERNAWDKLQEQFKCCGVNGYTDWFDSQW--FSNGVPFSCCNPSVS---CNS 169
Query: 188 TEEKAKSMKVQGCFKEILERIRNNAVTVGGVAVGVAALELAAMVVSMYLYCNL
240 +++ ++ +|| +++| + | + |||||+|+| ++| |++| | |++ Sbjct: 170
AQDEEDTIYQEGCLEKLLEWLEENLLIVGGVALGIALIQLLGMILSCCLCCSI 222
[0167] Members of the `transmembrane 4 superfamily` (TM4SF) are
cell-surface proteins presumed to have 4 transmembrane domains.
TM4SF proteins form complexes with integrins and other cell-surface
proteins. A number of eukaryotic cell surface antigens have been
shown to be related, including mammalian leukocyte antigen CD37,
mammalian lysosomal membrane protein CD63, human tumour-associated
antigen CD-029, and several others. These proteins are all type II
membrane proteins: they contain an N-terminal transmembrane (TM)
domain, which acts both as a signal sequence and a membrane anchor,
and 3 additional TM regions (hence the name `TM4`). The sequences
contain a number of conserved cysteine residues.
[0168] The disclosed NOV4 nucleic acid of the invention encoding a
TSPAN-1-like protein includes the nucleic acid whose sequence is
provided in Table 4A or a fragment thereof. The invention also
includes a mutant or variant nucleic acid any of whose bases may be
changed from the corresponding base shown in Table 4A while still
encoding a protein that maintains its TSPAN-1-like activities and
physiological functions, or a fragment of such a nucleic acid. The
invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, by way
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 20 percent of the
bases may be so changed.
[0169] The disclosed NOV4 protein of the invention includes the
TSPAN-1-like protein whose sequence is provided in Table 4B. The
invention also includes a mutant or variant protein any of whose
residues may be changed from the corresponding residue shown in
Table 4B while still encoding a protein that maintains its
TSPAN-1-like activities and physiological functions, or a
functional fragment thereof. In the mutant or variant protein, up
to about 69 percent of the residues may be so changed.
[0170] The protein similarity information, expression pattern, and
map location for the TSPAN-1-like protein and nucleic acid (NOV4)
disclosed herein suggest that this NOV4 protein may have important
structural and/or physiological functions characteristic of the
TSPAN-1 family. Therefore, the NOV4 nucleic acids and proteins of
the invention are useful in potential diagnostic and therapeutic
applications. 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, as well as 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), and (v) a
composition promoting tissue regeneration in vitro and in vivo.
[0171] The NOV4 nucleic acids and proteins of the invention are
useful in potential diagnostic and therapeutic applications
implicated in various diseases and disorders described below. For
example, the compositions of the present invention will have
efficacy for treatment of patients suffering from Hypercalceimia,
Ulcers, Inflammatory bowel disease, Diverticular disease,
Hirschsprung's disease, Crohn's Disease, Appendicitis, Fertility,
Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial
nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic
lupus erythematosus, Renal tubular acidosis, IgA nephropathy,
Hypercalceimia, Lesch-Nyhan syndrome, Cardiomyopathy,
Atherosclerosis, Hypertension, Congenital heart defects, Aortic
stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal
defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis,
Ventricular septal defect (VSD), valve diseases, Tuberous
sclerosis, Scleroderma, Obesity, Transplantation, and/or other
pathologies. The NOV4 nucleic acids, 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.
[0172] NOV4 nucleic acids and polypeptides 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. These antibodies may be generated according to
methods known in the art, using prediction from hydrophobicity
charts, as described in the "Anti-NOVX Antibodies" section below.
These novel proteins can be used in assay systems for functional
analysis of various human disorders, which will help in
understanding of pathology of the disease and development of new
drug targets for various disorders.
[0173] NOV5
[0174] A disclosed NOV5 nucleic acid of 469 nucleotides (also
referred to as CG56073-01) encoding a novel Fatty Acid-Binding
Protein, Epidermal-like protein is shown in Table 5A. An open
reading frame was identified beginning with an ATG initiation codon
at nucleotides 148-150 and ending with a TGA codon at nucleotides
395-397. Putative untranslated regions upstream from the initiation
codon and downstream from the termination codon are underlined in
Table 5A, and the start and stop codons are in bold letters.
TABLE-US-00045 TABLE 5A NOV5 Nucleotide Sequence (SEQ ID NO:23)
GCCATCTGCCAACCATGGTCACCACTCAGCAGCTGCTAGGAAGATGGCGCCCAGCACAGAGG
AAATACCTCAAAGAAACAGGGATGAGAATGGCCCTGCAAAAAATTGGTGCAATGACTAAACC
AGATGGTGCCATCTCTGATGGCAAAAGCTTCACTATAAAAACCAAGAGCACTCTGAAAACAA
CACGGTTTTCTTCTAAACTTGGAGAGAAGTATGAAAGAACTACAGGTGATGGCAGAAAAAAC
TCACTATTTGTCTGCAACTTTACAAAGCCTGCATTGGTTCAACACTGGGAATGGGATGAGGA
AAGAAAAACGAGAAGAAGAAAAGTGGGAGACAAAAAAGCAGGGATGGAATGCATTATGAACA
ATGTCACCTGTACTCAGATCTGTGAAAATAAAAAAAGCAGAATAAAAATTTCCTTACTGCTT
TGGAGAGCAATTAGCTGAGAGAAGGAACAATTTCA
[0175] The NOV5 nucleic acid was identified on chromosome 2 and has
313 of 411 bases (76%) identical to a
gb:GENBANK-ID:AF059507|acc:AF059507.1 mRNA from Bos taurus (Bos
taurus epidermal fatty acid-binding protein (E-FABP) mRNA, complete
cds) (E=4.5e.sup.-41)
[0176] A disclosed NOV5 polypeptide (SEQ ID NO:24) encoded by SEQ
ID NO:23 is 145 amino acid residues and is presented using the
one-letter code in Table 5B. Signal P, Psort and/or Hydropathy
results predict that NOV5 has no signal peptide and is likely to be
localized in the nucleus with a certainty of 0.9775. In other
embodiments, NOV5 may also be localized to the microbody
(peroxisome) with acertainty of 0.3925, the mitochondrial matrix
space with a certainty of 0.3600, or the lysosome (lumen) with a
certainty of 0.1000. TABLE-US-00046 TABLE 5B Encoded NOV5 protein
sequence (SEQ ID NO:24)
MVTTQQLLGRWRPAERKYLKETGMRMALQKIGAMTKPDGAISDGKSFTIKTKSTLKTTRFSSKLGEKYERTT
GDGRKNSLFVCNFTKRALVQHWEWDEERKTRRRKVGDKKAGMECIMNNVTCTQICENKKSRIKISLLLWRAI
S
[0177] The disclosed NOV5 amino acid sequence has 80 of 132 amino
acid residues (60%) identical to, and 96 of 132 amino acid residues
(72%) similar to, the 135 amino acid residue
ptnr:SWISSPROT-ACC:P55052 protein from Bos taurus (Bovine) (Fatty
Acid-Binding Protein, Epidermal (E-FABP)
(Differentiation-Associated Lipid Binding Protein LP2))
(E=0.0).
[0178] NOV5 is expressed in at least retina. This information was
derived by determining the tissue sources of the sequences that
were included in the invention including but not limited to
SeqCalling sources, Public EST sources, Literature sources, and/or
RACE sources.
[0179] NOV5 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 5C. TABLE-US-00047 TABLE 5C BLAST
results for NOV5 Gene Index/ Length Identity Positives Identifier
Protein/Organism (aa) (%) (%) Expect gi|6648071|sp|P55052| FATTY
ACID- 135 80/133 96/133 4e-32 FABE_BOVIN BINDING PROTEIN, (60%)
(72%) EPIDERMAL (E- FABP) gi|1293786|gb|AAB41297.1| LP2 [Bos
taurus] 135 80/133 96/133 7e-32 (U55188) (60%) (72%)
gi|4557581|ref|NP_001435.1| fatty acid 135 79/133 95/133 7e-31
(NM_001444) binding protein 5 (59%) (71%) (psoriasis- associated);
E- FABP [Homo sapiens] gi|13651882|ref|XP.sub.-- similar to fatty
135 73/133 93/133 2e-28 011655.5| acid binding (54%) (69%)
(XM_011655) protein 5 (psoriasis- associated); E- FABP (H. sapiens)
[Homo sapiens] gi|1836058|gb|AAB46848.1| DA11 [Rattus sp.] 135
65/133 88/133 2e-25 (S83247) (48%) (65%)
The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 5D.
[0180] The fatty acid-binding protein (FABP) family consists of
small, cytosolic proteins believed to be involved in the uptake,
transport, and solubilization of their hydrophobic ligands. Members
of this family have highly conserved sequences and tertiary
structures. Using an antibody against testis lipid-binding protein,
a member of the FABP family, a protein was identified from bovine
retina and testis that coeluted with exogenously added
docosahexaenoic acid during purification. Amino acid sequencing and
subsequent isolation of its cDNA revealed it to be nearly identical
to a bovine protein expressed in the differentiating lens and to be
the likely bovine homologue of the human epidermal fatty
acid-binding protein (E-FABP). From quantitative Western blot
analysis, it was estimated that bovine E-FABP comprised 0.9%, 0.1%,
and 2.4% of retina, testis, and lens cytosolic proteins,
respectively. Binding studies using the fluorescent probe ADIFAB
indicated that this protein bound fatty acids of differing levels
of saturation with relatively high affinities. Kd values ranged
from 27 to 97 nM. In addition, the protein was immunolocalized to
the Muller cells in the retina as well as to Sertoli cells in the
testis. The location of bovine E-FABP in cells known to be
supportive to other cell types in their tissues and the ability of
E-FABP to bind a variety of fatty acids with similar affinities
indicate that it may be involved in the uptake and transport of
fatty acids essential for the nourishment of the surrounding cell
types.
[0181] The disclosed NOV5 nucleic acid of the invention encoding a
Fatty Acid-Binding Protein, Epidermal-like protein includes the
nucleic acid whose sequence is provided in Table 5A or a fragment
thereof. The invention also includes a mutant or variant nucleic
acid any of whose bases may be changed from the corresponding base
shown in Table 5A while still encoding a protein that maintains its
Fatty Acid-Binding Protein, Epidermal-like activities and
physiological functions, or a fragment of such a nucleic acid. The
invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, by way
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 24 percent of the
bases may be so changed.
[0182] The disclosed NOV5 protein of the invention includes the
Fatty Acid-Binding Protein, Epidermal-like protein whose sequence
is provided in Table 5B. The invention also includes a mutant or
variant protein any of whose residues may be changed from the
corresponding residue shown in Table 5B while still encoding a
protein that maintains its Fatty Acid-Binding Protein,
Epidermal-like activities and physiological functions, or a
functional fragment thereof. In the mutant or variant protein, up
to about 52 percent of the residues may be so changed.
[0183] The NOV5 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in
psoriasis, basal and squamous cell carcinomas, obesity, diabetis,
and/or other pathologies and disorders involving fatty acid
transport of skin, oral mucosa, and/or other diseases, disorders
and conditions of the like. The NOV5 nucleic acid, 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.
[0184] NOV5 nucleic acids and polypeptides 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. These antibodies may be generated according to
methods known in the art, using prediction from hydrophobicity
charts, as described in the "Anti-NOVX Antibodies" section below.
For example the disclosed NOV5 protein have multiple hydrophilic
regions, each of which can be used as an immunogen. This novel
protein also has value in development of powerful assay system for
functional analysis of various human disorders, which will help in
understanding of pathology of the disease and development of new
drug targets for various disorders.
[0185] NOV6
[0186] A disclosed NOV6 nucleic acid of 816 nucleotides (also
referred to as CG50261-O.sub.2) encoding a novel Uncoupling Protein
I-like protein is shown in Table 6A. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
1-3 and ending with a TAA codon at nucleotides 814-816. The start
and stop codons are in bold letters in Table 6A. TABLE-US-00048
TABLE 6A NOV6 Nucleotide Sequence (SEQ ID NO:25)
ATGGGGGGCCTGACAGCCTCGGACGTACACCCGACCCTGGGGGTCCAGCTCTTCTCAGCTGGAATAGCGG
CGTGCTTGGCGGACGTGATCACCTTCCCGCTGGACACGGCCAAAGTCCGGCTCCAGGTCCAAGGTGAATG
CCCGACGTCCAGTGTTATTAGGTATAAAGGTGTCCTGGGAACAATCACCGCTGTGGTAAAAACAGAAGGG
CGGATGAAACTCTACAGCGGGCTGCCTGCGGGGCTTCAGCGGCAAATCAGCTCCGCCTCTCTCAGGATCG
GCCTCTACGACACGGTCCAGGAGTTCCTCACCGCAGGGAAAGAAACAGCACCTAGTTTAGGAAGCAAGAT
TTTAGCTGGTCTAACGACTGGAGGAGTGGCAGTATTCATTGGGCAACCCACAGAGGTCGTGAAAGTCAGA
CTTCAAGCACAGAGCCATCTCCACGGAATCAAACCTCGCTACACGGGGACTTATAATGCGTACAGAATAA
TAGCAACAACCGAAGGCTTGACGGGTCTTTGGAAAGGGACTACTCCCAATCTGATGAGAAGTGTCATCAT
CAATTGTACAGAGCTAGTAACATATGATCTAATGAAGGAGGCCTTTGTGAAAAACAACATATTAGCAGGA
CAGTACAAAAGTGTGCCCAACTGTGCAATGAAAGTGTTCACTAACGAAGGACCAACGGCTTTCTTCAAGG
GGTTGGTACCTTCCTTCTTGCGACTTGGATCCTGGAACGTCATTATGTTTGTGTGCTTTGAACAACTGAA
ACGAGAACTGTCAAAGTCAAGGCAGACTATGGACTGTGCCACATAA
[0187] The disclosed NOV6 nucleic acid sequence, located on
chromosome 4, has 628 of 628 bases (100%) identical to a
gb:GENBANK-ID:HSU28480|acc:U28480.1 mRNA from Homo sapiens (Human
uncoupling protein (UCP) mRNA, complete cds) (E=1.1e.sup.-176). A
disclosed NOV6 polypeptide (SEQ ID NO:26) encoded by SEQ ID NO:25
is 271 amino acid residues and is presented using the one-letter
amino acid code in Table 6B. Signal P, Psort and/or Hydropathy
results predict that NOV6 contains no signal peptide and is likely
to be localized extracellularly with a certainty of 0.4753. In
other embodiments, NOV6 is also likely to be localized to the
plasma membrane with a certainty of 0.1900, to the microbody
(peroxisome) with a certainty of 0.1544, or to the endoplasmic
reticulum (membrane) with a certainty of 0.1000 TABLE-US-00049
TABLE 6B Encoded NOV6 protein sequence (SEQ ID NO:26).
MGGLTASDVEPTLGVQLFSAGIAACLADVITFPLDTAKVRLQVQGECPTSSVIRYKGVLGTITAVVKTEGRMKL-
Y
SGLPAGLQRQISSASLRIGLYDTVQEFLTAGKETAPSLGSKILAGLTTGGVAVFIGQPTEVVKVRLQAQSHLHG-
I
KPRYTGTYNAYRIIATTEGLTGLWKGTTPNLMRSVIINCTELVTYDLMKEAFVKNNILAGQYKSVPNCAMKVFT-
N EGPTAFFKGLVPSFLRLGSWNVIMFVCFEQLKRELSKSRQTMDCAT
[0188] The disclosed NOV6 amino acid sequence has 209 of 209 amino
acid residues (100%) identical to, and 209 of 209 amino acid
residues (100%) similar to, the 307 amino acid residue
ptnr:pir-id:G01858 protein from human (uncoupling protein 1,
mitochondrial) (E=4.8e.sup.-140).
[0189] NOV6 is expressed in at least the following tissues: Adrenal
Gland/Suprarenal gland and Brown adipose. This information was
derived by determining the tissue sources of the sequences that
were included in the invention including but not limited to
SeqCalling sources, Public EST sources, Literature sources, and/or
RACE sources.
[0190] NOV6 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 6C. TABLE-US-00050 TABLE 6C BLAST
results for NOV6 Gene Index/ Length Identity Positives Identifier
Protein/Organism (aa) (%) (%) Expect gi|11225256|ref|NP.sub.--
uncoupling 307 271/307 271/307 e-144 068605.1| protein 1; (88%)
(88%) (NM_021833) thermogenin; mitochondrial brown fat uncoupling
protein [Homo sapiens] gi|1351353|sp|P25874| MITOCHONDRIAL 307
270/307 270/307 e-143 UCP1_HUMAN BROWN FAT (87%) (87%) UNCOUPLING
PROTEIN 1 (UCP 1) (THERMOGENIN) gi|136689|sp|P14271| MITOCHONDRIAL
306 226/307 245/307 e-117 UCP1_RABIT BROWN FAT (73%) (79%)
UNCOUPLING PROTEIN 1 (UCP 1) (THERMOGENIN) gi|109392|pir||A32446
uncoupling 306 225/307 245/307 e-117 protein - rabbit (73%) (79%)
gi|1351354|sp|P04575| MITOCHONDRIAL 307 216/303 238/303 e-115
UCP1_MESAU BROWN FAT (71%) (78%) UNCOUPLING PROTEIN 1 (UCP 1)
(THERMOGENIN)
[0191] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 6D.
[0192] Tables 6E-F list the domain description from DOMAIN analysis
results against NOV6. This indicates that the NOV6 sequence has
properties similar to those of other proteins known to contain this
domain. TABLE-US-00051 TABLE 6E Domain Analysis of NOV6
gnl|Pfam|pfam00153, mito_carr, Mitochondrial carrier protein. (SEQ
ID NO:92) CD-Length = 96 residues, 95.8% aligned Score = 81.6 bits
(200). Expect = 5e-17 Query: 111
PSLGSKILAGLTTGGVAVFIGQPTEVVKVRLQAQSHLHGIKPRYTGTYNAYRIIATTEGL 170 |
+ +||| | +| + | +||| ||| | +| | + ++ | || Sbjct: 3
LSFLASLLAGGIAGAIAALVTYPLDVVKTRLQVQGS----SSKYKGILDCFKKIVKEEGR 58
Query: 171 TGLWKGTTPNLMRSVIINCTELVTYDLMKEAFVKNN 206 ||+|| | |+| ||+
+|+ | Sbjct: 59 AGLYKGLGPTLLRVAPYAAIYFGTYEQLKKLLGKKL 94
[0193] TABLE-US-00052 TABLE 6F Domain Analysis of NOV6
gnl|Pfam|pfam00153, mito_carr, Mitochondrial carrier protein. (SEQ
ID NO 93) CD-Length = 96 residues, 99.0% aligned Score = 79.3 bits
(194), Expect = 3e-16 Query: 10
HPTLGVQLFSAGIAACLADVITFPLDTAKVRLQVQGECPTSSVIRYKGVLGTITAVVKTE 69 + |
+ ||| +| ++|+||| | |||||| +|||+| +|| | Sbjct: 2
PLSFLASLLAGGIAGAIAALVTYPLDVVKTRLQVQGSSS-----KYKGILDCFKKIVKEE 56
Query: 70 GRMKLYSGLPAGLQRQISSASLRIGLYDTVQEFLTAGKET 109 || || || | |
|++ | |+ +++ | Sbjct: 57 GRAGLYKGLGPTLLRVAPYAAIYFGTYEQLKKLLGKRLGE
96
[0194] The uncoupling protein (UCP) of mitochondria in brown
adipose tissue is a specific component unique to mammalian cells.
Complementary DNAs for rat and mouse UCP were isolated in several
laboratories (Jacobson et al., 1985; Bouillaud et al., 1986; Ridley
et al., 1986). The cDNAs have been used to determine the sequence
of rat UCP and to monitor changes in UCP mRNA levels under various
physiologic, pathologic, and pharmacologic circumstances. A
controversy exists concerning the physiologic significance of brown
adipose tissue in humans and its possible contribution to
resistance to obesity (see 601665). There is, however, a large
amount of evidence that this tissue is present in young infants and
also in human adults in certain pathologic and nonpathologic
situations.
[0195] Bouillaud et al. (1988) screened a human genomic library
with a cDNA corresponding to the UCP of rat brown adipose tissue
mitochondria. They succeeded in cloning a 0.5-kb fragment
containing 2 intronic regions and 2 exonic regions. The exonic
regions encode a sequence of 84 amino acids with a strong homology
to the central domain of rat UCP. Southern analysis experiments
suggested that there is 1 copy of the gene in the human, as there
is in rodents. In Northern analysis experiments, the probe detected
a specific 1.8-kb mRNA in human brown adipose tissue obtained from
6 patients with pheochromocytoma and from 1 patient with a
hibernoma.
[0196] Cassard et al. (1990) found that the human UCP gene spans 13
kb and contains a transcribed region that covers 9 kb. It has 6
exons. The uncoupling protein has 305 amino acids and a molecular
weight of 32,786.
[0197] Fletcher et al. (1991) mapped the Ucp gene to mouse
chromosome 8 in a location between a segment that carries genes
homologous to genes on human 8p, on the centromeric side, and a
segment that carries genes homologous to human genes on 16q, in the
telomeric direction. Thus, the human homolog of Ucp is probably on
either 8p or 16q. Using in situ hybridization, Cassard et al.
(1990) assigned the human UCP gene to 4q31. They found that the
primary structure of UCP is similar to that of ADP/ATP translocator
of skeletal muscle (103220), the gene for which is also located on
chromosome 4. Thus, the prediction from homology to the mouse did
not hold up.
[0198] Brown adipose tissue, because of its capacity for uncoupled
mitochondrial respiration, is an important site of facultative
energy expenditure. It has been speculated that this tissue
normally functions to prevent obesity. Surgical efforts to ablate
or denervate the brown adipose tissue have been unsuccessful
because of the diffuse deposits and substantial capacity for
regeneration and hypertrophy. Lowell et al. (1993) used a
transgenic toxigene approach to create 2 lines of transgenic mice
with primary deficiency of brown adipose tissue. In constructing
these transgenic mice, Lowell et al. (1993) used the regulatory
elements of the gene for uncoupling protein to drive expression of
the diphtheria toxin A chain (UCP-DTA) or an attenuated mutant. At
16 days, both lines had deficient brown fat and obesity. In one
line, brown fat subsequently regenerated and obesity resolved. In
the other line, the deficiency persisted and obesity, with its
morbid complications, advanced. Obesity developed in the absence of
hyperphagia, indicating that brown fat deficient mice have
increased metabolic efficiency. As obesity progressed, transgenic
animals developed hyperphagia. See also UCP2 (601693).
[0199] Uncoupling protein is a mitochondrial proton channel that is
not coupled to oxidative phosphorylation. Therefore, when a proton
gradient is established across the inner mitochondrial membrane,
activation of the uncoupling protein leads to the uncoupled passage
of protons through the channel and the generation of heat.
Expression and activation of uncoupling proteins is usually
mediated by the sympathetic nervous system and is directly
controlled by norepinephrine. This mechanism is part of the
adaptive response to cold temperatures. It also regulates energy
balance. Manipulation of thermogenesis could be an effective
strategy against obesity (Lowell et al., 1993). Enerback et al.
(1997) determined the role of UCP in the regulation of body mass by
targeted inactivation of the UCP gene in mice. They found that
UCP-deficient mice consumed less oxygen after treatment with a
beta-3-adrenergic receptor agonist and that they were sensitive to
cold, indicating that thermoregulation was defective. However, this
deficiency caused neither hyperphagia nor obesity in mice fed on
either a standard or a high-fat diet. Enerback et al. (1997)
proposed that the loss of UCP may be compensated by UCP2, a homolog
of UCP that is ubiquitously expressed and is induced in the brown
fat of UCP-deficient mice.
[0200] Adrenaline and noradrenaline, the main effectors of the
sympathetic nervous system and adrenal medulla, respectively, are
thought to control adiposity and energy balance through several
mechanisms. They promote catabolism of triglycerides and glycogen,
stimulate food intake when injected into the central nervous
system, activate thermogenesis in brown adipose tissue, and
regulate heat loss through modulation of peripheral
vasoconstriction and piloerection. Thermogenesis in brown adipose
occurs m response to cold and overeating, and there is an inverse
relationship between diet-induced thermogenesis and obesity both in
humans and animal models. As a potential model for obesity, Thomas
and Palmiter (1997) generated mice that could not synthesize
noradrenaline or adrenaline by inactivating the gene that encodes
dopamine beta-hydroxylase (DBH; 223360). These mice were cold
intolerant because they had impaired peripheral vasoconstriction
and were unable to induce thermogenesis in brown adipose tissue
through uncoupling protein (UCP1). The mutants had increased food
intake but did not become obese because their basal metabolic rate
(BMR) was also elevated. The unexpected increase in BMR was not due
to hyperthyroidism, compensation by the widely expressed UCP2, or
shivering.
[0201] The failure of UCP1 expressed in E. coli inclusion bodies to
carry out fatty acid-dependent H(+) transport activity inclusion
bodies made Echtay et al. (2000) seek a native UCP cofactor. They
identified coenzyme Q (CoQ, or ubiquinone) as such a cofactor. On
addition of CoQ(10) to reconstituted UCP1 from inclusion bodies,
fatty acid-dependent proton transport reached the same rate as with
native UCP1. The proton transport was highly sensitive to purine
nucleotides and was activated only by oxidized but not reduced CoQ.
Proton transport of native UCP1 correlated with the endogenous CoQ
content.
[0202] The disclosed NOV6 nucleic acid of the invention encoding a
Leucine-Rich Glioma-Inactivated Protein-like protein includes the
nucleic acid whose sequence is provided in Table 6A or a fragment
thereof. The invention also includes a mutant or variant nucleic
acid any of whose bases may be changed from the corresponding base
shown in Table 6A while still encoding a protein that maintains its
Leucine-Rich Glioma-Inactivated Protein-like activities and
physiological functions, or a fragment of such a nucleic acid. The
invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, by way
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 10% percent of
the bases may be so changed.
[0203] The disclosed NOV6 protein of the invention includes the
Leucine-Rich Glioma-Inactivated Protein-like protein whose sequence
is provided in Table 6B. The invention also includes a mutant or
variant protein any of whose residues may be changed from the
corresponding residue shown in Table 6B while still encoding a
protein that maintains its Leucine-Rich Glioma-Inactivated
Protein-like activities and physiological functions, or a
functional fragment thereof. In the mutant or variant protein, up
to about 29 percent of the residues may be so changed.
[0204] The above defined information for this invention suggests
that these Leucine-Rich Glioma-Inactivated Protein-like proteins
(NOV6) may function as a member of a "Leucine-Rich
Glioma-Inactivated Protein family". Therefore, the NOV6 nucleic
acids and proteins identified here may be useful in potential
therapeutic applications implicated in (but not limited to) various
pathologies and disorders as indicated below. The potential
therapeutic applications for this invention include, but are not
limited to: protein therapeutic, small molecule drug target,
antibody target (therapeutic, diagnostic, drug targeting/cytotoxic
antibody), diagnostic and/or prognostic marker, gene therapy (gene
delivery/gene ablation), research tools, tissue regeneration in
vivo and in vitro of all tissues and cell types composing (but not
limited to) those defined here.
[0205] The nucleic acids and proteins of NOV6 are useful in any
inflammatory diseases such as obesity, hyperphagia, and/or other
pathologies and disorders.
[0206] NOV6 nucleic acids and polypeptides 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. These antibodies may be generated according to
methods known in the art, using prediction from hydrophobicity
charts, as described in the "Anti-NOVX Antibodies" section below.
For example the disclosed NOV6 protein have multiple hydrophilic
regions, each of which can be used as an immunogen. This novel
protein also has value in development of powerful assay system for
functional analysis of various human disorders, which will help in
understanding of pathology of the disease and development of new
drug targets for various disorders.
[0207] NOV7
[0208] NOV7 includes three novel Leucine-Rich Glioma-Inactivated
Protein-like proteins disclosed below. The disclosed sequences have
been named NOV7a, and NOV7b.
[0209] NOV7a
[0210] A disclosed NOV7a nucleic acid of 1859 nucleotides (also
referred to CG56077-01) encoding a novel Leucine-Rich
Glioma-Inactivated Protein-like protein is shown in Table 7A. An
open reading frame was identified beginning with an ATG initiation
codon at nucleotides 101-103 and ending with a TGA codon at
nucleotides 1760-1762. In Table 7A, the 5' and 3' untranslated
regions are underlined and the start and stop codons are in bold
letters. TABLE-US-00053 TABLE 7A NOV7a Nucleotide Sequence (SEQ ID
NO:27)
GTAACTCCTCTTCATCCACTGGGGAGGAAGGTGAGGCAGGCGGGCCCAATTCATTCGCCTCCGGTACTTGC
AAGCCTCGCTCAGTCTTAAGCAAGAGGGGATGGATTCGCCCGCAGCACTGAGAATCCAGGGGCAGGCGGGA
TGGCGTTCAGGCGCTGTTGCTAGAAATCTCTGTCTTTACTCTGTTTTGGTCATTACGGAGGGAAGACAGCC
CCCAAAGGGAAAGTGTCCCCTGCGCTGCTCCTGCTCTAAAGACAGCGCCCTGTGTGAGGGCTCCCCGGACC
TGCCCGTCAGCTTCTCTCCGACCCTGCTGTCACTCTCACTCGTCAGGACGGGAGTCACCCAGCTGAAGGCC
GGCAGCTTCCTGAGAATTCCGTCTCTGCACCTGCTCCTCTTCACCTCCAACTCCTTCTCCGTGATTGAGGA
CGATGCATTTGCGGGCCTGTCCCACCTGCAGTACCTCTTCATCGAGGACAATGAGATTGGCTCCATCTCTA
AGAATGCCCTCAGAGGACTTCGCTCGCTTACACACCTAAGCCTGGCCAATAACCATCTGGAGACCCTCCCC
AGATTCCTGTTCCGAGGCCTGGACACCCTTACTCATGTGGACCTCCGCGGGAACCCGTTCCAGTGTGACTG
CCGCGTCCTCTGGCTCCTGCAGTGGATGCCCACCGTGAATGCCAGCGTGGGGACCGGCGCCTGTGCGGGCC
CCGCCTCCCTGAGCCACATGCAGCTCCACCACCTCGACCCCAAGACGTTCAAGTGCAGAGCCATAGAGCTG
TCCTGGTTCCAGACGGTGGGGGAGTCGGCACTGAGCGTAGAGCCCTTCTCCTACCAAGGGGAGCCTCACAT
TGTGCTGGCACAGCCCTTCGCCGGCCGCTGCCTGATTCTCTCCTGGGACTACAGCCTGCAGCGCTTCCGGC
CCGAGGAAGAGCTGCCCGCGGCCTCCGTGGTGTCCTGCAAGCCACTGGTGCTGGGCCCGAGCCTCTTCGTG
CTGGCTGCCCGCCTGTGGGGGGGCTCACAGCTGTGGGCCCGGCCCACTCCCGGCCTGCGCCTGGCCCCAAC
GCAGACCCTGGCCCCGCGGCGGCTGCTGCGGCCCAATGACGCCGAGCTCCTGTGGCTGGAAGGGCAACCCT
GCTTCGTGGTGGCCGATGCCTCCAAGGCGGGCAGCACCACGCTGCTGTGCCGCGACGGGCCCGGCTTTTAC
CCGCACCAGAGCCTGCACGCCTGGCACCGGGACACGGACGCTGAGGCCCTGGAGCTGGACGGCCGGCCCCA
CCTGCTGCTGGCCTCGGCTTCCCAGCGGCCCGTGCTCTTCCACTGGACCGGTGGCCGCTTCGAGAGACGCA
CAGACATCCCGAGGGCCGAGGATGTCTATGCCACACGCCACTTCCAGGCTGGTGGGGACGTGTTCCTGTGC
CTCACACGCTACATTGGGGACTCCATGGTCATGCGCTGGGACGGCTCCATGTTTCGTCTGCTGCAGCAACT
TCCCTCGCGCGGTGCCCACGTCTTCCAGCCACTGCTCATCGCCAGGGACCAGCTGGCCATCCTAGGCAGCG
ACTTCGCCTTCAGCCAGCTCCTCCGCCTTGAGCCTGACAAGGGGCTCCTGGAGCCACTGCAGGAGCTGGGG
CCTCCGGCCCTGGTGGCCCCCCGTGCCTTTGCCCACATCACTATGGCCGGCAGACGCTTCCTCTTTGCTGC
TTGCTTTAAGGGCCCCACACAGATCTACCAGCATCACGAGATCGACCTCAGTGCCTGAGACCACCAACGGG
ACTCTGGGCATGGCTGGGGCCCCTGGACGGCCCCTTGGCTGGCTCCTGGCCCTACTTGGGGTGATGGCCCG
CCTGTGAGCTGCT
[0211] The disclosed NOV7a nucleic acid sequence, localized to the
q 12 region of chromosome 19, has 940 of 1619 bases (58%) identical
to a gb:GENBANK-ID:HSU53204|acc:U53204.1 mRNA from Homo sapiens
(Human plectin (PLEC1) mRNA, complete cds) (E=7.0e.sup.-301).
[0212] A disclosed NOV7a polypeptide (SEQ ID NO:28) encoded by SEQ
ID NO:27 is 553 amino acid residues and is presented using the
one-letter amino acid code in Table 7B. Signal P, Psort and/or
Hydropathy results predict that NOV7a has a signal peptide and is
likely to be localized in the microbody (peroxisome) with a
certainty of 0.6562. In other embodiments, NOV7A is also likely to
be localized to the lysosome (lumen) with a certainty of 0.2474, or
to the mitochondrial matrix space with a certainty of 0.1000.
TABLE-US-00054 TABLE 7B Encoded NOV7a protein sequence (SEQ ID
NO:28).
MDSPAALRIQGQAGWRSGAVARNLCLYSVLVITEGRQPPKGKCPLRCSCSKDSALCEGSPDLPVSFSPTLL
SLSLVRTGVTQLKAGSFLRIPSLHLLLFTSNSFSVIEDDAFAGLSHLQYLFIEDNEIGSISKNALRGLRSL
THLSLANNHLETLPRFLFRGLDTLTHVDLRGNPFQCDCRVLWLLQWMPTVNASVGTGACAGPASLSHMQLH
HLDPKTFKCRAIELSWFQTVGESALSVEPFSYQGEPHIVLAQPFAGRCLILSWDYSLQRFRPEEELPAASV
VSCKPLVLGPSLFVLAARLWGGSQLWARPSPGLRLAPTQTLAPRRLLRPNDAELLWLEGQPCFVVADASKA
GSTTLLCRDGPGFYPEQSLHAWHRDTDAEALELDGRPHLLLASASQRPVLFHWTGGRFERRTDIPRAEDVY
ATRHFQAGGDVFLCLTRYIGDSMVMRWDGSMFRLLQQLPSRGAHVFQPLLIARDQLAILGSDFAFSQVLRL
EPDKGLLEPLQELGPPALVAPRAFAHITMAGRRFLFAACFKGPTQIYQHHEIDLSA
[0213] The disclosed NOV7a amino acid sequence has 274 of 557 amino
acid residues (49%) identical to, and 380 of 557 amino acid
residues (68%) similar to, the 557 amino acid residue
ptnr:SPTREMBL-ACC:Q9JIA1 protein from Mus musculus (Mouse)
(Leucine-Rich Glioma-Inactivated 1 Protein Precursor)
(E=2.5e.sup.-143).
[0214] NOV7a is expressed in at least the following tissues:
Adipose, Brain, Cervix, Heart, Hippocampus, Hypothalamus, Kidney
Cortex, Liver, Lung, Myometrium, Nasoepithelium, Pancreas,
Prostate, Retina, Small Intestine, Spinal Chord, Stomach,
Substantia Nigra, Testis, Thalamus, Thymus, Vein. This information
was derived by determining the tissue sources of the sequences that
were included in the invention including but not limited to
SeqCalling sources, Public EST sources, Literature sources, and/or
RACE sources.
[0215] In addition, the sequence is predicted to be expressed in
skin and muscle because of the expression pattern of (GENBANK-ID:
gb:GENBANK-ID:HSU53204|acc:U53204.1) a closely related Human
plectin (PLEC1) mRNA, complete cds homolog.
[0216] NOV7b
[0217] In the present invention, the target sequence identified
previously, NOV7a, was subjected to the exon linking process to
confirm the sequence. PCR primers were designed by starting at the
most upstream sequence available, for the forward primer, and at
the most downstream sequence available for the reverse primer. In
each case, the sequence was examined, walking inward from the
respective termini toward the coding sequence, until a suitable
sequence that is either unique or highly selective was encountered,
or, in the case of the reverse primer, until the stop codon was
reached. Such primers were designed based on in silico predictions
for the full length cDNA, part (one or more exons) of the DNA or
protein sequence of the target sequence, or by translated homology
of the predicted exons to closely related human sequences sequences
from other species. These primers were then employed in PCR
amplification based on the following pool of human cDNAs: adrenal
gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. Usually the resulting amplicons were gel purified, cloned
and sequenced to high redundancy. The resulting sequences from all
clones were assembled with themselves, with other fragments in
CuraGen Corporation's database and with public ESTs. Fragments and
ESTs were included as components for an assembly when the extent of
their identity with another component of the assembly was at least
95% over 50 bp. In addition, sequence traces were evaluated
manually and edited for corrections if appropriate. These
procedures provide the sequence reported below, which is designated
Accession Number NOV7b. This differs from the previously identified
sequence (Accession Number NOV7a) in being a splice variant.
[0218] A disclosed NOV7b nucleic acid of 1482 nucleotides (also
referred to CG56077-O.sub.2) encoding a novel Leucine-Rich
Glioma-Inactivated Protein-like protein is shown in Table 7C. An
open reading frame was identified beginning with an TTC initiation
codon at nucleotides 1-3 and ending with a TGA codon at nucleotides
1480-1482. In Table 7C, the 5' and 3' untranslated regions are
underlined and the start and stop codons are in bold letters.
Because the start codon for NOV7b is not a traditional initiation
codon, NOV7b may be a partial reading frame that extends further in
the 5' direction. TABLE-US-00055 TABLE 7C NOV7b Nucleotide Sequence
(SEQ ID NO:29)
TCCCGAGACTTTGGAAGTTCTCAGCTATTACTTTATTACATAGGATTTCTGTGTCTTTTCTCATCTCTTTT
CCTTTTGGAAATTGGAAGACCCCCAAAGGGAAAGTGTCCCCTGCGCTGCTCCTGCTCTAAAGACAGCGCCC
TGTGTGAGGGCTCCCCGGACCTGCCCGTCAGCTTCTCTCCGACCCTGCTGTCACTGACTGCCCACATCCCC
AGCTCACTCGTCAGGACGGGAGTCACCCAGCTGAAGGCCGGCAGCTTCCTGAGAATTCCGTCTCTGCACCT
GCTGCTCTTCACCTCCAACTCCTTCTCCGTGATTGAGGACGATGCATTTGCGGGCCTGTCCCACCTGCAGT
ACCTGTTCATCGAGGACAATGAGATTGGCTCCATCTCTAAGAATGCCCTCAGAGGACTTCGCTCGCTTACA
CACCTGAGCCTGGCCAATAACCATCTGGAGACCCTCCCCAGATTCCTGTTCCGAGGCCTGGACACCCTTAC
TCATGTGGACCTCCGCGGGAACCCGTTCCAGTGTGACTGCCGCGTCCTCTGGCTCCTGCAGTGGATGCCCA
CCGTGAATGCCAGCGTGGGGACCGGCGCCTGTGCGGGCCCCGCCTCCCTGAGCCACATGCAGCTCCACCAC
CTCGACCCCAAGACTTTCAAGTGCACAGCGGCCTCCGTGGTGTCCTGCAAGCCACTGGTGCTGGGCCCGAG
CCTCTTCGTGCTGGCTGCCCGCCTGTGGGGGGGCTCACAGCTGTGGGCCCGGCCCAGTCCCGGCCTGCGCC
TGGCCCCAACGCAGACCCTGGCCCCGCGGCGGCTGCTGCGGCCCAATGACGCCGAGCTCCTGTGGCTGGAA
GGGCAACCCTGCTTCGTGGTGGCCGATGCCTCCAAGGCGGGCAGCACCACGTGCAGCGCTTCCGGCCCGAG
GAAGAGCTGCCCGAGCCTGCACGCCTGGCACCGGGACACGGACGCTGAGGCCCTGGAGCTGGACGGCCGGC
CCCACCTGCTGCTGGCCTCGGCTTCCCAGCGGCCCGTGCTCTTCCACTGGACCGGTGGCCGCTTCGAGAGA
CGCACGGACATCCCCGAGGCCGAGGATGTCTATGCCACACGCCACTTCCAGGCTGGTGGGGACGTGTTCCT
GTGCCTCACACGCTACATTGGGGACTCCATGGTCATGCGCTGGGACGGCTCCATGTTTCGTCTGCTGCAGC
AACTTCCCTCGCGCGGTGCCCACGTCTTCCAGCCACTGCTCATCGCCAGGGACCAATTGGCCATCCTAGGC
AGCGACTTCGCCTTCAGCCAGGTCCTCCGCCTTGAGCCTGACAAGGGGCTCCTGGAGCCACTGCAGGAGCT
GGGGCCTCTGGCCCTGGTGGCCCCCCGTGCCTTTGCCCACATCACTATGGCCGGCAGACGCTTCCTCTTTG
CTGCTTGCTTTAAGGGCCCCACACAGATCTACCAGCATCACGAGATCGACCTCAGTGCCTGA
[0219] The disclosed NOV7b nucleic acid sequence, localized to the
q24 region of chromosome 10, has 559 of 919 bases (60%) identical
to a gb:GENBANK-ID:SC6D10|acc:AL138538.1 mRNA from Streptomyces
coelicolor A3(2) (Streptomyces coelicolor cosmid 6D10)
(E=9.8e.sup.-10).
[0220] A disclosed NOV7b polypeptide (SEQ ID NO:30) encoded by SEQ
ID NO:29 is 493 amino acid residues and is presented using the
one-letter amino acid code in Table 7D. Signal P, Psort and/or
Hydropathy results predict that NOV7b has a signal peptide and is
likely to be localized in the endoplasmic reticulum (membrane) with
a certainty of 0.8200. In other embodiments, NOV7b is also likely
to be localized to the microbody (peroxisome) with a certainty of
0.3264, to th plasma membrane with a certainty of 0.1900, or to the
endoplasmic reticulum (lumen) with a certainty of 0.1000. The most
likely cleavage site for NOV7b is between positions 25 and 26:
LFL-LE. TABLE-US-00056 TABLE 7D Encoded NOV7b protein sequence (SEQ
ID NO:30).
SRDFGSSQLLLYYIGFLCLFSSLFLLEIGRPPKGKCPLRCSCSKDSALCEGSPDLPVSFSPTLLSLTAHIP
SSLVRTGVTQLKAGSFLRIPSLHLLLFTSNSFSVIEDDAFAGLSHLQYLFIEDNEIGSISKNALRGLRSLT
HLSLANNHLETLPRFLPRGLDTLTHVDLRGNPFQCDCRVLWLLQWMPTVNASVGTGACAGPASLSHMQLHH
LDPKTFKCTAASVVSCKPLVLGPSLFVLAARLWGGSQLWARPSPGLRLAPTQTLAPRRLLRPNDAELLWLE
GQPCFVVADASKAGSTTCSASGPRKSCPSLHAWNRDTDAEALELDGRPHLLLASASQRPVLFHWTGGRFER
RTDIPEAEDVYATRHFQAGGDVFLCLTRYIGDSMVMRWDGSMFRLLQQLPSRGAHVFQPLLIARDQLAILG
SDFAFSQVLRLEPDKGLLEPLQELGPLALVAPRAFAHITMAGRRFLFAACFKGPTQIYQHHEIDLSA
[0221] The disclosed NOV7b amino acid sequence has 137 of 295 amino
acid residues (46%) identical to, and 191 of 295 amino acid
residues (64%) similar to, the 557 amino acid residue
ptnr:SPTREMBL-ACC:Q9JIA1 protein from Mus musculus (Mouse)
(Leucine-Rich Glioma-Inactivated 1 Protein Precursor)
(E=5.7e.sup.-122).
[0222] NOV7b is expressed in at least brain. Expression information
was derived from the tissue sources of the sequences that were
included in the derivation of the sequence of CuraGen Acc. No.
CG56077-02.
[0223] NOV7 also has homology to the amino acid sequence shown in
the BLASTP data listed in Table 7E. TABLE-US-00057 TABLE 7E BLAST
results for NOV7 Gene Index/ Length Identity Positives Identifier
Protein/Organism (aa) (%) (%) Expect gi|9938002|ref|NP_064674.1|
leucine-rich, 557 271/561 376/561 e-157 (NM_020278) glioma (48%)
(66%) inactivated 1 [Mus musculus] gi|4826816|ref|NP_005088.1|
leucine-rich, 557 265/542 364/542 e-151 (NM_005097) glioma (48%)
(66%) inactivated 1 precursor [Homo sapiens]
gi|15722102|emb|CAC78757.1| bA512J3.1 461 225/464 308/464 e-129
(AL358154) (leucine-rich, (48%) (65%) glioma inactivated 1) [Homo
sapiens] gi|15620891|dbj|BAB67809.1| KIAA1916 protein 542 211/513
312/513 e-113 (AB067503) [Homo sapiens] (41%) (60%)
gi|4590406|gb|AAD26567.1| slit protein 1504 58/187 89/187 2e-18
AF126540_1 [Drosophila (31%) (47%) (AF126540) melanogaster]
[0224] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 7F.
[0225] Tables 7E-G list the domain description from DOMAIN analysis
results against NOV7. This indicates that the NOV7 sequence has
properties similar to those of other proteins known to contain this
domain. TABLE-US-00058 TABLE 7E Domain Analysis of NOV7
gnl|Smart|smart00082, LRRCT, Leucine rich repeat C-terminal domain
(SEQ ID NO:99) CD-Length = 51 residues, 100.0% aligned Score = 46.6
bits (109), Expect = 3e-06 Query: 173
NPFQCDCRVLWLLQWMPTVNASVGTGA--CAGPASLSHMQLHHLDPKTFKCT 222 ||| ||| +
|||+|+ || | || | | | +||| Sbjct: 1
NPFICDCELRWLLRWLQANRHLQDPVDLRCASPESL-RGPLLLLLPSSFKCP 51
[0226] TABLE-US-00059 TABLE 7F Domain Analysis of NOV7
gnl|Pfam|pfam01463, LRRCT, Leucine rich repeat C-terminal domain.
Leucine Rich Repeats pfam00560 are short sequence motifs present in
a number of proteins with diverse functions and cellular locations.
Leucine Rich Repeats are often flanked by cysteine rich domains.
This domain is often found at the C-terminus of tandem leucine rich
repeats. (SEQ ID NO:100) CD-Length = 51 residues, 98.0% aligned
Score = 45.4 bits (106), Expect = 7e-06 Query: 173
NPFQCDCRVLWLLQWMPTVNASVGTGA--CAGPASLSHMQLHHLDPKTFKC 221 ||| ||| +
|||+|+ || | || | | | | | Sbjct: 1
NPFICDCELRWLLRWLREPRRLEDPEDLRCASPESL-RGPLLELLPSDFSC 50
[0227] TABLE-US-00060 TABLE 7G Domain Analysis of NOV7
gnl|Smart|smart00369, LRR_TYP, Leucine-rich repeats, typical (most
populated) subfamily (SEQ ID NO:101) CD-Length = 24 residues.
100.0% aligned Score = 35.8 bits (81). Expect = 0.006 Query: 138
LRSLTHLSLANNHLETLPRFLFRG 161 | +| | |+|| | +|| |+| Sbjct: 1
LPNLRELDLSNNQLSSLPPGAFQG 24
[0228] Loss of heterozygosity for 10q23-26 is seen in over 80% of
glioblastoma multiforme tumors. A novel gene, LGI1 (Leucine-rich
gene-Glioma Inactivated), is rearranged as a result of the
t(10;19)(q24;q13) balanced translocation in the T98G glioblastoma
cell line lacking any normal chromosome 10. Rearrangement of the
LGI1 gene was also detected in the A172 glioblastoma cell line and
several glioblastoma tumors. These rearrangements lead to a
complete absence of LGI1 expression in glioblastoma cells. The LGI1
gene encodes a protein with a calculated molecular mass of 60 kD
and contains 3.5 leucine-rich repeats (LRR) with conserved flanking
sequences. In the LRR domain, LGI1 has the highest homology with a
number of transmembrane and extracellular proteins which function
as receptors and adhesion proteins. LGI1 is predominantly expressed
in neural tissues, especially in brain; its expression is reduced
in low grade brain tumors and it is significantly reduced or absent
in malignant gliomas. Its localization to the 10q24 region, and
rearrangements or inactivation in malignant brain tumors, suggest
that LGI1 is a candidate tumor suppressor gene involved in
progression of glial tumors.
[0229] The disclosed NOV7 nucleic acid of the invention encoding a
Leucine-Rich Glioma-Inactivated Protein-like protein includes the
nucleic acid whose sequence is provided in Table 7A, 7C or a
fragment thereof. The invention also includes a mutant or variant
nucleic acid any of whose bases may be changed from the
corresponding base shown in Table 7A or 7C while still encoding a
protein that maintains its Leucine-Rich Glioma-Inactivated
Protein-like activities and physiological functions, or a fragment
of such a nucleic acid. The invention further includes nucleic
acids whose sequences are complementary to those just described,
including nucleic acid fragments that are complementary to any of
the nucleic acids just described. The invention additionally
includes nucleic acids or nucleic acid fragments, or complements
thereto, whose structures include chemical modifications. Such
modifications include, by way of nonlimiting example, modified
bases, and nucleic acids whose sugar phosphate backbones are
modified or derivatized. These modifications are carried out at
least in part to enhance the chemical stability of the modified
nucleic acid, such that they may be used, for example, as antisense
binding nucleic acids in therapeutic applications in a subject. In
the mutant or variant nucleic acids, and their complements, up to
about 40 percent of the bases may be so changed.
[0230] The disclosed NOV7 protein of the invention includes the
Leucine-Rich Glioma-Inactivated Protein-like protein whose sequence
is provided in Table 7B or 7D. The invention also includes a mutant
or variant protein any of whose residues may be changed from the
corresponding residue shown in Table 7B or 7D while still encoding
a protein that maintains its Leucirie-Rich Glioma-Inactivated
Protein-like activities and physiological functions, or a
functional fragment thereof. In the mutant or variant protein, up
to about 69 percent of the residues may be so changed.
[0231] The protein similarity information, expression pattern, and
map location for the Leucine-Rich Glioma-Inactivated Protein-like
protein and nucleic acid (NOV7) disclosed herein suggest that NOV7
may have important structural and/or physiological functions
characteristic of the Leucine-Rich Glioma-Inactivated Protein-like
family. Therefore, the NOV7 nucleic acids and proteins of the
invention are useful in potential diagnostic and therapeutic
applications. 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, as well as 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), and (v) a
composition promoting tissue regeneration in vitro and in vivo.
[0232] The NOV7 nucleic acids and proteins of the invention are
useful in potential diagnostic and therapeutic applications
implicated in various diseases and disorders described below and/or
other pathologies. For example, the compositions of the present
invention will have efficacy for treatment of patients suffering
from cardiomyopathy, atherosclerosis, hypertension, congenital
heart defects, aortic stenosis, atrial septal defect (ASD),
atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary
stenosis, subaortic stenosis, ventricular septal defect (VSD),
valve diseases, tuberous sclerosis, scleroderma, obesity,
transplantation, anemia, bleeding disorders, scleroderma, diabetes,
Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, fertility,
cirrhosis, inflammatory bowel disease, diverticular disease,
hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura,
immunodeficiencies, graft versus host disease, Alzheimer's disease,
stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease,
Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan
syndrome, multiple sclerosis, ataxia-telangiectasia,
leukodystrophies, behavioral disorders, addiction, anxiety, pain,
neuroprotection, multiple sclerosis, leukodystrophies,
neuroprotection, systemic lupus erythematosus, autoimmune disease,
asthma, emphysema, scleroderma, allergy, ARDS, autoimmune disease,
renal artery stenosis, interstitial nephritis, glomerulonephritis,
polycystic kidney disease, systemic lupus erythematosus, renal
tubular acidosis, IgA nephropathy Cholesteryl ester storage
disease; Corneal dystrophy, Thiel-Behnke type; Dubin-Johnson
syndrome; Leukemia, T-cell acute lymphocytic; Retinol binding
protein, deficiency of; SEMD, Pakistani type; Spinocerebellar
ataxia, infantile-onset, with sensory neuropathy; Split hand/foot
malformation, type 3; Tolbutamide poor metabolizer; Urofacial
syndrome; Warfarin sensitivity; Wolman disease, and/or other
pathologies/disorders. The NOV7 nucleic acid, 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.
[0233] NOV7 nucleic acids and polypeptides 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. These antibodies may be generated according to
methods known in the art, using prediction from hydrophobicity
charts, as described in the "Anti-NOVX Antibodies" section below.
For example the disclosed NOV7 protein have multiple hydrophilic
regions, each of which can be used as an immunogen. This novel
protein also has value in development of powerful assay system for
functional analysis of various human disorders, which will help in
understanding of pathology of the disease and development of new
drug targets for various disorders.
[0234] NOV8
[0235] A disclosed NOV8 nucleic acid of 430 nucleotides (also
referred to as AL163195_dal) encoding a novel RNase-like protein is
shown in Table 8A. An open reading frame was identified beginning
with an ATG initiation codon at nucleotides 16-18 and ending with a
TAA codon at nucleotides 408-410. A putative untranslated region
upstream from the initiation codon is underlined in Table 8A. The
start and stop codons are in bold letters. TABLE-US-00061 TABLE 8A
NOV8 nucleotide sequence (SEQ ID NO:31).
GGGGAATTCGCCCTTATGATATGTCTTCCACATTACTGACATTCAGAAGTTTACATTATAATGACCCCAAGG
GAAACAGTTCGGGTAATGACAAAGAGTGTTGCAATGACATGACAGTCTGGAGAAAAGTTTCAGAAGCAAACG
GATCGTGCAAGTGGAGCAATAACTTCATCCGCAGCTCCACAGAAGTGATGCGCAGGGTCCACAGGGCCCCCA
GCTGCAAGTTTGTACAGAATCCTGGCATAAGCTGCTGTGAGAGCCTAGAACTGGAAAATACAGTGTGCCAGT
TCACTACAGGCAAACAATTCCCCAGGTGCCAATACCATAOTGTTACCTCATTAGAGAAGATATTGACAGTGC
TGACAGGTCATTCTCTGATGAGCTGGTTAGTTTGTGGCTCTAAGTTGTAAATCCCACAGAGCTTTAGGAC
[0236] The NOV8 nucleic acid sequence is located on chromsome
14.
[0237] The disclosed NOV8 polypeptide (SEQ ID NO:32) encoded by SEQ
ID NO:31 has 129 amino acid residues and is presented in Table 8B
using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV8 has a signal peptide and is
likely to be localized to the microbody (peroxisome) with a
certainty of 0.8000. In other embodiments, NOV8 may also be
localized to the mitochondrial matrix space with a certainty of
0.1000, or the lysosome (lumen) with a certainty of 0.1000.
TABLE-US-00062 TABLE 8B Encoded NOV8 protein sequence (SEQ ID
NO:32). ##STR1## ##STR2##
[0238] NOV8 also has homology to the amino acid sequence shown in
the BLASTP data listed in Table 8C. TABLE-US-00063 TABLE 8C BLAST
results for NOV8 Gene Index/ Length Identity Positives Identifier
Protein/Organism (aa) (%) (%) Expect gi|17476497|ref| similar to
199 129/129 129/129 5e-67 XP_058653.1| RIBONUCLEASE (100%) (100%)
(XM_058653) PANCREATIC (RNASE 1) (RNASE A) >gi|133196|sp|P00673|
RIBONUCLEASE 124 33/116 55/116 0.006 RNP_BALAC PANCREATIC (RNASE
(28%) (46%) 1) (RNASE A)
[0239] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 8D.
[0240] Pancreatic ribonuclease (EC 3.1.27.5) is one of the
digestive enzymes secreted in abundance by the pancreas. Elliott et
al. (Cytogenet. Cell Genet. 42: 110-112, 1986) mapped the mouse
gene to chromosome 14 by Southern blot analysis of genomic DNA from
recombinant inbred strains of mice, using a probe isolated from a
pancreatic cDNA library with the rat cDNA. A polymorphic BamHI site
was used to demonstrate complete concordance of the Rib-1 locus
with Tcra and Np-2, encoding the alpha subunit of the T-cell
receptor (186880) and nucleoside phosphorylase (164050),
respectively. The assignment to mouse 14 and the close linkage to
the other 2 loci was confirmed by study of one of Snen's congenic
strains: the 3 loci went together. Elliott et al. (Cytdgenet. Cell
Genet. 42: 110-112, 1986) predicted that the homologous human gene
RIB1 is on chromosome 14.
[0241] Human pancreatic RNase is monomeric and is devoid of any
biologic activity other than its RNA degrading ability. Piccoli et
al. (Proc. Nat. Acad. Sci. 96: 7768-7773, 1999) engineered the
monomeric form into a dimeric protein with cytotoxic action on
mouse and human tumor cells, but lacking any appreciable toxicity
on human and mouse normal cells. The dimeric variant of human
pancreatic RNase selectively sensitized cells derived from a human
thyroid tumor to apoptotic death. Because of its selectivity for
tumor cells, and because of its human origin, this protein was
thought to represent an attractive tool for anticancer therapy.
[0242] The disclosed NOV8 nucleic acid of the invention encoding a
RNase-like protein includes the nucleic acid whose sequence is
provided in Table 8A, or a fragment thereof. The invention also
includes a mutant or variant nucleic acid any of whose bases may be
changed from the corresponding base shown in Table 8A while still
encoding a protein that maintains its RNase-like activities and
physiological functions, or a fragment of such a nucleic acid. The
invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, by way
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 10% percent of
the bases may be so changed.
[0243] The disclosed NOV8 protein of the invention includes the
RNase-like protein whose sequence is provided in Table 8B. The
invention also includes a mutant or variant protein any of whose
residues may be changed from the corresponding residue shown in
Table 2 while still encoding a protein that maintains its
RNase-like activities and physiological functions, or a functional
fragment thereof. In the mutant or variant protein, up to about 72
percent of the residues may be so changed.
[0244] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0245] The above defined information for this invention suggests
that this RNase-like protein (NOV8) may function as a member of a
"RNase family". Therefore, the NOV8 nucleic acids and proteins
identified here may be useful in potential therapeutic applications
implicated in (but not limited to) various pathologies and
disorders as indicated below. The potential therapeutic
applications for this invention include, but are not limited to:
protein therapeutic, small molecule drug target, antibody target
(therapeutic, diagnostic, drug targeting/cytotoxic antibody),
diagnostic and/or prognostic marker, gene therapy (gene
delivery/gene ablation), research tools, tissue regeneration in
vivo and in vitro of all tissues and cell types composing (but not
limited to) those defined here.
[0246] The NOV8 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in
Diabetes, Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Obesity,
Hyperthyroidism and Hypothyroidism and Cancers including, but no
limited to Thyroid and Pancreas, and/or other diseases or
pathologies.
[0247] NOV8 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV8 substances for use in therapeutic or diagnostic methods.
These antibodies may be generated according to methods known in the
art, using prediction from hydrophobicity charts, as described in
the "Anti-NOVX Antibodies" section below. The disclosed NOV8
protein has multiple hydrophilic regions, each of which can be used
as an immunogen. These novel proteins can be used in assay systems
for functional analysis of various human disorders, which will help
in understanding of pathology of the disease and development of new
drug targets for various disorders.
[0248] NOV9
[0249] A disclosed NOV9 nucleic acid of 1860 nucleotides (also
referred to as CG56069-01) encoding a novel Insulin like growth
factor binding protein-like protein is shown in Table 9A. An open
reading frame was identified beginning with an ATG initiation codon
at nucleotides 1-3 and ending with a TGA codon at nucleotides
1858-1860. The start and stop codons are in bold letters in Table
9A. TABLE-US-00064 TABLE 9A NOV9 nucleotide sequence (SEQ ID
NO:33).
ATGGACAGGCACTTGCTGTTGCCTGGTCTGCTCCTGTCCCTTCCTCTGACCGCAGGCTGGACCATCTCCAAT
AGTTTAGTGACTGAAGGCTCCCGGCTGTCTATGGTCTCCCGCTTCTTCCTGATTTGCCTCTTGGACTCCAGC
CTGCCTTTCCTCACCACATGCCTCTCAGTGATCAACTTGGTGCGGGCCTTGGAAACTGTGCTGCAGAACGTG
GAGGGTCTCTGTCAATCTGGTTCCACTTCTGCTCTGCCTCAGGATGCCTTCTCCCGCTTTCCTGGGCTCAAG
GCTGAAGCTGGCCAGTCCTGGAGCCTTCCAGGTCCTCAAGCTGGGGACTCTGAATCTGGACCACACAAAGAT
GAAGGCAGATGCACTGGTGGGACGGGGGCTGCAGAGATTGGATGCCCTGTGACACTCACTGACATGGCTGAG
CTGCCTGCCAGGATGGTTGCCCATTTTGAGCTTCAGGAGCTGAATTTGGGGATTAATCGGACAAGGCACATA
GCCCTGGAAGGCCTGGCTTCCTGTCACAGCCTGAAGAGCTCGGGTCTTCGGAGCAATGGCCTGATTGAGTTA
CCACGAGGTTTCCTGGCTGCCATGCCCAGGCTTCAGAGACTGAACCTGGCCAACAACCAACTGAGGAGCGCC
ATGTGGTGTATGAATGAGACAGGGTTTGTGTCAGGATTGTGGGCCCTGGATCTGTCCAAGAATAGGCTGTGT
ACCCTGTCCCCAGTCATCTTCTCCTGTTTGCCCCACCTGCGGGAGCTGCTACTTCAAGGGAACCAACTGGTT
TGCTTGAAAGACCAGGTATTCCAGGGCCTACAGAGGCTACAGACCTTGAACTTGGGCAATAATCCACTGGTA
ACCCTGGGTGAGGGCTGGCTGGCTCCTCTGCCTACACTGACCACCCAAAACCTGGTAGGTACTCACATGGTG
CTGAGCCCAACCTGGGGCTTCCGGGGCCCAGAAAGTCTGCACAGCTTGAGAATACAGTTTCCCTTTGGCCCT
GCGGGAGTAGCATTTTCCCTGCTCACAAGACTGACTAGCTTGGAGCTCCACGCAGTTTCAGGCATGAAGCAT
TGGAGGTTGTCTCCTAATGTCTTTCCAGTCTTGCAGATCCTGACTTTAAAGGGCTGGGGACTGCAGCTAGAG
ACCCAGAATATCTCCAAGATCTTCCCTGCCCTTCATCAACTCTCCCTGCTTGGCACTCCCGAAGCTCAAGTC
CTTGAAGGATGGGGAAACAGGCATAGCCCTAGGCCCTACTGCATCACGGGACTGCCCAGTCTACAGGAGCTG
AAGCTGCAGGCACTGCAGTCTCAAGCATGCCCCTGCCCAGTGCGGCTTGAGGAGCTGGTGGGGTTGGAGACA
CTGTCTGCTGCTGCTTTTGGGGGCCTCGGCAGTCTCCAGGTCTTAGTACTAGACAGGGAGAAAGACTTCATG
CTGGATGACAGCCTCCAGGAGCACAGTCCTCGGATGCCCCAGTACATCTATATTCTGACCTCATCCTTGGCC
TGCCAGTGTGCCAATGCCTGCCTCTGCCCTGCTGCTTCTGCTGGTCTCCTTGCCCTTCCTAAAGGAAGCCAG
GAATTCCTGGATCCTCTAACTCAAGGCCTTGCTCAGGGTTTGGTTCCAGAGTCTGAGGAGTCAGAAGGGCAA
GACCAGGGCTGGATGGTGCAGGAGCTGCTGCCTGCTCTAGAGGACTGCCCTCCAGCTGGCCGGGGGCTGCCA
CTCTGCCTCCATGAGTGGGATTTTGAGCCAGGCAAGGATGTGGCTGACAATGCAGCAGACAGCATGATTGGC
CTGGTTGCTCCGCTGAAGAGACTATTGCATGTGGCCCAAGGAAGAGGAAAGAAAGAATGA
[0250] The disclosed NOV9 nucleic acid sequence, localized to the q
12 region of chromosome 1, has 410 of 699 bases (58%) identical to
a gb:GENBANK-ID:MMU91967|acc:U91967.1 mRNA from Mus musculus (Mus
musculus platelet glycoprotein Ib-alpha gene, complete cds)
(E=0.031).
[0251] The disclosed NOV9 polypeptide (SEQ ID NO:34) encoded by SEQ
ID NO:33 has 619 amino acid residues is presented in Table 9B using
the one-letter amino acid code. Signal P, Psort and/or Hydropathy
results predict that NOV9 has a signal peptide and is likely to be
localized in the lysosome (lumen) with a certainty of 0.6400. In
other embodiments, NOV9 is predicted to be localized
extracellularly with a certainty of 0.5087, to the endoplasmic
reticulum (membrane) with a certainty of 0.1000, or to the
endoplasmic reticulum (lumen) with a certainty of 0.1000. The most
likely ceavage site for NOV9 is between positions 19 and 20,
TAG-WT. TABLE-US-00065 TABLE 9B Encoded NOV9 protein sequence (SEQ
ID NO:34).
MDRHLLLPGLLLSLPLTAGWTISNSLVTEGSRLSMVSRFFLICLLDSSLPFLTTCLSVINLVRALETVLQNV
EGLCQSGSTSALPQDAFSRFPGLKAEAGQSWSLPGPQAGDSESGPHKDEGRCTGGTGAAEIGCPVTLTDMAE
LPARMVAHEELQELNLGINRTRHIALEGLASCHSLKSSGLRSNGLIELPRGFLAAMPRLQRLNLANNQLRSA
MLCMNETGFVSGLWALDLSKNRLCTLSPVIFSCLPHLRELLLQGNQLVCLKDQVFQGLQRLQTLNLGNNPLV
TLGEGWLAPLPTLTTQNLVGTHMVLSPTWGFRGPESLHSLRIQFPFGPAGVAFSLLTRLTSLELHAVSGMKH
WRLSPNVFPVLQILTLKGWGLQLETQNISKIFPALHQLSLLGTPEAQVLEGWGNRHSPRPYCITGLPSLQEL
KLQALQSQACPCPVRLEELVGLETLSAAAFGGLGSLQVLVLDREKDFMLDDSLQEHSPRMPQYIYILTSSLA
CQCANACLCPAASAGLLALPKGSQEFLDFLTQGLAQGLVPESEESEGQDQGWMVQELLPALEDCPPAGRGLP
LCLHEWDFEPGKDVADNAADSMIGLVAPLKRLLHVAQGRGKKE
[0252] A BLASTX of NOV9 shows that it has 77 of 253 amino acid
residues (30%) identical to, and 114 of 253 amino acid residues
(45%) similar to, the 605 amino acid residue
ptnr:SWISSNEW-ACC:002833 protein from Papio hamadryas (Hamadryas
baboon) (Insulin-Like Growth Factor Binding Protein Complex Acid
Labile Chain Precursor (ALS)) (E=1.1e.sup.-9)
[0253] NOV9 is expressed in at least brain and liver. This
information was derived by determining the tissue sources of the
sequences that were included in the invention including but not
limited to SeqCalling sources, Public EST sources, Literature
sources, and/or RACE sources.
[0254] In addition, the sequence is predicted to be expressed in
B-cells and blood cells because of the expression pattern of
(GENBANK-ID: gb:GENBANK-ID:MMU91967|acc:U91967.1) a closely related
Mus musculus platelet glycoprotein Ib-alpha gene, complete cds
homolog.
[0255] The disclosed NOV9 polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 9C.
TABLE-US-00066 TABLE 9C BLAST results for NOV9 Gene Index/ Length
Identity Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi|5669800|gb|AAD46477.1| Toll-like 503 41/138 71/138 2e-07
AF113614_1 receptor 2 (29%) (50%) (AF113614) [Cricetulus griseus]
gi|6449037|gb|AAF08787.1| platelet 567 63/206 84/206 4e-07
(AF163101) glycoprotein V (30%) (40%) [Mus musculus]
gi|6680055|ref|NP_032174.1| glycoprotein V 567 63/206 84/206 6e-07
(NM_008148) (platelet); GP V (30%) (40%) [Mus musculus]
gi|6678754|ref|NP_032559.1| lymphocyte 661 72/269 112/269 1e-06
(NM_008533) antigen 78 [Mus (26%) (40%) musculus]
gi|112908|sp|P02750| LEUCINE-RICH 312 60/196 82/196 2e-06
A2GL_HUMAN ALPHA-2- (30%) (41%) GLYCOPROTEIN (LRG)
[0256] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 9D. In the
ClustalW alignment of the NOV9 protein, as well as all other
ClustalW analyses herein, the black outlined amino acid residues
indicate regions of conserved sequence (i.e., regions that may be
required to preserve structural or functional properties), whereas
non-highlighted amino acid residues are less conserved and can
potentially be altered to a much broader extent without altering
protein structure or function.
[0257] Table 9E lists the domain description from DOMAIN analysis
results against NOV9. This indicates that the NOV9 sequence has
properties similar to those of other proteins known to contain this
domain. TABLE-US-00067 TABLE 9E Domain Analysis of NOV9
gnl|Pfam|pfam01582, TIR, TIR domain. The TIR domain is an
intracellular signaling domain found in MyD88, interleukin 1
receptor and the Toll receptor. Called TIR (by SMART?) for
Toll-Interleukin-Resistance. (SEQ ID NO:109) CD-Length = 141
residues, 29.1% aligned Score = 38.9 bits (89), Expect = 9e-04
Query: 549 SEGQDQGWMVQELLPALEDCPPAGRGLPLCLHEWDPEPGKDVADN 593 Sbjct:
6 SGKDDRDTFVSHLLKELEE----KPGIKLFIDDRDELPGESILEN 46
[0258] The predicted sequence described here belongs to the
leucine-rich repeat protein family. It is homologous to insulin
like growth factor binding protein (IGFBP) and RP105, a novel B
cell surface molecule. It contains five leucine-rich repeat
domains. Leucine-rich repeats (LRRs) are relatively short motifs
(22-28 residues in length) found in a variety of cytoplasmic,
membrane and extracellular proteins (1). Although these proteins
are associated with widely different functions, a comunon property
involves protein-protein interaction. Other functions of
LRR-containing proteins include, for example, binding to enzymes
and vascular repair (1). LRRs form elongated non-globular
structures and are often flanked by cysteine rich domains. The
circulating insulin-like growth factors (IGF-I and -II) occur
largely as components of a 140 kDa protein complex with IGF binding
protein-3 and the acid-labile subunit (ALS). This ternary complex
regulates the metabolic effects of the serum IGFs by limiting their
access to tissue fluids. A cDNA for baboon ALS was isolated by
Delhanty and Baxter (2) and used to screen Northern blots of total
RNA from the lung, liver, kidney, adrenal, muscle, intestine, and
spleen of adult baboons. The expression of the single approximately
2.2 kb baboon ALS mRNA transcript was restricted to the liver,
suggesting that serum ALS levels are controlled by regulation of
hepatic expression of this peptide in primates (2).
[0259] The RP105 Ag is a murine B cell surface molecule that
transmits an activation signal into B cells or
dexamethasone-induced apoptosis, and to B cell proliferation. A
cDNA encoding the RP105 Ag was isolated by Miyake, et al (3). An
encoded protein is a type I transmembrane protein consisting of 641
amino acids in a mature form. Northern hybridization with a probe
specific for the cDNA clone detected a transcript with a size of
approximately 3 kb. The transcript was observed in spleen, but not
in thymus, kidney, muscle, heart, brain, or liver. Stable
transfection of the cDNA clone conferred the expression of the
RP105 Ag on a pro-B cell line, which was confirmed by
immunofluorescence staining and immunoprecipitation with anti-RP105
mAb. The RP105 molecule possesses 22 tandem repeats of a
leucine-rich motif. These repeated motifs are observed in members
of the leucine-rich repeat protein family, and have been implicated
in protein-protein interactions, such as cell adhesion or
receptor-ligand binding. Amino- and carboxyl-flanking regions that
are characteristically conserved among members of the family are
located on both sides of tandemly repeated leucine-rich motifs in
RP105 molecule. These results demonstrate that RP105 is a novel
member of the leucine-rich repeat protein family, and the first
member that is specifically expressed on B cells (3).
[0260] Because of the presence of the Leucine rich repeat domains
and the homology to the IGFBP and RP105, we anticipate that the
novel sequence described here will have useful properties and
functions similar to these genes.
[0261] References: 1. Artavanis-Tsakonas S., Goodman C. S.,
Rothberg J. M., Jacobs J. R. (1990) Slit: an extracellular protein
necessary for development of midline glia and commissural axon
pathways contains both EGF and LRR domains. Genes Dev. 4:
2169-2187. 2. Delhanty P, Baxter R C. (1992) The cloning and
expression of the baboon acid-labile subunit of the insulin-like
growth factor binding protein complex. Biochem Biophys Res Commun.
227 (3):897-902.3. Miyake K, Yamashita Y, Ogata M, Sudo T, Kimoto
M. (1995) RP105, a novel B cell surface molecule implicated in B
cell activation, is a member of the leucine-rich repeat protein
family. J Immunol. 154 (7):3333-40
[0262] The disclosed NOV9 nucleic acid of the invention encoding a
Insulin like growth factor binding protein-like protein includes
the nucleic acid whose sequence is provided in Table 9A, or a
fragment thereof. The invention also includes a mutant or variant
nucleic acid any of whose bases may be changed from the
corresponding base shown in Table 9A while still encoding a protein
that maintains its Insulin like growth factor binding protein-like
activities and physiological functions, or a fragment of such a
nucleic acid. The invention further includes nucleic acids whose
sequences are complementary to those just described, including
nucleic acid fragments that are complementary to any of the nucleic
acids just described. The invention additionally includes nucleic
acids or nucleic acid fragments, or complements thereto, whose
structures include chemical modifications. Such modifications
include, by way of nonlimiting example, modified bases, and nucleic
acids whose sugar phosphate backbones are modified or derivatized.
These modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject.
[0263] The disclosed NOV9 protein of the invention includes the
Insulin like growth factor binding protein-like protein whose
sequence is provided in Table 9B. The invention also includes a
mutant or variant protein any of whose residues may be changed from
the corresponding residue shown in Table 2 while still encoding a
protein that maintains its Insulin like growth factor binding
protein-like activities and physiological functions, or a
functional fragment thereof. In the mutant or variant protein, up
to about 74 percent of the residues may be so changed.
[0264] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0265] The above defined information for this invention suggests
that this Insulin like growth factor binding protein-like protein
(NOV9) may function as a member of a "Insulin like growth factor
binding protein family". Therefore, the NOV9 nucleic acids and
proteins identified here may be useful in potential therapeutic
applications implicated in (but not limited to) various pathologies
and disorders as indicated below. The potential therapeutic
applications for this invention include, but are not limited to:
protein therapeutic, small molecule drug target, antibody target
(therapeutic, diagnostic, drug targeting/cytotoxic antibody),
diagnostic and/or prognostic marker, gene therapy (gene
delivery/gene ablation), research tools, tissue regeneration in
vivo and in vitro of all tissues and cell types composing (but not
limited to) those defined here.
[0266] The NOV9 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in
diabetes, obesity, Von Hippel-Lindau (VHL) syndrome, Alzheimer's
disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's
disease, Huntington's disease, cerebral palsy, epilepsy,
Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia,
leukodystrophies, behavioral disorders, addiction, anxiety, pain,
neuroprotection, cirrhosis, transplantation, hemophilia,
hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume
disease, allergies, immunodeficiencies, graft versus host disease
(GVHD), lymphaedema, and other diseases, disorders and conditions
of the like.
[0267] NOV9 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV9 substances for use in therapeutic or diagnostic methods.
These antibodies may be generated according to methods known in the
art, using prediction from hydrophobicity charts, as described in
the "Anti-NOVX Antibodies" section below. The disclosed NOV9
protein has multiple hydrophilic regions, each of which can be used
as an immunogen. These novel proteins can be used in assay systems
for functional analysis of various human disorders, which will help
in understanding of pathology of the disease and development of new
drug targets for various disorders.
[0268] NOV10
[0269] A disclosed NOV10 nucleic acid of 4660 nucleotides (also
referred to as SC133419534_A) encoding a novel pregnancy zone
protein precursor-like protein is shown in Table 10A. An open
reading frame was identified beginning with an ATG initiation codon
at nucleotides 141-143 and ending with a TAA codon at nucleotides
45784580. A putative untranslated region upstream from the
initiation codon and downstream from the termination codon is
underlined in Table 10A. The start and stop codons are in bold
letters. TABLE-US-00068 TABLE 10A NOV10 nucleotide sequence (SEQ ID
NO:35).
AATTCTGAACTCTATTCAGAGGCCTTGGGGTGGGGCTTGTTTGTACATAGTGGCCACATAAAGCCTGGTAAA
CTCAGCTGGTCTCCAGGGCGAGGCTGAGGGCAGAGAGTTGGACACAACCCTGAGATTTATCCCTCACAATGC
GGAAAGACAGACTTCTTCATTTATGTCTTGTGCTACTTCTTATCCTGCTTTCTGCCAGTGACTCAAACTCTA
CAGAACCGCAGTATATGGTGCTGGTCCCCTCCCTGCTCCACACTGAGGCCCCTAAGAAGGGCTGTGTCCTTC
TGAGCCACCTGAATGAGACAGTGACTGTAAGTGCTTCCTTGGAGTCTGGCAGGGAAAACAGGAGCCTCTTCA
CTGACCTGGTGGCGGAGAAGGACTTATTCCACTGTGTCTCCTTCACTGTGCCAAGGATCTCAGCCTCTTCAG
AGGTGGCATTCCTTAGCATCCAGATAAAGGGGCCTACGCAAGATTTCAGGAAGAGGAACACAGTTCTGGTAC
TGAACACCCAAAGTCTGGTCTTTGTCCAGACAGACAAACCCATGTATAAACCAGGACAGACAGGTAAGGTAA
GATTCCGTGTTGTCTCCGTGGATGAAAATTTTCGCCCTCGAAATGAACTGGTAAGCCTTGTTTCCCTTCAGA
ACCCAAGAAGAAATCGAATTGCACAATGGCAGAGTCTCAAGCTAGAAGCTGGCATCAATCAGTTGTCCTTTC
CCCTCTCATCAGAGCCCATTCAGGGCTCCTACAGGGTGGTGGTACAGACAGAATCAGGTGGAAGGATACAGC
ACCCCTTCACCGTGGAGGAATTTGTGCTTCCCAAGTTTGAGGTCAAAGTTCAGGTGCCAAAGATAATCAGTA
TCATGGATGAAAAAGTGAACATAACAGTCTGTGGATGTTATAGGTACACATATGGAGAGCCTGTCCCTGGTC
TGGTGACACTTAGTGTATGCAGAAGATATTCACTATGCCGTTCCGACTGCCACAACACACATTCACAGCTTA
ACAGCAATGGCTGCATCACCCAACAAGTACACACCAAAATGCTCCAGATTACAAATACGGGCTTTGAAATGA
AGCTTAGAGTGGAAGCCAGGATCAGAGAAGAGGGGACAGGTGTGGAAGTCACTGCAAACAGGATCAGTGAAA
TCACAAACATTGTATCCAAACTCAAATTCGTGAAAGTGGATTCACACTTTAGACAAGGAATCCCCTTTTTTG
CACAGGTAAGACTGGTGGATGGAAAAGGTGTGCCCATCCCCAATAAACTCTTCTTCATCTCTGTGAATGACG
CCAATTATTACTCCAATGCAACCACCAATGAGCAGGGTCTTGCACAGTTTTCAATCAATACTACCAGTATCT
CGGTTAATAAACTTTTTGTCCGGGTAAGTTACAAAGAGAGTAACAATTGTTCTGATAACTGGTGGCTTGATG
AATTTCATACGCAAACATCTCATACTGCAAAGCATTTTTTTTCCCCAAGCAAGAGTTATATTCACCTCAAAC
CTATTATTGGTACTTTGACCTGTGGACAAACCCAGGAGATTCAAGCACACTACATTCTGAATAAACAGATTC
TCAGGGATGAAAAAGAATTAACCTTCTACTATTTGGTAAAAGCAAGAGGAAAAATCTCCCAATCAGGAATCC
ATGTGTTATCCATTGAACAAGGAAACAGTAAAGGCAGTTTTGCCTTATCCTTCCCTGTGGAGTCAGACGTTG
CCCCCATTGCACGAATGTTCATCTTTGCCATTTTACCAGATGGAGAAGTTGTTGGAGACTCTGAAAAATTTG
AGATTGAAAACTGTCTAGCCAACAAGGTGGATTTGAGCTTCAGCCCAGCACAAAGTCCCCCAGCCTCACATG
CCCACCTGCAAGTAGCAGCTGCTCCGCAGTCCCTCTGTGCCCTTCGTGCTGTGGACCAAAGTGTGCTGCTCA
TGAAGCCTGAGGCTGAGCTCTCTGTGTCCTCAGTGTATAATCTGCTAACTGTGAAGGATCTCACCAATTTTC
CTGACAATGTGGACCAGCAGGAGGAAGAACAAGGACACTGTCCCCGTCCTTTCTTCATTCATAATGGAGCCA
TCTATGTTCCCTTATCAAGTAATGAAGCAGATATTTATAGCTTCCTCAAGGGGATGGGATTGAAGGTGTTCA
CTAACTCAAAAATCCGAAAACCAAAGTCGTGTTCAGTCATCCCTTCCGTGTCTGCAGGAGCAGTAGGTCAAG
GATACTATGGAGCAGGTCTAGGAGTAGTAGAGAGACCATATGTTCCTCAATTAGGCACATATAATGTGATAC
CCTTAAATAATGAACAAAGTTCAGGGCCAGTCCCTGAAACGGTGCGAAGCTATTTTCCTGAGACTTGGATCT
GGGAGTTGGTGGCAGTGAGCTCATCAGGTGTGGCTGAGGTAGGAGTAACAGTCCCTGACACCATCACCGAGT
GGAAGGCAGGGGCCTTCTGCCTGTCCGAAGATGCTGGACTTGGTATCTCTTCCACTGCCTCTCTCCGAGCCT
TCCAGCCCTTCTTTGTGGAGCTCACAATGCCTTACTCTGTGATTCGTGGAGAGGTCTTCACACTCAAGGCCA
CGGTCCTAAACTACCTTCCCAAATGCATCCGGGTAGTTGTGCAGCTGGAGGTCTCTTCCGCTTTCCTGGCTG
TTCCAACAGAGAAGAATGAAGAATCTCACTGTGTCTGTAGAAATGGGCGGAAAACCGTGTCCTGGGTTGTGA
CTCCGAAGTCACTGGGTAATGTGAACTTCTCAGTGAGTGCAGAGGCAATGCAGTCCTTAGAACTCTGTGGAA
ATGAGGTTGTTGAGGTCCCTGAGATTAAAAGAAAAGACACAGTCATCAAAACCCTGTTGGTGGAGCCTGAAG
GAATAGCAAAGGAGGAAACTTTCAACACGCTGCCCTGTGCATCAGGTGCTAATGTGTCTGAGCAGTTGTCCT
TGAAGCTCCCATCAAATGTGGTCAAAGAATCTGCCAGAGCTTCTTTCTCAGTTCTGGGTGGTGACATATTAG
GTTCTGCTATGCAAAATATACAAAATCTCCTCCAGATGCCATATGGCTGTGGAGAACAGAACATGGTCCTAT
TTGCTCCTAACATCTATGTCTTGAACTATCTGAATGAAACCCAGCAGCTGACGCAGGAGATCAAGGCCAAGG
CCGTTGGCTATCTCATCACTGGTTACCAGAGACAGCTGAACTACAAACACCAAGATGGCTCCTACAGCACCT
TTGGGGAACGATATGGCAGGAACCAGGGCAACACTTGGCTCACAGCTTTTGTACTGAAGACTTTCGCCCAGG
CTCGATCCTACATCTTCATTGATGAAGCACACATTACCCAATCTCTCACGTGGCTCTCCCAGATGCAGAAGG
ACAATGGCTGTTTCAGGAGCTCTGGGTCACTGCTCAACAATGCCATAAAGGGAGGTGTAGAAGATGAAGCGA
CCCTCTCCGCCTATGTTACTATTGCCCTTCTGGAAATTCCTCTCCCAGTCACTAACCCTATTGTTCGCAATG
CCCTGTTCTGCCTGGAGTCAGCCTGGAATGTAGCAAAGGAGGGGACCCATGGGAGCCATGTCTACACCAAGG
CATTGCTGGCCTATGCTTTTTCCCTACTGGGAAAGCAAAATCAGAATAGAGAAATACTGAACTCACTTGATA
AGGAAGCTGTGAAAGACAACCTCGTCCATTGGGAGCGCCCTCAGAGACCCAAGGCACCAGTGGGGCATCTTT
ACCAAACCCAGGCTCCCTCTGCTGAGGTGGAGATGACATCCTATGTGCTCCTCGCTTATCTCACGGCCCAGC
CAGCCCCCACCTCAGGGGACCTGACCTCTGCAACTAACATTGTGAAGTGGATCATGAAGCAGCAGAACGCCC
AAGGTGGTTTCTCCTCCACCCAGGACACAGTGGTGGCTCTCCATGCCCTGTCCAGGTATGGAGCAGCCACTT
TCACCAGAACTGAGAAAACTGCACAGGTCACCGTTCAGGATTCACAGACCTTTTCTACAAATTTCCAAGTAG
ACAACAACAACCTCCTATTACTGCAGCAGATCTCATTGCCAGAGCTCCCTGGAGAATATGTCATAACAGTAA
CTGGGGAAAGATGTGTGTATCTTCAGACATCCATGAAATACAATATTCTTCCAGAGAAAGAGGACTCCCCAT
TTGCTTTAAAAGTGCAGACTGTGCCCCAAACTTGCGATGGACACAAAGCCCACACCAGCTTTCAGATCTCAC
TGACCATCAGTTACACAGGAAACCGTCCTGCTTCCAATATGGTGATTGTTGATGTAAAGATGGTATCTGGTT
TTATTCCCCTGAAACCAACAGTAAAAATGCTTGAAAGATCTAGCTCTGTGAGCCGGACAGAAGTGAGCAACA
ACCATGTCCTCATTTATGTGGAACAGGTGCTAACCCATCAAACCCTGCATTTTTCCTTCTTTGTGGAACAAG
ACATCCAAATAAAGAATTTAAAACCAGCTACAGTAAAAGCCTATGATTATTATGAGACATCAGATGAATTCA
CCTTTGAAGAATACAATGCCCCTTGCAGTGCTGGTAAAGTATAAATGATTCAATCTAATGCCACTTGAAAGA
AAATAAATAAGCATCTCAGTTAAACAGTAAAGTCTAATCCCAACTTCAAAAT
[0270] In a search of public sequence databases, the NOV10 nucleic
acid sequence, localized to chromosome 12, has 4170 of 4478 bases
(93%) identical to a gb:GENBANK-ID:HSPZHEP|acc:X54380 mRNA from
Homo sapiens (Human mRNA for pregnancy zone protein (E=0.0). Public
nucleotide databases include all GenBank databases and the GeneSeq
patent database.
[0271] The disclosed NOV10 polypeptide (SEQ ID NO:36) encoded by
SEQ ID NO:35 has 1479 amino acid residues and is presented in Table
10B using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV10 has no signal peptide and is
likely to be localized extracellularly with a certainty of 0.8200.
In other embodiments, NOV10 may also be localized to the lysosome
(lumen) with a certainty of 0.1900, the endoplasmic reticulum
(membrane) with a certainty of 0.1000, or in the endoplasmic
reticulum (lumen) with a certainty of 0.1000. The most likely
cleavage site for NOV10 is between positions 23 and 24: SDS-NS.
TABLE-US-00069 TABLE 10B Encoded NOV10 protein sequence (SEQ ID
NO:36).
MRKDRLLHLCLVLLLILLSASDSNSTEPQYMVLVPSLLETEAPKKGCVLLSHLWETVTVSASLESGRENESL
FTDLVAEKDLFHCVSFTVPRISASSEVAFLSIQIKGPTQDFRKRNTVLVLNTQSLVFVQTDKPMYKPGQTGK
VRFRVVSVDENFRPRNELVSLVSLQNPRRNRIAQWQSLKLEAGINQLSFPLSSEPIQGSYRVVVQTESGGRI
QHPFTVEEFVLPKFEVKVQVPKIISINDEKVNITVCGCYRYTYGEPVPGLVTLSVCRRYSLCRSDCHNTHSQ
LNSNGCITQQVHTKMLQITNTGFEMKLRVEARIREEGTGVEVTANRISEITNIVSKLKFVKVDSHFRQGIPF
FAQVRLVDGKGVPIPNKLFFISVNDANYYSNATTNEQGLAQFSINTTSISVNKLFVRVSYKESNNCSDNWWL
DEFHTQTSHTAKHFFSPSKSYIHLKPIIGTLTCGQTQEIQAHYILNKQILRDEKELTFYYLVKARGKISQSG
IHVLSIEQGNSKGSFALSFPVESDVAPIARMFIFAILPDGEVVGDSEKFEIENCLANKVDLSFSPAQSPPAS
HAHLQVAAAPQSLCALRAVDQSVLLMKPEAELSVSSVYNLLTVKDLTNFPDNVDQQEEEQGHCPRPFFIHNG
AIYVPLSSNEADIYSFLKGMGLKVFTNSKIRKPKSCSVTPSVSAGAVGQGYYGAGLGVVERPYVPQLGTYNV
IPLNNEQSSGPVPETVRSYFPETWIWELVAVSSSGVAEVGVTVPDTITEWKAGAFCLSEDAGLGISSTASLR
AFQPFFVELTMPYSVIRGEVFTLKATVLNYLPKCIRVVVQLEVSSAFLAVPTEKNEESHCVCRNGRKTVSWV
VTPKSLGNVNFSVSAEAMQSLELCGNEVVEVPEIKRKDTVIKTLLVEPEGIAKEETFNTLPCASGANVSEQL
SLKLPSNVVKESARASFSVLGGDILGSAMQNIQNLLQMPYGCGEQNMVLFAPNIYVLNYLNETQQLTQEIKA
KAVGYLITGYQRQLNYKHQDGSYSTFGERYGRNQGNTWLTAFVLKPFAQARSYIFIDEAHITQSLTWLSQMQ
KDNGCFRSSGSLLNNAIKGGVEDEATLSAYVTIALLEIPLPVTNPIVRNALFCLESAWNVAKEGTHGSEVYT
KALLAYAFSLLGKQNQNREILNSLDKEAVKDNLVHWERPQRPKAPVGHLYQTQAPSAEVEMTSYVLLAYLTA
QPAPTSGDLTSATNIVKWIMKQQNAQGGFSSTQDTVVALHALSRYGAATFTRTEKTAQVTVQDSQTFSTNEQ
VDNNNLLLLQQISLPELPGEYVITVTGERCVYLQTSMKYNILPEKEDSPFALKVQTVPQTCDGHKAHTSFQI
SLTISYTGNRPASNMVIVDVKMVSGFIPLKPTVKMLERSSSVSRTEVSNNHVLIYVEQVLTEQTLHFSFFVE
QDIQIKNLKPATVKAYDYYETSDEFTFEEYNAPCSAGKV
[0272] A search of sequence databases reveals that the NOV10 amino
acid sequence has 1348 of 1475 amino acid residues (91%) identical
to, and 1387 of 1475 amino acid residues (94%) similar to, the 1482
amino acid residue ptnr:SWISSPROT-ACC:P20742 protein from Homo
sapiens (Human) (Pregnancy Zone Protein Precursor (E=0.0). Public
amino acid databases include the GenBank databases, SwissProt, PDB
and PIR.
[0273] NOV10 is predicted to be expressed in late-pregnancy sera
because of the expression pattern of (GENBANK-ID:
gb:GENBANK-ID:HSPZHEP|acc:X54380) a closely related mRNA for
pregnancy zone protein homolog in species Homo sapiens.
[0274] The disclosed NOV10 polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 10C.
TABLE-US-00070 TABLE 10C BLAST results for NOV10 Gene Index/ Length
Identity Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi|13651966|ref|XP.sub.-- pregnancy-zone 1242 1063/1239 1097/1239
0.0 006924.3| protein [Homo (85%) (87%) (XM_006924) sapiens]
gi|6680608|ref|NP_031402.1| alpha-2- 1495 826/1479 1048/1479 0.0
(NM_007376) macroglobulin (55%) (70%) [Mus musculus]
gi|1171932|sp|P20740| OVOSTATIN 1473 612/1468 900/1468 0.0
OVOS_CHICK PRECURSOR (41%) (60%) (OVOMACROGLOBULIN)
gi|224053|prf||1009174A macroglobulin 1450 1012/1469 1164/1469 0.0
alpha2 [Homo (68%) (78%) sapiens] gi|6678964|ref|NP_032671.1|
murinoglobulin 1 1476 802/1466 1034/1466 0.0 [Mus musculus] (54%)
(69%) (NM_008645)
[0275] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 10D. In the
ClustalW alignment of the NOV10 protein, as well as all other
ClustalW analyses herein, the black outlined amino acid residues
indicate regions of conserved sequence (i.e., regions that may be
required to preserve structural or functional properties), whereas
non-highlighted amino acid residues are less conserved and can
potentially be altered to a much broader extent without altering
protein structure or function.
[0276] Tables 10E-10F lists the domain description from DOMAIN
analysis results against NOV10. This indicates that the NOV10
sequence has properties similar to those of other proteins known to
contain this domain. TABLE-US-00071 TABLE 10E Domain Analysis of
NOV10 gnl|Pfam|pfam00207, A2M, Alpha-2-macroglobulin family. This
family includes the C-terminal region of the alpha-2-macroglobulin
family. (SEQ ID NO:115) CD-Length = 751 residues, 99.9% aligned
Score = 785 bits (2028). Expect = 0.0 Query: 730
GPVPETVRSYFPETWIWELVAVSSSGVAEVGVTVPDTITEWKAGAFCLSEDAGLGISSTA 789
|+||||||+|+||+ | | | | +|+||+|| |+ | || || ++ Sbjct: 2
DEDDITIRSYFPESWLWEVEEVDRSPVLTVNITLPDSITTWEILAVSLSNTKGLCVADPV 61
Query: 790
SLRAFQPFFVELTMPYSVIRGEVFTLKATVLNYLP-KCIRVVVQLEVSSAFLAVPTEKNE 848 |
|| ||+|| +||||+||| |+| + |||| + |+||||||| + Sbjct: 62
ELTVFQDFFLELRLPYSVVRGEQVELRAVLYNYLPSQDIKVVVQLEVEP--------LCQ 113
Query: 849
ESHCVCRNGRKTVSWVVTPKSLGNVNFSVSAEAMQSLELCGNEVVEVPEIKRKDTVIKTL 908 |
| | | |||| +|+| | + | | ||| || |+||| Sbjct: 114
AGFCSLATQRTRSSQSVRPKSLSSVSFPVVVVPLASGLSLVEVVASVPEFFVKDAVVKTL 173
Query: 909
LVEPEGIAKEETFNTLPC-----ASGANVSEQLSLKLPSNVVKESARASFSVLGGDILGS 963
||||| |||| ++| | ||| +|||| +| | | || || + Sbjct: 174
KVEPEGARKEETVSSLLLPPEHLGGGLEVSEVPALKLPDDVPDTEAEAVTSVQ-GDPVAQ 232
Query: 964
AMQN------IQNLLQMPYGCGEQNMVLFAPNIYVLNYLNETQQLTQ---EIKAKAVGYL 1014
|+|| + |||++| |||||||+ || +|||+||+|| | + + | ||+ + Sbjct: 233
AIQNTLSGEGLNNLLRLPSGCGEQNMIYMAPTVYVLHYLDETWQWEKPGTKKKQKAIDLI 292
Query: 1015
ITGYQRQLNYKHQDGSYSTFGERYGRNQGNTWLTAFVLKTFAQARSYIFIDEAHITQSLT 1074
||||||||+ ||||+ | | +||||||||| |+|||+|+|||| || ++ Sbjct: 293
NKGYQRQLNYRKADGSYAAFLHR----ASSTWLTAFVLKVFSQARNYVFIDEEHICGAVK 348
Query: 1075
WLS-QMQKDNGCFRSSGSLLNNAIKGGVED----EATLSAYVTIALLEIPLPVTNPIVRN 1129
|| |||+| || || +++| +|||| | | ||+|++|||||| | +|+| | Sbjct: 349
WLILNQQKDDGVFRESGPVIHNEMKGGVGDDAEVEVTLTAFITIALLEAKLVCISPVVAN 408
Query: 1130
ALFCLESAWNVAKEGTHGSHVYTKALLAYAFSLLGKQNQNREILNSLDKEAVK-DNLVHW 1188
|| |+++ + + +| ||| || ||| +| | ++ +||| || +| | || Sbjct: 409
ALSILKASDYLLENYANGQRVYTLALTAYALALAGVLEKLKEILKSLKEELYKALVKGHW 468
Query: 1189
ERPQRPKAPVGHLYQTQAPSAEVEMTSYVLLAYLTAQPAPTSGDLTSATNIVKWIMKQQN 1248
||||+|| || | | +| |||||| ||| || | | | +|||+ +|| Sbjct: 469
ERPQKPKDAPGHPYSPQPQAAAVEMTSYALLALLTLLPFPKVE---MAPKVVKWLTEQQY 525
Query: 1249
AQGGFSSTQDTVVALHALSRYGAATFTRTEKTAQVTVQ-DSQTFSTNFQVDNNNLLLLQQ 1307
||| ||||||+|| |||+|| || | || ||+| | +| ++||+ ||| ||+ Sbjct: 526
YGGGFGSTQDTVMALQALSKYGIATPTHKEKNLSVTIQSPSGSFKSHFQILNNNAFLLRP 585
Query: 1308
ISLPE-LPGEYVITVTGERCVYLQTSMKYNILPEKEDSPFALKVQTVPQTCDGHK-AHTS 1365 +
|| |||+ + | |+ +| +| +| | ||++||| || | | | Sbjct: 586
VELPLNEGFTVTAKVTGQGTLTLVTTYRYKVLDKKNTFCFDLKIETVPDTCVEPKGAKNS 645
Query: 1366
FQISLTISYTGNRPASNMVIVDVKMVSGFIPLKPTVKML--ERSSSVSRTEVSNNHVLIY 1423
+|+ | |+| | | | |+ |++||||||| +| | ||+ |+ ||||+| Sbjct: 646
DYLSICTRYAGSRSDSGMAIADISMLTGFIPLKPDLRKLENGVDRYVSKYEIDGNHVLLY 705
Query: 1424 VEQVLTHQTLHFSFFVEQDIQIKNLKPATVKAYDYYETSDEFTFEEY 1470
+++| +| | + || ++ |+||+|| ||||| || | Sbjct: 706
LDKVSESETECVGFKIHQDFEVGLLQPASVKVYDYYEP-DEQCTAFY 751
[0277] TABLE-US-00072 TABLE 10F Domain Analysis of NOV10
gnl|Pfam|pfam01835, A2M_N, Alpha-2-macroglobulin family N-terminal
region. This family includes the N-terminal region of the alpha-2-
macroglobulin family. (SEQ ID NO:116) CD-Length = 620 residues,
98.4% aligned Score = 617 bits (1592), Expect = 1e-177 Query: 19
SASDSNSTEPQYMVLVPSLLHTEAPKKGCVLLSNLNETVTVSASLESGRENR---SLFTD 75 ||+
+|+|||+|||+| || |+| || | |||||||+ || | | |||| Sbjct: 11
LFFDSSLQKPRYMVIVPSILRTETPEKVCVQLHDLNETVTVTVSLHSFPGKRNLSSLFTV 70
Query: 76
LVAEKDLFHCVSFTVPRI----SASSEVAFLSIQIKGPTQDFRKRNTVLVLNTQSLVFVQ 131
|++ ||||||||||||+ |+ | +|+ +|+|||| |+++ |||| + + |||+| Sbjct: 71
LLSSKDLFNCVSFTVPQPGLFKSSKGEESFVVVQVKGPTHTFKEKVTVLVSSRRGLVFIQ 130
Query: 132
TDKPMYKPGQTGKVRFRVVSVDENFRPRNELVSLVSLQNPRHNRIAQWQSLKLEAGINQL 191
||||+| |||| ||+|| ||||| || |||+ || +++| ||+ ||+ ||| || || Sbjct:
131 TDKPIYTPGQT--VRYRVFSVDENLRPLNELI-LVYIEDPEGNRVDQWEVNKLEGGIFQL
187 Query: 192
SFPLSSEPIQGSYRVVVQTESGGR--IQHPFTVEEFVLPKFEVKVQVPKIISIMDEKVNI 249
|||+ ||||||++++| + ||| | | |+|+||| ||| + || | Sbjct: 188
SFPIPSEPIQGTWKIVARYESGPESNYTHYFEVKEYVLPSFEVSITPPKPFIYYDNFKEF 247
Query: 250
TVCGCYRYTYGEPVPGLVTLSVCRRYSLCRSD----CHNTHSQLNSNG--CITQQVNTKM 303 |
| |||||+||||+ + + + + |+ || |++|+| | Sbjct: 248
EVTICARYTYGKPVPGVAYVRFGVKDEDGKKELLAGLEERAKLLDGNGEICLSQEVLLKE 307
Query: 304
LQITNTGFEMK--LRVEARIREEGTGVEVTANRISEITNIVSKLKFVKVDSHFRQGIPFF 361
||+ | | | | | || +| +| |||||| |||+ ||||| Sbjct: 308
LQLKNEDLEGKSLYVAVAVIESEGGDMEEAELGGIKIVRSPYKLKFVKTPSNPKPGIPFF 367
Query: 362
AQVRLVDGKGVPIPNKLFFISVNDANYYSNATTNEQGLAQFSINTTSISVNKLFVRVSYK 421 +|
+|| | | || +| ||+|||| ||+| |||||||||+ || + || ++| Sbjct: 368
LKVLVVDPDGSPAPNVPVKVSAQDASYYSNGTTDEDGLAQPSINTSGISSLSITVRTNHK 427
Query: 422
ESNNCSDNWWLDEFHTQTSHTAKHFFSPSKSYIHLKPIIGTLTCGQTQEIQAHYILNKQI 481 |
+ | + || | ||||||| | || || ||++|| + Sbjct: 428
ELP------EEVQAHAEAQATAYSTVSLSKSYIHLS-IERTLPCGPGVGEQANFILRGKS 480
Query: 482
LRDEKELTFYYLVKARGKISQSGIHVLSIEQGNSKGSFALSFPVESDVAPIARMFIFAIL 541 |
+ | | ||||+ ++||| ++| || |+|| || |+|| |+ + || Sbjct: 481
LGELKILHFYYLIMSKGKIVKTGREPREPGQGL----FSLSIPVTPDLAPSFRLVAYYIL 536
Query: 542
PDGEVVGDSEKFEIENCLANKVDLSFSPAQS--PPASHAHLQVAAAPQSLCALRAVDQSV 599 |
|||| || ++|+| |||+||||||++ || |+| | |||| |||||||+| Sbjct: 537
PQGEVVADSVWIDVEDCCANKLDLSFSPSKDYRLPAQQVKLRVEADPQSLVALRAVDQAV 596
Query: 600 LLMKPEAELSVSSVYNLLTVKDLT 623 |+||+|+||+| ||+|| || Sbjct:
597 YLLKPKAKLSMSKVYDLLEKSDLG 620
[0278] Pregnancy zone protein (PZP), one of the major
pregnancy-associated plasma proteins (see 260100 for another
example), was described by Smithies (1959) who used
zone-electrophoresis in starch gels. PZP is a prominent constituent
of late-pregnancy sera. In healthy, nonpregnant females and in
males, PZP is present in trace amounts only: females, 10-30 mg/l;
males, less than 10 mg/l. During pregnancy, PZP levels may reach
1000-1400 mg/l just before term. Sottrup-Jensen et al. (1984)
showed that PZP closely resembles alpha-2-macroglobulin (103950) in
structure. Both have a quaternary structure of 2 covalently bound
180-kD subunits which are further noncovalently assembled into a
tetramer of 720 kD. Amino acid sequence of the 2 proteins are
extensively homologous. Marynen et al. (1989) used in situ
hybridization and somatic cell hybrid DNA analysis to demonstrate
that PZP, alpha-2-macroglobulin, and an alpha-2-macroglobulin
pseudogene mapped to human chromosome 12p13-p12.2. Although the
function of PZP in pregnancy is largely unknown, its close
structural relationship to alpha 2M suggests analogous proteinase
binding properties and a potential for being taken up in cells by
receptor-mediated endocytosis. I
[0279] The disclosed NOV10 nucleic acid of the invention encoding a
Pregnancy Zone Protein Precursor-like protein includes the nucleic
acid whose sequence is provided in Table 10A or a fragment thereof.
The invention also includes a mutant or variant nucleic acid any of
whose bases may be changed from the corresponding base shown in
Table 10A while still encoding a protein that maintains its
Pregnancy Zone Protein Precursor-like activities and physiological
functions, or a fragment of such a nucleic acid. The invention
further includes nucleic acids whose sequences are complementary to
those just described, including nucleic acid fragments that are
complementary to any of the nucleic acids just described. The
invention additionally includes nucleic acids or nucleic acid
fragments, or complements thereto, whose structures include
chemical modifications. Such modifications include, by way of
nonlimiting example, modified bases, and nucleic acids whose sugar
phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 7 percent of the
bases may be so changed.
[0280] The disclosed NOV10 protein of the invention includes the
Pregnancy Zone Protein Precursor-like protein whose sequence is
provided in Table 10B. The invention also includes a mutant or
variant protein any of whose residues may be changed from the
corresponding residue shown in Table 10B while still encoding a
protein that maintains its Pregnancy Zone Protein Precursor-like
activities and physiological functions, or a functional fragment
thereof. In the mutant or variant protein, up to about 46 percent
of the residues may be so changed.
[0281] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0282] The above defined information for this invention suggests
that this Pregnancy Zone Protein Precursor-like protein (NOV10) may
function as a member of a "Pregnancy Zone Protein Precursor
family". Therefore, the NOV10 nucleic acids and proteins identified
here may be useful in potential therapeutic applications implicated
in (but not limited to) various pathologies and disorders as
indicated below. The potential therapeutic applications for this
invention include, but are not limited to: protein therapeutic,
small molecule drug target, antibody target (therapeutic,
diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or
prognostic marker, gene therapy (gene delivery/gene ablation),
research tools, tissue regeneration in vivo and in vitro of all
tissues and cell types composing (but not limited to) those defined
here.
[0283] The NOV10 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in
pregnancy, hypertensive toxemia, pre-eclampsia/eclampsia
(gestational proteinuric hypertension), glomerular endotheliosis,
cholestasis, and pruritic urticarial papules and plaques of
pregnancy, and/or other pathologies and disorders.
[0284] NOV10 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV10 substances for use in therapeutic or diagnostic
methods. These antibodies may be generated according to methods
known in the art, using prediction from hydrophobicity charts, as
described in the "Anti-NOVX Antibodies" section below. The
disclosed NOV10 protein has multiple hydrophilic regions, each of
which can be used as an immunogen. These novel proteins can be used
in assay systems for functional analysis of various human
disorders, which will help in understanding of pathology of the
disease and development of new drug targets for various
disorders.
[0285] NOV11
[0286] A disclosed NOV11 nucleic acid of 2895 nucleotides (also
referred to as SC139725617_A) encoding a novel Transmembrane
Receptor UNC5H2-like protein is shown in Table 11A. An open reading
frame was identified beginning with an ATG initiation codon at
nucleotides 31-33 and ending with a TGA codon at nucleotides
2866-2868. A putative untranslated region upstream from the
initiation codon is underlined in Table 11A. The start and stop
codons are in bold letters. TABLE-US-00073 TABLE 11A NOV11
nucleotide sequence (SEQ ID NO:37).
CTCCAGGTCCGCCTGTCCCTGGAATGTTCTATGCAGGCCAGGGGTTATGGTAGAAATAGCGTCTTGTTGGCC
GGGCTCCTCTGCTGGACCCCTTACCCAGCCTTAGCAGGCACTGATTCTGGCAGCGAGGTGCTCCCTGACTCC
TTCCCGTCAGCGCCAGCAGAGCCGCTGCCCTACTTCCTGCAGGAGCCACAGGACGCCTACATTGTGAAGAAC
AAGCCTGTGGAGCTCCGCTGCCGCGCCTTCCCCGCCACACAGATCTACTTCAAGTGCAACGGCGAGTGGGTC
AGCCAGAACGACCACGTCACACAGGAAGGCCTGGATGAGGCCACCGGTCTGCGGGTGCGCGAGGTGCAGATC
GAGGTGTCGCGGCAGCAGGTGGAGGAGCTCTTTGGGCTGGAGGATTACTGGTGCCAGTGCGTGGCCTGGAGC
TCCGCGGGCACCACCAAGAGTCGCCGAGCCTACGTCCGCATCGCCTGTCTGCGCAAGAACTTCGATCAGGAG
CCTCTGGGCAAGGAGGTGCCCCTGGACCATGAGGTTCTCCTGCAGTGCCGCCCGCCGGAGGGOGTGCCTGTG
GCCGAGGTGGAATGGCTCAAGAATGAGGATGTCATCGACCCCACCCAGGACACCAACTTCCTGCTCACCATC
GACCACAACCTCATCATCCGCCAGGCCCGCCTGTCGGACACTGCCAACTATACCTGCGTGGCCAAGAACATC
GTGGCCAAACGCCGGAGCACCACTGCCACCGTCATCGTCTACGTGAATGGCGGCTGGTCCAGCTGGGCAGAG
TGGTCACCCTGCTCCAACCGCTGTGGCCGAGGCTGGCAGAAGCGCACCCGGACCTGCACCAACCCCGCTCCA
CTCAACGGAGGGGCCTTCTGCGAGGGCCAGGCATTCCAGAAGACCGCCTGCACCACCATCTGCCCAGTCGAT
GGGGCGTGGACGGAGTGGAGCAAGTGGTCAGCCTGCAGCACTGAGTGTGCCCACTGGCGTAGCCGCGAGTGC
ATGGCGCCCCCACCCCAGAACGGAGGCCGTGACTGCAGCGGGACGCTGCTCGACTCTAAGAACTGCACAGAT
GGGCTGTGCATGCAAAGTGAGTCACAGTGTGGTCCTCCTGTCCCCGCAGTGCTGGAGGCCTCAGGGGATGCG
GCGCTGTATGCGGOGCTCGTGGTGGCCATCTTCGTGGTCGTOGCAATCCTCATGGCGGTGGGGGTGGTGGTG
TACCGCCGCAACTGCCGTGACTTCGACACAGACATCACTGACTCATCTGCTGCCCTGACTGGTGGTTTCCAC
CCCGTCAACTTTAAGACGGCAAGGCCCAGTAACCCGCAGCTCCTACACCCCTCTGTGCCTCCTGACCTGACA
GCCAGCGCCGGCATCTACCGCGGACCCGTGTATGCCCTGCAGGACTCCACCGACAAAATCCCCATGACCAAC
TCTCCTCTGCTGGACCCCTTACCCAGCCTTAAGGTCAAGGTCTACAGCTCCAGCACCACGGGCTCTGGGCCA
GGCCTGGCAGATGGGGCTGACCTGCTGGGGGTCTTGCCGCCTGGCACATACCCTAGCGATTTCGCCCGGCAC
ACCCACTTCCTGCACCTGCGCAGCGCCAGCCTCGGTTCCCAGCAGCTCTTGGGCCTGCCCCGAGACCCAGGG
AGCAGCGTCAGCGGCACCTTTGGCTGCCTGGGTGGGAGGCTCAOCATCCCCGGCACAGGTGTCAGCTTGCTG
GTGCCCAATGGAGCCATTCCCCAGGGCAAGTTCTACGAGATGTATCTACTCATCAACAAGGCAGAAAGTACC
CTGCCGCTTTCAGAACGGACCCAGACAGTATTGAGCCCCTCGGTGACCTGTGGACCCACAGGCCTCCTGCTG
TGCCGCCCCGTCATCCTCACCATGCCCCACTGTGCCGAAGTCAGTGCCCGTGACTGGATCTTTCAGCTCAAG
ACCCAGGCCCACCAGGGCCACTGGGAGGAGGTGCTGACCCTGGATGAGGAGACCCTGAACACACCCTGCTAC
TGCCAGCTGGAGCCCAGCGCCTGTCACATCCTGCTGGACCAGCTGGGCACCTACGTGTTCACGGGCGAGTCC
TATTCCCGCTCAGCAGTCAAGCGGCTCCAGCTGGCCGTCTTCGCCCCCGCCCTCTGCACCTCCCTGGAGTAC
AGCCTCCGGGTCTACTGCCTGGAGGACACGCCTGTAGCACTGAAGGAGGTGCTGGAGCTGGAGCGGACTCTG
GGCGGATACTTGGTGGAGGAGCCGAAACCGCTAATGTTCAAGGACAGTTACCACAACCTGCGCCTCTCCCTC
CATGACCTCCCCCATGCCCATTGGAGGAGCAAGCTGCTGGCCAAATACCAGGAGATCCCCTTCTATCACATT
TGGAGTGGCAGCCAGAAGGCCCTCCACTGCACTTTCACCCTGGAGAGGCACAGCTTGGCCTCCACAGAGCTC
ACCTGCAAGATCTGCGTGCGGCAAGTGGAAGGGGAGGGCCAGATATTCCAGCTGCATACCACTCTGGCAGAG
ACACCTGCTGGCTCCCTGGACACTCTCTGCTCTGCCCCTGGCAGCACTGTCACCACCCAGCTGGGACCTTAT
GCCTTCAAGATCCCACTGTCCATCCGCCAGAAGATATGCAACAGCCTAGATGCCCCCAACTCACGGGGCAAT
GACTGGCGGATGTTAGCACAGAAGCTCTCTATGGACCGGTACCTGAATTACTTTGCCACCAAAGCGAGCCCC
ACGGGTGTGATCCTGGACCTCTGGGAAGCTCTGCAGCAGGACGATGGGGACCTCAACAGCCTGGCGAGTGCC
TTGGAGGAGATGGGCAAGAGTGAGATGCTGGTGGCTGTGGCCACCGACGGGGACTGCTGAGCCTCCTGGGAC
AGCGGGCTGGCAGGG
[0287] In a search of public sequence databases, the NOV11 nucleic
acid sequence, located on chromosome 10 has 2425 of 2811 bases
(86%) identical to a gb:GENBANK-ID:RNU87306|acc:U87306 mRNA from
Rattus norvegicus (Rattus norvegicus transmembrane receptor Unc5H2
mRNA, complete cds (E=0.0). Public nucleotide databases include all
GenBank databases and the GeneSeq patent database.
[0288] The disclosed NOV11 polypeptide (SEQ ID NO:38) encoded by
SEQ ID NO:37 has 945 amino acid residues and is presented in Table
11B using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV11 has a signal peptide and is
likely to be localized in the plasma membrane with a certainty of
0.4600. In other embodiments, NOV11 may also be localized to the
endoplasmic reticulum (membrane) with a certainty of 0.1000, the
endoplasmic reticulum (lumen) with a certainty of 0.1000, or
extracellularly with a certainty of 0.1000. The most likely
cleavage site for NOV10 is between positions 26 and 27: ALA-GT
TABLE-US-00074 TABLE 11B Encoded NOV11 protein sequence (SEQ ID
NO:38).
MQARGYGRNSVLLAGLLCWTPYPALAGTDSGSEVLPDSFPSAPAEPLPYFLQEPQDAYIVKNKPVELRCRAF
PAPQIYFKCNGEWVSQNDHVTQEGLDEATGLRVREVQIEVSRQQVEELFGLEDYWCQCVAWSSAGTTKSRRA
YVRIACLRKNFDQEPLGKEVPLDHEVLLQCRPPEGVPVAEVEWLKNEDVIDPTQDTNFLLTIDHNLIIRQAR
LSDTANYTCVAKNIVAKRRSTTATVIVYVNGGWSSWAEWSPCSNRCGRGWQKRTRTCTNPAPLNGGAFCEGQ
AFQKTACTTICPVDGAWTEWSKWSACSTECAHWRSRECMAPPPQNGGRDCSGTLLDSKNCTDGLCMQSESQC
GPPVPAVLEASGDAALYAGLVVAIFVVVAILMAVGVVVYRRNCRDFDTDITDSSAALTGGFHPVNFKTARPS
NPQLLHPSVPPDLTASAGIYRGPVYALQDSTDKIPMTNSPLLDPLPSLKVKVYSSSTTGSGPGLADGADLLG
VLPPGTYPSDFARDTHFLHLRSASLGSQQLLGLPRDPGSSVSGTFGCLGGRLSIPGTGVSLLVPNGAIPQGK
FYEMYLLINKAESTLPLSEGTQTVLSPSVTCGPTGLLLCRPVILTMPHCAEVSARDWIFQLKTQAHQGHWEE
VVTLDEETLNTPCYCQLEPRACHILLDQLGTYVFTGESYSRSAVKRLQLAVFAPALCTSLEYSLRVYCLEDT
PVALKEVLELERTLGGYLVEEPKPLMFKDSYHNLRLSLHDLPHAHWRSKLLAKYQEIPFYHIWSGSQKALHC
TFTLERHSLASTELTCKICVRQVEGEGQIFQLHTTLAETPAGSLDTLCSAPGSTVTTQLGPYAFKIPLSIRQ
KICNSLDAPNSRGNDWRMLAQKLSMDRYLNYFATKASPTGVILDLWEALQQDDGDLNSLASALEEMGKSEML
VAVATDGDC
[0289] A search of sequence databases reveals that the NOV11 amino
acid sequence has 855 of 945 amino acid residues (90%) identical
to, and 892 of 945 amino acid residues (94%) similar to, the 945
amino acid residue ptnr:SPTREMBL-ACC:008722 protein from Rattus
norvegicus (Rat) (transmembrane receptor UNC5H2 (E=9.7e.sup.-307).
Public amino acid databases include the GenBank databases,
SwissProt, PDB and PIR.
[0290] NOV11 is expressed in at least Epidermis. This information
was derived by determining the tissue sources of the sequences that
were included in the invention.
[0291] In addition, the sequence is predicted to be expressed in
brain because of the expression pattern of (GENBANK-ID: RNU87306) a
closely related {Rattus norvegicus transmembrane receptor Unc5H2
mRNA, complete cds homolog.
[0292] The disclosed NOV11 polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 11C.
TABLE-US-00075 TABLE 11C BLAST results for NOV11 Gene Index/ Length
Identity Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi|6678505|ref|NP_033498.1| UNC-5 homolog (C. 931 592/942 694/942
0.0 (NM_009472) elegans) 3 [Mus (62%) (72%) musculus]
gi|3789765|gb|AAC67491.1| transmembrane 931 581/942 690/942 0.0
(AF055634) receptor UNC5C (61%) (72%) [Homo sapiens]
gi|16933525|ref|NP.sub.-- unc5 (C. elegans 931 581/942 690/942 0.0
003719.2| homolog) c; (61%) (72%) (NM_003728) homolog of C. elegans
transmembrane receptor Unc5 [Homo sapiens]
gi|16159681|ref|XP.sub.-- unc5 (C. elegans 931 582/942 690/942 0.0
042940.3| homolog) c [Homo (61%) (72%) (XM_042940) sapiens]
gi|11559982|ref|NP.sub.-- transmembrane 945 815/945 848/945 0.0
071543.1| receptor Unc5H2 (86%) (89%) (NM_022207) [Rattus
norvegicus]
[0293] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 11D. In the
ClustalW alignment of the NOV11 protein, as well as all other
ClustalW analyses herein, the black outlined amino acid residues
indicate regions of conserved sequence (i.e., regions that may be
required to preserve structural or functional properties), whereas
non-highlighted amino acid residues are less conserved and can
potentially be altered to a much broader extent without altering
protein structure or function.
[0294] Tables 1E-M list the domain descriptions from DOMAIN
analysis results against NOV11. This indicates that the NOV11
sequence has properties similar to those of other proteins known to
contain this domain. TABLE-US-00076 TABLE 11E Domain Analysis of
NOV11 gnl|Smart|smart00218, ZU5, Domain present in ZO-1 and
Unc5-like netrin receptors; Domain of unknown function. (SEQ ID
NO:122) CD-Length = 104 residues, 100.0% aligned Score = 149 bits
(376), Expect = 7e-37 Query: 541
PGSSVSGTFGCLGGRLSIPGTGVSLLVPNGAIPQGKFYEMYLLINKAESTLPLSEGTQTV 600 |
||||| |||| | ||| |++| |||||| | ||+++ || | | +|+ Sbjct: 1
PSFLVSGTFDARGGRLRGPRTGVRLIIPPGAIPQGTRYTCYLVVHDKLSTPPPLEEGETL 60
Query: 601 LSPSVTCGPTGLLLCRPVILTMPHCAEVSARDWIFQLKTQAHQG 644 ||| |
||| | | ||||| +|||| + ||| | + | Sbjct: 61
LSPVVECGPHGALFLRPVILEVPECASLRPRDWEIVLLRSENGG 104
[0295] TABLE-US-00077 TABLE 11F Domain Analysis of NOV11
gnl|Pfam|pfam00791, ZU5, ZU5 domain. Domain present in ZO-1 and
Unc5- like netrin receptors Domain of unknown function. (SEQ ID
NO:123) CD-Length = 104 residues, 100.0% aligned Score = 147 bits
(371), Expect = 3e-36 Query: 541
PGSSVSGTFGCLGGRLSIPGTGVSLLVPNGAIPQGKFYEMYLLINKAESTLPLSEGTQTV 600 |
||||| |||| | ||| |++| |||||| | ||+++ || | | +|+ Sbjct: 1
SGFLVSGTFDARGGRLRGPRTGVRLIIPPGAIPQGTRYTCYLVVHDKLSTPPPLEEGETL 60
Query: 601 LSPSVTCGPTGLLLCRPVILTMPHCAEVSARDWIFQLKTQAHQG 644 ||| |
||| | | ||||| +|||| + ||| | + | Sbjct: 61
LSPVVECGPHGALFLRPVILEVPHCASLRPRDWELVLLRSENGG 104
[0296] TABLE-US-00078 TABLE 11G Domain Analysis of NOV11
gnl|Smart|smart00005, DEATH, DEATH domain, found in proteins
involved in cell death (apoptosis).; Alpha-helical domain present
in a variety of proteins with apoptotic functions. Some (but not
all) of these domains form homotypic and heterotypic dimers. (SEQ
ID NO:124) CD-Length = 96 residues, 99.0% aligned Score = 64.7 bits
(156), Expect = 2e-11 Query: 852
GPYAFKIPLSIRQKICNSLDAPNSRGNDWRMLAQKLSM-DRYLNYFATKAS-----PTGV 905 |
| + |+|+ || + |+||| ||+|| + + ++ |++ + Sbjct: 1
PPGAASLTELTREKLAKLLD--HDLGDDWRELARKLGLSEADIDQIETESPRDLAEQSYQ 58
Query: 906 ILDLWEALQQDDGDLNSLASALEEMGKSEMLVAVATD 942 +| ||| + + |
+| || +||+ + + + ++ Sbjct: 59 LLRLWEQREGKNATLGTLLEALRKMGRDDAVELLRSE
95
[0297] TABLE-US-00079 TABLE 11H Domain Analysis of NOV11
gnl|Smart|smart00209, TSP1, Thrombospondin type 1 repeats; Type 1
repeats in thrombospondin-1 bind and activate TGF-beta. (SEQ ID
NO:125) CD-Length = 51 residues, 100.0% aligned Score = 62.4 bits
(150). Expect = 1e-10 Query: 249
WSSWAEWSPCSNRCGRGWQKRTRTCTNPAPLNGGAFCEGQAFQKTACTT-ICP 300 |
|+|||||| || | | ||| | | ||| | | + || || Sbjct: 1
WGEWSEWSPCSVTCGGGVQTRTRCCNPPP--NGGGPCTGPDTETRACNEQPCP 51
[0298] TABLE-US-00080 TABLE 11I Domain Analysis of NOV11
gnl|Smart|smart00209, TSP1, Thrombospondin type 1 repeats; Type 1
repeats in thrombospondin-1 bind and activate TGF-beta. (SEQ ID
NO:125) CD-Length = 51 residues, 98.0% aligned Score = 49.3 bits
(116), Expect = 1e-06 Query: 305
WTEWSKWSACSTECAH-WRSRECMAPPPQNGGRDCSGTLLDSKNCTDGLC 353 | |||+|| ||
| ++| || ||| |+| +++ | + | Sbjct: 1
WGEWSEWSPCSVTCGGGVQTRTRCCNPPPNGGGPCTGPDTETRACNEQPC 50
[0299] TABLE-US-00081 TABLE 11J Domain Analysis of NOV11
gnl|Pfam|pfam00531, death, Death domain. (SEQ ID NO:126) CD-Length
= 83 residues, 98.8% aligned Score = 59.7 bits (143), Expect =
7e-10 Query: 864
QKICNSLDAPNSRGNDWRMLAQKLSM-DRYLNYFATKA----SPTGVILDLWEALQQDDG 918
+++| || | | ||| ||+|| + + ++ + ||| +||||| + Sbjct: 1
RELCKLLDDP--LGRDWRRLARKLGLSEEEIDQIEHENPRLASPTYQLLDLWEQRGGKNA 58
Query: 919 DLNSLASALEEMGKSEMLVAVATD 942 + +| || +||+ + + + + Sbjct:
59 TVGTLLEALRKMGRDDAVELLESA 82
[0300] TABLE-US-00082 TABLE 11K Domain Analysis of NOV11
gnl|Pfam|pfam00090, tsp_1, Thrombospondin type 1 domain. (SEQ ID
NO:127) CD-Length = 48 residues, 91.7% aligned Score = 49.3 bits
(116), Expect = 1e-06 Query: 250
SSWAEWSPCSNRCGRGWQKRTRTCTNPAPLNGGAFCEGQAFQKTACT 296 | |+|||||| ||+|
+ | ||| +|| || | | | + || Sbjct: 1
SPWSEWSPCSVTCGKGIRTRQRTCNSPA---GGKPCTGDAQETEACM 44
[0301] TABLE-US-00083 TABLE 11L Domain Analysis of NOV11
gnl|Pfam|pfam00090, tsp_1, Thrombospondin type 1 domain. (SEQ ID
NO:127) CD-Length = 48 residues, 100.0% aligned Score = 36.2 bits
(82), Expect = 0.009 Query: 306
TEWSKWSACSTEC---AHWRSRECMAPPPQNGGRDCSGTLLDSKNCTDGLC 353 + ||+|| ||
| | | | +| ||+ |+| +++ | | Sbjct: 1
SPWSEWSPCSVTCGKGIRTRQRTCNSPA---GGKPCTGDAQETEACMMDPC 48
[0302] TABLE-US-00084 TABLE 11M Domain Analysis of NOV11
gnl|Smart|smart00408, IGc2, Immunoglobulin C-2 Type (SEQ ID ID:128)
CD-Length = 63 residues, 87.3% aligned Score = 42.7 bits (99),
Expect = 9e=05 Query: 170
VLLQCRPPEGVPVAEVEWLKNEDVIDPTQDTNFLLTIDHNLIIRQARLSDTANYTCVAKN 229 |
| | | | || + |||+ + ++ ++ | |+ | |+ |||||+| Sbjct: 6
VTLTC-PASGDPVPNITWLKDGKPLPESR----VVASGSTLTIKNVSLEDSGLYTCVARN 60
[0303] PMID: 10908620, UI: 20370928 Netrin-1 promotes thalamic axon
growth and is required for proper development of the
thalamocortical projection. Braisted J E, Catalano S M, Stimac R,
Kennedy T E, Tessier-Lavigne M, Shatz C J, O'Leary D D Molecular
Neurobiology Laboratory, The Salk Institute, La Jolla, Calif.
92037, USA.
[0304] The thalamocortical axon (TCA) projection originates in
dorsal thalamus, conveys sensory input to the neocortex, and has a
critical role in cortical development. We show that the secreted
axon guidance molecule netrin-1 acts in vitro as an attractant and
growth promoter for dorsal thalamic axons and is required for the
proper development of the TCA projection in vivo. As TCAs approach
the hypothalamus, they turn laterally into the ventral
telencephalon and extend toward the cortex through a population of
netrin-1-expressing cells. DCC and neogenin, receptors implicated
in mediating the attractant effects of netrin-1, are expressed in
dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors
implicated in repulsion, are not. In vitro, dorsal thalamic axons
show biased growth toward a source of netrin-1, which can be
abolished by netrin-1-blocking antibodies. Netrin-1 also enhances
overall axon outgrowth from explants of dorsal thalamus. The biased
growth of dorsal thalamic axons toward the internal capsule zone of
ventral telencephalic explants is attenuated, but not
significantly, by netrin-1-blocking antibodies, suggesting that it
releases another attractant activity for TCAs in addition to
netrin-1. Analyses of netrin-1-/- mice reveal that the TCA
projection through the ventral telencephalon is disorganized, their
pathway is abnormally restricted, and fewer dorsal thalamic axons
reach cortex. These findings demonstrate that netrin-1 promotes the
growth of TCAs through the ventral telencephalon and cooperates
with other guidance cues to control their pathfinding from dorsal
thalamus to cortex.
[0305] PMID: 10366627, UI: 99296863 Netrin-3, a mouse homolog of
human NTN2L, is highly expressed in sensory ganglia and shows
differential binding to netrin receptors. Wang H, Copeland N G,
Gilbert D J, Jenkins N A, Tessier-Lavigne M Departments of Anatomy,
and Biochemistry and Biophysics, Howard Hughes Medical Institute,
University of California, San Francisco, Calif. 94143-0452,
USA.
[0306] The netrins comprise a small phylogenetically conserved
family of guidance cues important for guiding particular axonal
growth cones to their targets. Two netrin genes, netrin-1 and
netrin-2, have been described in chicken, but in mouse so far a
single netrin gene, an ortholog of chick netrin-1, has been
reported. We report the identification of a second mouse netrin
gene, which we name netrin-3. Netrin-3 does not appear to be the
ortholog of chick netrin-2 but is the ortholog of a recently
identified human netrin gene termed NTN2L ("netrin-2-like"), as
evidenced by a high degree of sequence conservation and by
chromosomal localization. Netrin-3 is expressed in sensory ganglia,
mesenchymal cells, and muscles during the time of peripheral nerve
development but is largely excluded from the CNS at early stages of
its development. The murine netrin-3 protein binds to netrin
receptors of the DCC (deleted in colorectal cancer) family [DCC and
neogenin] and the UNC5 family (UNC5H1, UNC5H2 and UNC5H3). Unlike
chick netrin-1, however, murine netrin-3 binds to DCC with lower
affinity than to the other four receptors. Consistent with this
finding, although murine netrin-3 can mimic the outgrowth-promoting
activity of netrin-1 on commissural axons, it has lower specific
activity than netrin-1. Thus, like netrin-1, netrin-3 may also
function in axon guidance during development but may function
predominantly in the development of the peripheral nervous system
and may act primarily through netrin receptors other than DCC.
[0307] PMID: 10399920, UI: 99325507 A ligand-gated association
between cytoplasmic domains of UNC5 and DCC family receptors
converts netrin-induced growth cone attraction to repulsion. Hong
K, Hinck L, Nishiyama M, Poo M M, Tessier-Lavigne M, Stein E
Department of Biology, University of California, San Diego, 92093,
USA.
[0308] Netrins are bifunctional: they attract some axons and repel
others. Netrin receptors of the Deleted in Colorectal Cancer (DCC)
family are implicated in attraction and those of the UNC5 family in
repulsion, but genetic evidence also suggests involvement of the
DCC protein UNC-40 in some cases of repulsion. To test whether
these proteins form a receptor complex for repulsion, we studied
the attractive responses of Xenopus spinal axons to netrin-1, which
are mediated by DCC. We show that attraction is converted to
repulsion by expression of UNC5 proteins in these cells, that this
repulsion requires DCC function, that the UNC5 cytoplasmic domain
is sufficient to effect the conversion, and that repulsion can be
initiated by netrin-1 binding to either UNC5 or DCC. The isolated
cytoplasmic domains of DCC and UNC5 proteins interact directly, but
this interaction is repressed in the context of the full-length
proteins. We provide evidence that netrin-1 triggers the formation
of a receptor complex of DCC and UNC5 proteins and simultaneously
derepresses the interaction between their cytoplasmic domains,
thereby converting DCC-mediated attraction to UNC5/DCC-mediated
repulsion.
[0309] PMID: 10341242, UI: 99274743 Floor plate and netrin-1 are
involved in the migration and survival of inferior olivary neurons.
Bloch-Gallego E, Ezan F, Tessier-Lavigne M, Sotelo C Institut
National de la Sante et de la Recherche Medicale U106, Hopital de
la Salpetriere, 75013 Paris, France.
[0310] During their circumferential migration, the nuclei of
inferior olivary neurons translocate within their axons until they
reach the floor plate where they stop, although their axons have
already crossed the midline to project to the contralateral
cerebellum. Signals released by the floor plate, including
netrin-1, have been implicated in promoting axonal growth and
chemoattraction during axonal pathfinding in different midline
crossing systems. In the present study, we report experiments that
strongly suggest that the floor plate could also be involved in the
migration of inferior olivary neurons. First, we show that the
pattern of expression of netrin receptors DCC (for deleted in
colorectal cancer), neogenin (a DCC-related protein), and members
of the Unc5 family in wild-type mice is consistent with a possible
role of netrins in directing the migration of precerebellar neurons
from the rhombic lips. Second, we have studied mice deficient in
netrin-1 production. In these mice, the number of inferior olivary
neurons is remarkably decreased. Some of them are located
ectopically along the migration stream, whereas the others are
located medioventrally and form an atrophic inferior olivary
complex: most subnuclei are missing. However, axons of the
remaining olivary cell bodies located in the vicinity of the floor
plate still succeed in entering their target, the cerebellum, but
they establish an ipsilateral projection instead of the normal
contralateral projection. In vitro experiments involving ablations
of the midline show a fusion of the two olivary masses normally
located on either side of the ventral midline, suggesting that the
floor plate may function as a specific stop signal for inferior
olivary neurons. These results establish a requirement for netrin-1
in the migration of inferior olivary neurons and suggest that it
may function as a specific guidance cue for the initial steps of
the migration from the rhombic lips and then later in the
development of the normal crossed projection of the inferior
olivary neurons. They also establish a requirement for netrin-1,
either directly or indirectly, for the survival of inferior olivary
neurons.
[0311] PMID: 9126742 Vertebrate homologues of C. elegans UNC-5 are
candidate netrin receptors. Leonardo E D, Hinck L, Masu M,
Keino-Masu K, Ackerman S L, Tessier-Lavigne M Howard Hughes Medical
Institute, Department of Anatomy, Programs in Cell and
Developmental Biology and Neuroscience, University of California,
San Francisco 94143-0452, USA.
[0312] In the developing nervous system, migrating cells and axons
are guided to their targets by cues in the extracellular
environment. The netrins are a family of phylogenetically conserved
guidance cues that can function as diffusible attractants and
repellents for different classes of cells and axons. In
vertebrates, insects and nematodes, members of the DCC subfamily of
the immunoglobulin superfamily have been implicated as receptors
that are involved in migration towards netrin sources. The
mechanisms that direct migration away from netrin sources (presumed
repulsions) are less well understood. In Caenorhabditis elegans,
the transmembrane protein UNC-5 (ref. 14) has been implicated in
these responses, as loss of unc-5 function causes migration defects
and ectopic expression of unc-5 in some neurons can redirect their
axons away from a netrin source. Whether UNC-5 is a netrin receptor
or simply an accessory to such a receptor has not, however, been
defined. We now report the identification of two vertebrate
homologues of UNC-5 which, with UNC-5 and the product of the mouse
rostral cerebellar malformation gene (rcm), define a new subfamily
of the immunoglobulin superfamily, and whose messenger RNAs show
prominent expression in various classes of differentiating neurons.
We provide evidence that these two UNC-5 homologues, as well as the
rcm gene product, are netrin-binding proteins, supporting the
hypothesis that UNC-5 and its relatives are netrin receptors.
[0313] The disclosed NOV11 nucleic acid of the invention encoding a
Transmembrane Receptor UNC5H2-like protein includes the nucleic
acid whose sequence is provided in Table 11A or a fragment thereof.
The invention also includes a mutant or variant nucleic acid any of
whose bases may be changed from the corresponding base shown in
Table 11A while still encoding a protein that maintains its
Transmembrane Receptor UNC5H2-like activities and physiological
functions, or a fragment of such a nucleic acid. The invention
further includes nucleic acids whose sequences are complementary to
those just described, including nucleic acid fragments that are
complementary to any of the nucleic acids just described. The
invention additionally includes nucleic acids or nucleic acid
fragments, or complements thereto, whose structures include
chemical modifications. Such modifications include, by way of
nonlimiting example, modified bases, and nucleic acids whose sugar
phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 14 percent of the
bases may be so changed.
[0314] The disclosed NOV11 protein of the invention includes the
Transmembrane Receptor UNC5H2-like protein whose sequence is
provided in Table 11B. The invention also includes a mutant or
variant protein any of whose residues may be changed from the
corresponding residue shown in Table 11B while still encoding a
protein that maintains its Transmembrane Receptor UNC5H2-like
activities and physiological functions, or a functional fragment
thereof. In the mutant or variant protein, up to about 39 percent
of the residues may be so changed.
[0315] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0316] The above defined information for this invention suggests
that this Transmembrane Receptor UNC5H2-like protein (NOV11) may
function as a member of a "Transmembrane Receptor UNC5H2 family".
Therefore, the NOV11 nucleic acids and proteins identified here may
be useful in potential therapeutic applications implicated in (but
not limited to) various pathologies and disorders as indicated
below. The potential therapeutic applications for this invention
include, but are not limited to: protein therapeutic, small
molecule drug target, antibody target (therapeutic, diagnostic,
drug targeting/cytotoxic antibody), diagnostic and/or prognostic
marker, gene therapy (gene delivery/gene ablation), research tools,
tissue regeneration in vivo and in vitro of all tissues and cell
types composing (but not limited to) those defined here.
[0317] The NOV11 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in various
diseases and pathologies.
[0318] NOV11 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV11 substances for use in therapeutic or diagnostic
methods. These antibodies may be generated according to methods
known in the art, using prediction from hydrophobicity charts, as
described in the "Anti-NOVX Antibodies" section below. The
disclosed NOV11 protein has multiple hydrophilic regions, each of
which can be used as an immunogen. These novel proteins can be used
in assay systems for functional analysis of various human
disorders, which will help in understanding of pathology of the
disease and development of new drug targets for various
disorders.
[0319] NOV12
[0320] A disclosed NOV12 nucleic acid of 192 nucleotides (also
referred to as Curagen Accession No. SC134999661_A) encoding a
novel Thymosin-like protein is shown in Table 12A. An open reading
frame was identified beginning with an ATG initiation codon at
nucleotides 31-33 and ending with a TAA codon at nucleotides
175-177. Putative untranslated regions upstream from the initiation
codon and downstream of the termination codon are underlined in
Table 12A. The start and stop codons are in bold letters.
TABLE-US-00085 TABLE 12A NOV12 nucleotide sequence (SEQ ID NO:39).
TTTTTCTTTTCAGGCTTTCTTCTAGTCAAGATGAGTGATAAACCAGACTTGTCGGAAGTGGAGAAGTTTGAC
AGGTCAAAACTGAAGAAAACTAATACTGAAGAAAAAAATACTCTTCCCTCAAAGGAAAGTAAGTCATGTGGG
GTTCTACTGGAAACAAACAATAGAGGAAGTTAATAGGTTCAGTAAATA
[0321] In a search of public sequence databases, the NOV12 nucleic
acid sequence, localized to the X chromosome, has 192 of 192 bases
(100%) identical to a gb:GENBANK-ID:HSV362H12|acc:Z70227 mRNA from
Homo sapiens (E=2.8e.sup.-36). Public nucleotide databases include
all GenBank databases and the GeneSeq patent database.
[0322] The disclosed NOV12 polypeptide (SEQ ID NO:40) encoded by
SEQ ID NO:39 has 48 amino acid residues and is presented in Table
12B using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV12 has no signal peptide and is
likely to be localized to the nucleus with a certainty of 0.5392.
In other embodiments, NOV12 may also be localized to the microbody
(peroxisome) with acertainty of 0.3000, the mitochondrial membrane
space with a certainty of 0.1000, or to the lysosome (lumen) with a
certainty of 0.1000. TABLE-US-00086 TABLE 12B Encoded NOV12 protein
sequence (SEQ ID NO:40).
MSDKPDLSEVEKFDRSKLKKTNTEEKNTLPSKESKSCGVLLETNNRGS
[0323] A search of sequence databases reveals that the NOV12 amino
acid sequence has 33 of 34 amino acid residues (97%) identical to,
and 34 of 34 amino acid residues (100%) similar to, the 45 amino
acid residue ptnr:pir-id:JC5274 protein from human thymosin beta
(E=1.6e.sup.-12). Public amino acid databases include the GenBank
databases, SwissProt, PDB and PIR.
[0324] NOV12 is expressed in at least the following tissues: Brain,
Foreskin, Heart, Kidney, Lung, Mammary gland/Breast, Muscle,
Parathyroid Gland, Peripheral Blood, Prostate, Testis, Uterus. This
information was derived by determining the tissue sources of the
sequences that were included in the invention. In addition, the
sequence is predicted to be expressed in Brain, Breast, and
Prostate because of the expression pattern of (GENBANK-ID:
HSV362H12) a closely related {Human DNA sequence from cosmid
V362H12, between markers DXS366 and DXS87 on chromosome X
homolog.
[0325] The disclosed NOV12a polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 12C.
TABLE-US-00087 TABLE 12C BLAST result for NOV12a Gene Index/ Length
Identity Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi|2143995| thymosin beta-4 56 16/34 21/34 6.4 pir||I52084
precursor - rat (47%) (61%) (fragment)
[0326] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 12D. In the
ClustalW alignment of the NOV12 protein, as well as all other
ClustalW analyses herein, the black outlined amino acid residues
indicate regions of conserved sequence (i.e., regions that may be
required to preserve structural or functional properties), whereas
non-highlighted amino acid residues are less conserved and can
potentially be altered to a much broader extent without altering
protein structure or function.
[0327] Thymosin beta-4 is a small polypeptide whose exact
physiological role is not yet known [1]. It was first isolated as a
thymic hormone that induces terminal deoxynucleotidyltransferase.
It is found in high quantity in thymus and spleen but is widely
distributed in many tissues. It has also been shown to bind to
actin monomers and thus to inhibit actin polymerization [2].cndot.
function: exact physiological role is not yet known thymic hormone
that induces terminal deoxynucleotidyltransferase can bind to actin
monomers and thus to inhibit actin polymerization. .cndot.
function: hematopoietic system regulatory peptide has inhibitory
activity on the proliferation of hematopoeitic pluripotent stem
cells. .cndot. subcellular location: cytoplasmic. .cndot. tissue
specificity: originally found in thymus but it is widely
distributed in many tissues. .cndot. induction: by
alpha-interferon, nerve and fibroblast growth factors. .cndot.
similarity: belongs to the thymosin beta family. Blocks protein
family: BL00500 Thymosin beta-4 family proteins.
[0328] PMID: 2325669, UI: 90220652 Thymosin beta 4 is expressed in
ROS 17/2.8 osteosarcoma cells in a regulated manner. Atkinson M J,
Freeman M W, Kronenberg H M Endocrine Unit, Massachusetts General
Hospital, Boston.
[0329] The differential expression of mRNAs between the closely
related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was
used to identify genes whose expression is associated with the
osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS
17/2.8 complimentary DAN library on the basis of its differential
hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and
ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta
4, hither to a putative immunodulatory hormone, was indeed
differentially expressed. Steady state mRNA levels were several
fold higher in ROS 17/2.8 cells exhibiting an osteoblast-like
phenotype, compared with the less osteoblast-like ROS 25/1.
Thymosin beta 4 transcripts were also detected in rat UMR 106
osteosarcoma cells and in intact neonatal and fetal rat calvaria.
Sequence analysis of the cDNA indicated that thymosin beta 4
transcripts may arise by processing at a more distal
polyadenylation signal. Treatment of ROS 17/2.8 cells with
dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3
decreased thymosin beta 4 mRNA. The phenotype-dependent expression
in the ROS cells and the response to steroid hormone suggest that
thymosin beta 4 expression contributes to the osteoblast
phenotype.
[0330] PMID: 10777749, UI: 20241883 De La Cruz E M, Ostap E M,
Brundage R A, Reddy K S, Sweeney H L, Safer D Pennsylvania Muscle
Institute and Department of Physiology, University of Pennsylvania
School of Medicine, Philadelphia, Pa. 19104 USA.
enriquem@mail.med.upenn.edu
[0331] Thymosin-beta(4) (Theta(4)) binds actin monomers
stoichiometrically and maintains the bulk of the actin monomer pool
in metazoan cells. Theta(4) binding quenches the fluorescence of
N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)
conjugated to Cys(374) of actin monomers. The K(d) of the
actin-Tbeta(4) complex depends on the cation and nucleotide bound
to actin but is not affected by the AEDANS probe. The different
stabilities are determined primarily by the rates of dissociation.
At 25 degrees C., the free energy of Theta(4) binding MgATP-actin
is primarily enthalpic in origin but entropic for CaATP-actin.
Binding is coupled to the dissociation of bound water molecules,
which is greater for CaATP-actin than MgATP-actin monomers.
Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on
subdomain 2 is >12 times faster when Theta(4) is bound. The C
terminus of Theta(4) contacts actin near this cleavage site, at
His(40). By tritium exchange, Tbeta(4) slows the exchange rate of
approximately eight rapidly exchanging amide protons on actin. We
conclude that Theta(4) changes the conformation and structural
dynamics ("breathing") of actin monomers. The conformational change
may reflect the unique ability of Tbeta(4) to sequester actin
monomers and inhibit nucleotide exchange.
[0332] PMID: 10581087, UI: 20048164 Young J D, Lawrence A J,
MacLean A G, Leung B P, McInnes I B, Canas B, Pappin D J, Stevenson
R D Division of Infection and Immunity, Institute of Biomedical and
Life Sciences, University of Glasgow, Glasgow, G128QQ, UK.
[0333] The possibility that glucocorticoids upregulate the
expression of anti-inflammatory mediators is an exciting prospect
for therapy in inflammatory diseases, because these molecules could
give the therapeutic benefits of steroids without toxic side
effects. Supernatants from monocytes and macrophages cultured in
the presence of glucocorticoids increase the dispersion of
neutrophils from a cell pellet in the capillary tube migration
assay. This supernatant factor, unlike other neutrophil agonists,
promotes dispersive locomotion of neutrophils at uniform
concentration, lowers their adhesion to endothelial cells, inhibits
their chemotactic response to fMLP and induces distinctive
morphological changes. Here we show that thymosin beta4 sulfoxide
is generated by monocytes in the presence of glucocorticoids and
acts as a signal to inhibit an inflammatory response. In vitro,
thymosin beta4 sulfoxide inhibited neutrophil chemotaxis, and in
vivo, the oxidized peptide, but not the native form, was a potent
inhibitor of carrageenin-induced edema in the mouse paw. Thymosin
beta4 is unique, because oxidation attenuates its intracellular
G-actin sequestering activity, but greatly enhances its
extracellular signaling properties. This description of methionine
oxidation conferring extracellular function on a cytosolic protein
may have far-reaching implications for future strategies of
anti-inflammatory therapy.
[0334] PMID: 10469335, UI: 99398473 Malinda K M, Sidhu G S, Mani H,
Banaudha K, Maheshwari R K, Goldstein A L, Kleinman H K
Craniofacial Developmental Biology and Regeneration Branch,
National Institute of Dental and Craniofacial Research, NIH,
Bethesda, Md. 20892-4370, USA.
[0335] Angiogenesis is an essential step in the repair process that
occurs after injury. In this study, we investigated whether the
angiogenic thymic peptide thymosin beta4 (Tbeta4) enhanced wound
healing in a rat full thickness wound model. Addition of Theta4
topically or intraperitoneally increased reepithelialization by 42%
over saline controls at 4 d and by as much as 61% at 7 d
post-wounding. Treated wounds also contracted at least 11% more
than controls by day 7. Increased collagen deposition and
angiogenesis were observed in the treated wounds. We also found
that Theta4 stimulated keratinocyte migration in the Boyden chamber
assay. After 4-5 h, migration was stimulated 2-3-fold over
migration with medium alone when as little as 10 pg of Theta4 was
added to the assay. These results suggest that Tbeta4 is a potent
wound healing factor with multiple activities that may be useful in
the clinic.
[0336] The disclosed NOV12 nucleic acid of the invention encoding a
Thymosin-like protein includes the nucleic acid whose sequence is
provided in Table 12A or a fragment thereof. The invention also
includes a mutant or variant nucleic acid any of whose bases may be
changed from the corresponding base shown in Table 12A while still
encoding a protein that maintains its Thymosin-like activities and
physiological functions, or a fragment of such a nucleic acid. The
invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, by way
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 10 percent of the
bases may be so changed.
[0337] The disclosed NOV12 protein of the invention includes the
Thymosin-like protein whose sequence is provided in Table. 12B. The
invention also includes a mutant or variant protein any of whose
residues may be changed from the corresponding residue shown in
Table 12B while still encoding a protein that maintains its
Thymosin-like activities and physiological functions, or a
functional fragment thereof. In the mutant or variant protein, up
to about 53 percent of the residues may be so changed.
[0338] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0339] The above defined information for this invention suggests
that this Thymosin-like protein (NOV12) may function as a member of
a "Thymosin family". Therefore, the NOV12 nucleic acids and
proteins identified here may be useful in potential therapeutic
applications implicated in (but not limited to) various pathologies
and disorders. The potential therapeutic applications for this
invention include, but are not limited to: protein therapeutic,
small molecule drug target, antibody target (therapeutic,
diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or
prognostic marker, gene therapy (gene delivery/gene ablation),
research tools, tissue regeneration in vivo and in vitro of all
tissues and cell types composing (but not limited to) those defined
here.
[0340] The NOV12 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in various
pathologies and disorders.
[0341] NOV12 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV12 substances for use in therapeutic or diagnostic
methods. These antibodies may be generated according to methods
known in the art, using prediction from hydrophobicity charts, as
described in the "Anti-NOVX Antibodies" section below. These novel
proteins can be used in assay systems for functional analysis of
various human disorders, which will help in understanding of
pathology of the disease and development of new drug targets for
various disorders.
[0342] NOV13
[0343] A disclosed NOV13 nucleic acid of 594 nucleotides (also
referred to as Curagen Accession No. AC025256_da7) encoding a novel
neuromodulin-like protein is shown in Table 13A. An open reading
frame was identified beginning with an ATG initiation codon at
nucleotides 193-195 and ending with a TAA codon at nucleotides
535-537. The untranslated regions are underlined and the start and
stop codons are in bold letters in Table 13A. TABLE-US-00088 TABLE
13A NOV13 nucleotide sequence (SEQ ID NO:41).
CTGGGTTTTGGCGGCCGATCAGGCGCAGCCGGTGTACCTGCGTGACCAGGTCGCCACGCCGAAAGCACCGCC
GGCATGACAACTGGTCGGATGCTTGGCGTCAGGATTCCTTCGGCATTTGCCAAGGACGCAGCCCGCCGCTAC
ATTGCCAGCACTGTCCCTTGTTCAGCCGAGCCCACCGAGCAGTCCTAGATGCGGATCGACGGTTACCTACCT
TCCTACTCGCCAGATCGTGGCCCCCGTTCGGGGACTGCGGTCACGCCCTATCGAGAGGCGCAGCGGGAGGTC
GAGGCTCAGCGTGAACAGCCGGCTGCCCCAGCCAGCAGCCAGGGGCTGGAGCAGGCGCCGCAGATTCGCCGC
GTGCAGGCCAGCAGCAGTAACACCGATAGCCTGCCGACCCGCTCGCAGGACCTCGGTTATCAACAACCTACG
TTGAGCAACCGTGCCGCTCAGGCGTTGGCCAGCTACAGCACCACCGCCGCTTACGCCAGCGAGTACGATGCG
CAGGAAGTGCTCGGCCTCGATCTCTACGCGTAACCCCGTTTCACGGCGTGGGTCAGCCCCTCAGCTGGACCG
TCGCATAGATCGATGAGC
[0344] In a search of public sequence databases, the NOV13 nucleic
acid sequence, located on the q13 region of chromosome 12, has 126
of 204 bases (61%) identical to a
gb:GENBANK-ID:AF072132|acc:AF072132.1 mRNA from Pseudomonas
aeruginosa Hypothetical 12.1 Kda Protein (E=0.0073). Public
nucleotide databases include all GenBank databases and the GeneSeq
patent database.
[0345] The disclosed NOV13 polypeptide (SEQ ID NO 42) encoded by
SEQ ID NO:41 has 114 amino acid residues and is presented in Table
13B using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV13 has no signal peptide and is
likely to be localized in the cytoplasm with a certainty of 0.6500.
TABLE-US-00089 TABLE 13B Encoded NOV13 protein sequence (SEQ ID
NO:42).
MRIDGYLPSYSPDRGPRSGTAVTPYREAQREVEAQREQPAAPASSQGLEQAPQIRRVQASSSNTDSLPTRSQ
DLGYQQPTLSNRAAQALASYSTTAAYASEYDAQEVLGLDLYA
[0346] A search of sequence databases reveals that the NOV13 amino
acid sequence has 68 of 115 amino acid residues (59%) identical to,
and 87 of 115 amino acid residues (75%) similar to, the 115 amino
acid residue ptnr:TREMBLNEW-ACC: AAG07072 protein from Pseudomonas
aeruginosa Hypothetical 12.1 KDA Protein (E=1.1e.sup.-27). Public
amino acid databases include the GenBank databases, SwissProt, PDB
and PIR.
[0347] Neuromodulin (GAP-43), neurogranin (RC3), and PEP-19 are
small acid-stable proteins that bind calcium-poor calmodulin
through a loosely conserved IQ-motif. Even though these proteins
have been known for many years, much about their function in cells
is not understood. It has recently become appreciated that
calmodulin activity in cells is tightly controlled and that pools
of otherwise free calmodulin are sequestered so as to restrict its
availability for activating calcium/calmodulin-dependent enzymes.
Neuromodulin, neurogranin, and PEP-19 appear to be major
participants in this type of regulation. One way in which they do
this is by providing localized increases in the concentration of
calmodulin in cells so that the maximal level of target activation
is increased. Additionally, they can function as calmodulin
antagonists by directly inhibiting the association of
calcium/calmodulin with enzymes and other proteins. Although
neuromodulin, neurogranin, and PEP-19 were early representatives of
the small IQ-motif-containing protein family, newer examples have
come to light that expand the number of cellular systems through
which the IQ-peptide/calmodulin interaction could regulate
biological processes including gene transcription. It is the
purpose of this review to examine the behavior of neuromodulin,
neurogranin, and PEP-19 in paradigms that include both in vitro and
in situ systems in order to summarize possible biological
consequences that are linked to the expression of this type of
protein. The use of protein:protein interaction chromatography is
also examined in the recovery of a new calmodulin-binding peptide,
CAP-19 (ratMBF1). Consistent with earlier predictions, at least one
function of small IQ-motif proteins appears to be that they lessen
the extent to which calcium-calmodulin-dependent enzymes become or
stay activated. It also appears that these polypeptides can
function to selectively inhibit activation of intracellular targets
by some agonists while simultaneously permitting activation of
these same targets by other agonists. Much of the mechanism for how
this occurs is unknown, and possible explanations are examined. One
of the biological consequences for a cell that expresses a
calmodulin-regulatory protein could be an increased resistance to
calcium-mediated toxicity. This possibility is examined for cells
expressing PEP-19 and both anatomical and cell-biological data is
described. The study of IQ-motif-containing small proteins has
stimulated considerable thought as to how calcium signaling is
refined in neurons. Current evidence suggests that signaling
through calmodulin is not a fulminating and homogenous process but
a spatially limited and highly regulated one. Data from studies on
neuromodulin, neurogranin, and PEP-19 suggest that they play an
important role in establishing some of the processes by which this
regulation is accomplished.
[0348] The disclosed NOV13 nucleic acid of the invention encoding a
Neuromodulin-like protein includes the nucleic acid whose sequence
is provided in Table 13A or a fragment thereof. The invention also
includes a mutant or variant nucleic acid any of whose bases may be
changed from the corresponding base shown in Table 13A while still
encoding a protein that maintains its Neuromodulin-like activities
and physiological functions, or a fragment of such a nucleic acid.
The invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid
fragments that are complementary to any of the nucleic acids just
described. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications. Such modifications include, by way
of nonlimiting example, modified bases, and nucleic acids whose
sugar phosphate backbones are modified or derivatized. These
modifications are carried out at least in part to enhance the
chemical stability of the modified nucleic acid, such that they may
be used, for example, as antisense binding nucleic acids in
therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their complements, up to about 39 percent of the
bases may be so changed.
[0349] The disclosed NOV13 protein of the invention includes the
Neuromodulin-like protein whose sequence is provided in Table 13B.
The invention also includes a mutant or variant protein any of
whose residues may be changed from the corresponding residue shown
in Table 13B while still encoding a protein that maintains its
Neuromodulin-like activities and physiological functions, or a
functional fragment thereof. In the mutant or variant protein, up
to about 30 percent of the residues may be so changed.
[0350] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0351] The above defined information for this invention suggests
that this Neuromodulin-like protein (NOV13) may function as a
member of a "Neuromodulin family". Therefore, the NOV13 nucleic
acids and proteins identified here may be useful in potential
therapeutic applications implicated in (but not limited to) various
pathologies and disorders as indicated below. The potential
therapeutic applications for this invention include, but are not
limited to: protein therapeutic, small molecule drug target,
antibody target (therapeutic, diagnostic, drug targeting/cytotoxic
antibody), diagnostic and/or prognostic marker, gene therapy (gene
delivery/gene ablation), research tools, tissue regeneration in
vivo and in vitro of all tissues and cell types composing (but not
limited to) those defined here.
[0352] The NOV13 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in various
pathologies and disorders.
[0353] NOV13 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV13 substances for use in therapeutic or diagnostic
methods. These antibodies may be generated according to methods
known in the art, using prediction from hydrophobicity charts, as
described in the "Anti-NOVX Antibodies" section below. The
disclosed NOV13 protein has multiple hydrophilic regions, each of
which can be used as an immunogen. These novel proteins can be used
in assay systems for functional analysis of various human
disorders, which will help in understanding of pathology of the
disease and development of new drug targets for various
disorders.
[0354] NOV14
[0355] NOV14 includes three novel Prostatin Precursor-like proteins
disclosed below. The disclosed sequences have been named NOV14a and
NOV14b.
[0356] NOV14a
[0357] A disclosed NOV14a nucleic acid of 1102 nucleotides (also
referred to as CG56075-01) encoding a novel Prostatin
Precursor-like protein is shown in Table 14A. An open reading frame
was identified beginning with an ATG initiation codon at
nucleotides 19-21 and ending with a TGG codon at nucleotides
1051-1053. A putative untranslated region upstream from the
initiation codon is underlined in Table 14A. The start and stop
codons are in bold letters. Because the stop codon is not a
traditional termination codon, NOV14a could be a partial reading
frame. Therefore, it could extend further in the 3' direction.
TABLE-US-00090 TABLE 14A NOV14a nucleotide sequence (SEQ ID NO:43).
GGGCCCTTGTCCTGGGCCATGGCCCAGAAGGGGGTCCTGCGGCCTGGGCAGCTGGGGGCTGTGGCCAATTCT
GACTCATACTCACTTTACGGGTTGGTGCCGTCCGGACCCGCTAGGGGCCCCCCGTACTGCGGGCGCCCTGAG
CCCTCGGCCCGCATCGTGGGGGGCTCAAACGCGCAGCCGGGCACCTGGCCTTGGCAAGTGAGCCTGCACCAT
GGAGGTGGCCACATCTGCGGGGGCTCCCTCATCGCCCCCTCCTGGGTCCTCTCCGCTGCTCACTGTTTCATG
ACGAATGGGACGTTGGAGCCCGCGGCCGAGTGGTCGGTACTGCTGGGCGTGCACTCCCAGGACGGGCCCCTG
GACGGCGCGCACACCCGCGCAGTGCCCGCCATCGTGGTGCCGGCCAACTACAGCCAAGTGGAGCTGGGCGCC
GACCTGGCCCTGCTGCGCCTGGCCTCACCCGCCAGCCTGGGCCCCGCCGTGTGGCCTGTCTGCCTGCCCCGC
GCCTCACACCGCTTCGTGCACGGCACCGCCTGCTGGGCCACCGGCTGGGGAGACGTCCAGGAGGCAGATCCT
CTGCCTCTCCCCTGGGTGCTACAGGAAGTGGAGCTAAGGCTGCTGGGCGAGGCCACCTGTCAATGTCTCTAC
AGCCAGCCCGGTCCCTTCAACCTCACTCTCCAGATATTGCCAGGGATGCTGTGTGCTGGCTACCCAGAGGGC
CGCAGGGACACCTGCCAGGGTGACTCTGGGGGGCCCCTGGTCTGTGAGGAAGGCGGCCGCTGGTTCCAGGCA
GGAATCACCAGCTTTGGGTTTGGCTGTGGACGGAGAAACCGCCCTGGAGTTTTCACTGCTGTGGCTACCTAT
GAGGCATGGATACGGGAGCAGGTGATGGGTTCAGAGCCTGGGCCTGCCTTTCCCACCCAGCCCCAGAAGACC
CAGTCAGATTGTTTACATCAAACGGCATTCCTGGATTCTGCCAGAATCCTTTTGAGGCCCTTGTCCCATATA
TCAGTAGGAGTCTCAACTGGGACCAAAAGCCTTGTCCTCCCCTGGCTCTCTCCACACTCTCTCCTGGGCCTC
TGGGGGTTCTGATGGGGCCTCC
[0358] In a search of public sequence databases, the NOV14a nucleic
acid sequence, located on chromosome 16 has 469 of 795 bases (58%)
identical to a gb:GENBANK-ID:BTTRYPTMR|acc:X94982.1 mRNA from Bos
taurus (B. taurus mRNA for tryptase) (E=2.7e.sup.-21). Public
nucleotide databases include all GenBank databases and the GeneSeq
patent database.
[0359] The disclosed NOV14a polypeptide (SEQ ID NO:44) encoded by
SEQ ID NO:43 has 344 amino acid residues and is presented in Table
14B using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV14a has no signal peptide and is
likely to be localized extracellularly with a certainty of 0.4500.
In other embodiments, NOV14a may also be localized to the microbody
(peroxisome) with a certainty of 0.4370, the lysosome (lumen) with
a certainty of 0.3047, or to the mitochondrial matrix space with a
certainty of 0.1000. TABLE-US-00091 TABLE 14B Encoded NOV14a
protein sequence (SEQ ID NO:44).
MAQKGVLGPGQLGAVANSDSYSLYGLVPSGPARGPPYCGRPEPSARIVGGSNAQPGTWPWQVSLHHGGGHIC
GGSLIAPSWVLSAAHCFMTNGTLEPAAEWSVLLGVHSQDGPLDGAHTRAVAAIVVPANYSQVELGADLALLR
LASPASLGPAVWPVCLPRASHRFVHGTACWATGWGDVQFADPLPLPWVLQEVELRLLGEATCQCLYSQPGPF
NLTLQILPGMLCAGYPEGRRDTCQGDSGGPLVCEEGGRWFQAGITSFGFGCGRRNRPGVFTAVATYEAWIRE
QVMGSEPGPAFPTQPQKTQSDCLHQTAFLDSARILLRPLSHISVGVSTGTKSLVLP
[0360] A search of sequence databases reveals that the NOV14a amino
acid sequence has 149 of 340 amino acid residues (43%) identical
to, and 197 of 340 amino acid residues (57%) similar to, the 343
amino acid residue ptnr:SWISSPROT-ACC:Q16651 protein from Homo
sapiens (Human) (Prostasin Precursor (EC 3.4.21.-))
(E=3.9e.sup.-65). Public amino acid databases include the GenBank
databases, SwissProt, PDB and PIR.
[0361] NOV14a is expressed in at least Heart. This information was
derived by determining the tissue sources of the sequences that
were included in the invention including but not limited to
SeqCalling sources, Public EST sources, Literature sources, and/or
RACE sources.
[0362] In addition, the sequence is predicted to be expressed in
Heart because of the expression pattern of (GENBANK-ID:
gb:GENBANK-ID:BTrRYPTMR|acc:X94982.1) a closely related B. taurus
mRNA for tryptase homolog.
[0363] NOV14b
[0364] A disclosed NOV14b nucleic acid of 1102 nucleotides (also
referred to as CG56075-01) encoding a novel Prostatin
Precursor-like protein is shown in Table 14C. An open reading frame
was identified beginning with an ATG initiation codon at
nucleotides 19-21 and ending with a TGA codon at nucleotides
1090-1092. A putative untranslated region upstream from the
initiation codon is underlined in Table 14C. The start and stop
codons are in bold letters. TABLE-US-00092 TABLE 14C NOV14b
nucleotide sequence (SEQ ID NO:45).
GGGCCCTTGTCCTGCGCCATGGCCCAGAAGGCGGTCCTGGGGCCTGGGCAGCTGGGGGCTGTGGCCAATTCT
GACTCATACTCACTTTACGGGTTGGTGCCGTCCGGACCCGCTAGGGGCCCCCCGTACTGCGGGCGCCCTGAG
CCCTCGGCCCGCATCGTGGGGGGCTCAAACGCGCAGCCGGGCACCTGGCCTTGGCAAGTGAGCCTGCACCAT
GGAGGTGGCCACATCTGCGGGGGCTCCCTCATCGCCCCCTCCTGGGTCCTCTCCGCTGCTCACTGTTTCATG
ACGAATGGGACGTTGGAGCCCGCGGCCGAGTGGTCGGTACTGCTGGGCGTGCACTCCCAGGACGGGCCCCTG
GACGGCGCGCACACCCGCGCAGTGGCCGCCATCGTGGTGCCGGCCAACTACAGCCAAGTGCAGCTGGGCGCC
GACCTGGCCCTGCTGCGCCTGGCCTCACCCGCCAGCCTGGGCCCCGCCGTGTGGCCTGTCTGCCTGCCCCGC
GCCTCACACCGCTTCGTGCACGGCACCGCCTGCTGGGCCACCGGCTGGGGAGACGTCCAGGAGGCAGATCCT
CTGCCTCTCCCCTGGGTGCTACAGGAAGTGGAGCTAAGGCTGCTGGGCGAGGCCACCTGTCAATGTCTCTAC
AGCCAGCCCGGTCCCTTCAACCTCACTCTCCAGATATTGCCAGGGATGCTGTGTGCTGGCTACCCAGAGGGC
CGCAGGGACACCTGCCAGGGTGACTCTGGGGGGCCCCTGGTCTGTGAGGAAGGCGGCCGCTGGTTCCAGGCA
GGAATCACCAGCTTTGGGTTTGGCTGTGGACGGAGAAACCGCCCTGGAGTTTTCACTGCTGTGGCTACCTAT
GAGGCATGGATACGGGAGCAGGTGATGGGTTCAGAGCCTGGGCCTGCCTTTCCCACCCAGCCCCAGAAGACC
CAGTCAGATTGTTTACATCAAACGGCATTCCTGGATTCTGCCAGAATCCTTTTGAGGCCCTTGTCCCATATA
TCAGTAGGAGTCTCAACTGGGACCAAAAGCCTTGTCCTCCCCTGGCTCTCTCCACACTCTCTCCTGGGCCTC
TGGGGGTTCTGATGGGGCCTCC
[0365] The disclosed NOV14b polypeptide (SEQ ID NO:46) encoded by
SEQ ID NO:45 has 357 amino acid residues and is presented in Table
14D using the one-letter amino acid code. TABLE-US-00093 TABLE 14D
Encoded NOV14b protein sequence (SEQ ID NO:46).
MAQKGVLGPGQLGAVANSDSYSLYGLVPSGPARGPPYCGRPEPSARIVGGSNAQPGTWPWQVSLHHGGGHIC
GGSLIAPSWVLSAAHCFMTNGTLEPAAEWSVLLGVHSQDGPLDGAHTRAVAAIVVPANYSQVELGADLALLR
LASPASLGPAVWPVCLPRASHRFVHGTACWATGWGDVQEADPLPLPWVLQEVELRLLGEATCQCLYSQPGPF
NLTLQILPGNLCAGYPEGRRDTCQGDSGGPLVCEEGGRWFQAGITSFGFGCGRRNRPGVFTAVATYEAWIRE
QVMGSEPGPAFPTQPQKTQSDCLHQTAFLDSARILLRPLSHISVGVSTGTKSLVLPWLSPHSLLGLWGF
[0366] The disclosed NOV14 polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 14E.
TABLE-US-00094 TABLE 14E BLAST results for NOV14 Gene Index/ Length
Identity Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi|4506153|ref|NP_002764.1| protease, serine, 343 140/333 180/333
1e-51 (NM_002773) 8 (prostasin) (42%) (54%) [Homo sapiens]
gi|6009515|dbj|BAA84941.1| epidermis 389 104/265 144/265 2e-49
(AB018694) specific serine (39%) (54%) protease [Xenopus laevis]
gi|12249015|dbj|BAB prostamin [Homo 855 103/249 140/249 4e-47
20376.1| (AB030036) sapiens] (41%) (55%) gi|11181573|gb|AAG32641.1|
prostasin [Rattus 342 130/347 175/347 4e-47 AF202076_1 norvegicus]
(37%) (49%) (AF202076) gi|13632973|sp|Q9ES Prostasin 342 130/347
175/347 6e-47 87|PSS8_RAT precursor (37%) (49%)
[0367] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 14F. In the
ClustalW alignment of the NOV14 protein, as well as all other
ClustalW analyses herein, the black outlined amino acid residues
indicate regions of conserved sequence (i.e., regions that may be
required to preserve structural or functional properties), whereas
non-highlighted amino acid residues are less conserved and can
potentially be altered to a much broader extent without altering
protein structure or function.
[0368] Tables 14G-H lists the domain descriptions from DOMAIN
analysis results against NOV14. This indicates that the NOV14
sequence has properties similar to those of other proteins known to
contain this domain. TABLE-US-00095 TABLE 14G Domain Analysis of
NOV14 gnl|Smart|smart00020, Tryp_SPc, Trypsin-like serine protease;
Many of these are synthesised as inactive precursor zymogens that
are cleaved during limited proteolysis to generate their active
forms. A few, however, are active as single chain molecules, and
others are inactive due to substitutions of the catalytic triad
residues. (SEQ ID NO:135) CD-Length = 230 residues, 100.0% aligned
Score = 209 bits (531), Expect = 3e-55 Query: 46
RIVGGSNAQPGTWPWQVSLH-HGGGHICGGSLIAPSWVLSAAHCFMTNGTLEPAAEWSVL 104
|||||| | |++|||||| || | ||||||+| |||+|||| + + | Sbjct: 1
RIVGGSEANIGSFPWQVSLQYRGGRHFCGGSLISPRWVLTAAHCVYGS----APSSIRVR 56
Query: 105
LGVHSQDGPLDGAHTRAVAAIVVPANYSQVELGADLALLRLASPASLGPAVWPVCLPRAS 164 ||
| + | |+ ++| ||+ |+|||+|+ | +| | |+||| + Sbjct: 57
LGSHDLSSG-EETQTVKVSKVIVHPNYNPSTYDNDIALLKLSEPVTLSDTVRPICLPSSG 115
Query: 165
HRFVHGTACWATGWGDVQEADPLPLPWVLQEVELRLLGEATCQCLYSQPGPFNLTLQILP 224 +
|| | +||| |+ || |||| + ++ |||+ || | Sbjct: 116
YNVPAGTTCTVSGWGRTSESSG-SLPDTLQEVNVPIVSNATCRRAYSGGPA------ITD 168
Query: 225
GMLCAGYPEGRRDTCQGDSGGPLVCEEGGRWFQAGITSFG-FGCGRRNRPGVFTAVATYE 283
||||| || +| ||||||||||| || || |+| +|| | |+|||+| |++| Sbjct: 169
NMLCAGGLEGGKDACQGDSGGPLVC-NDPRWVLVGIVSWGSYGCARPNKPGVYTRVSSYL 227
Query: 284 AWI 286 || Sbjct: 228 DWI 230
[0369] TABLE-US-00096 TABLE 14H Domain Analysis of NOV14
gnl|Pfam|pfam00089, trypsin, Trypsin. Proteins recognized include
all proteins in families S1, S2A, S2B, S2C, and S5 in the
classification of peptidases. Also included are proteins that are
clearly members, but that lack peptidase activity, such as
haptoglobin and, protein Z (PRTZ*). (SEQ ID NO:136) CD-Length = 217
residues, 100.0% aligned Score = 165 bits (417), Expect = 5e-42
Query: 47
IVGGSNAQPGTWPWQVSLHHGGGHICGGSLIAPSWVLSAAHCFMTNGTLEPAAEWSVLLG 106
|||| || |++|||||| || ||||||+ +|||+|||| |+ |+|| Sbjct: 1
IVGGREAQAGSFPWQVSLQVSSGHFCGGSLISENWVLTAAHCVSG------ASSVRVVLG 54
Query: 107
VHSQDGPLDGAHTRAVAAIVVPANYSQVELGADLALLRLASPASLGPAVWPVCLPRASHR 166 |+
| |+| ||+ |+|||+| || +|| | |+||| || Sbjct: 55
EHNLGTTEGTEQKFDVKKIIVHPNYNP--DTNDIALLKLKSPVTLGDTVRPICLPSASSD 112
Query: 167
FVHGTACWATGWGDVQEADPLPLPWVLQEVELRLLGEATCQCLYSQPGPFNLTLQILPGM 226 ||
| +||| + |||| + ++ ||+ | + | Sbjct: 113
LPVGTTCSVSGWGRTKNLGT---SDTLQEVVVPIVSRETCRSAYGGT--------VTDTM 161
Query: 227
LCAGYPEGRRDTCQGDSGGPLVCEEGGRWFQAGITSFGFGCGRRNRPGVFTAVATYEAWI 286
+||| | +| ||||||||||| +| || |+|+|| | |||+| |+ | || Sbjct: 162
ICAGALGG-KDACQGDSGGPLVCSDG---ELVGIVSWGYGCAVGNYPGVYTRVSRYLDWI
217
[0370] Human seminal fluid contains a variety of proteolytic
enzymes, including prostate-specific antigen and acrosin. These
enzymes are involved in the postejaculatory hydrolysis of proteins
and in semen coagulation and liquefaction. Yu et al. (1995)
obtained partial amino acid sequence of a 40-kD protein isolated
from seminal fluid originally by Yu et al. (1994). Yu et al. (1995)
designed degenerate primers based on the amino acid sequence and
used to screen a human prostate cDNA library by PCR. The 3-prime
end of the cDNA was obtained by the RACE (rapid amplification of
cDNA ends) method. A 1.8-kb cDNA sequence was assembled encoding a
predicted protein of 343 amino acids which contains a 32-amino acid
signal peptide. The protein, designated serine protease-8 (gene
symbol .dbd.PRSS8), was called prostasin by the authors. The
precursor, proprostasin, is cleaved between residues 12 and 13 to
produce a 12-amino acid light chain and a 299-amino acid heavy
chain which are associated through a disulfide bond. The predicted
amino acid sequence is between 34 and 42% identical to human
acrosin, plasma kallikrein, and hepsin. The deduced protein has a
hydrophobic domain at the C terminus, indicating to the authors
that it may be membrane anchored. The authors showed that the
hydrophobic region is cleaved between residues 290 and 291 during
secretion. Expression levels of the prostasin mRNA were assayed by
Southern blots of RT-PCR products. Expression was noted in a wide
variety of tissues. In the prostate gland, expression was localized
to the epithelial cells. Yu et al. (1996) isolated and
characterized the full length PRSS8 gene. They found that it
consists of 6 exons and 5 introns. The authors characterized the
5-prime flanking region of the gene and found a number of potential
regulatory elements, including an AP2 site, 2 erythroid-specific
promoter elements, and a sterol regulatory element, although no
TATA box was found. In addition, there were a variant GC box and a
variant AP1 site in the promoter region. Prostasin, denoted as
PRSS8, is a newly identified human serine proteinase that shares
high sequence identity with acrosin, plasma kallikrein, and hepsin
(Yu et al., 1994, 1995). In the present study, a full-length PRSS8
gene has been isolated and characterized. A 7-kb PRSS8 gene
fragment has been sequenced, including a 1.4-kb 5'-flanking region,
the 4.4-kb PRSS8 gene, and a 1.2-kb 3'-flanking region. The gene
consists of six exons and five introns based on comparison with its
cDNA sequence. The sizes of these exons are 417, 18, 163, 272, 167,
and 899 bp, while those of the introns are 243, 1763, 271, 85, and
92 bp. A number of potential regulatory elements have been revealed
in the 5'-flanking region, including an AP2 site, two
erythroid-specific promoter elements, and a sterol regulatory
element. In addition, there are a variant GC box and a variant AP1
site in the promoter region. The transcription initiation site of
the PRSS8 gene has been defined at the G residue and its adjacent A
residue in a sequence CTCATGACT, which is similar to an initiator
element CTCANTCT. Between the transcription initiation site and
these putative regulatory elements, there is an AC-rich repetitive
sequence that spans over 300 bp. Human PRSS8 is a single-copy gene
and has been localized on chromosome 16p11.2 by in situ
hybridization.
[0371] The disclosed NOV14 nucleic acid of the invention encoding a
Prostatin Precursor-like protein includes the nucleic acid whose
sequence is provided in Table 14A, 14C or a fragment thereof. The
invention also includes a mutant or variant nucleic acid any of
whose bases may be changed from the corresponding base shown in
Table 14A, or 14C while still encoding a protein that maintains its
Prostatin Precursor-like activities and physiological functions, or
a fragment of such a nucleic acid. The invention further includes
nucleic acids whose sequences are complementary to those just
described, including nucleic acid fragments that are complementary
to any of the nucleic acids just described. The invention
additionally includes nucleic acids or nucleic acid fragments, or
complements thereto, whose structures include chemical
modifications. Such modifications include, by way of nonlimiting
example, modified bases, and nucleic acids whose sugar phosphate
backbones are modified or derivatized. These modifications are
carried out at least in part to enhance the chemical stability of
the modified nucleic acid, such that they may be used, for example,
as antisense binding nucleic acids in therapeutic applications in a
subject. In the mutant or variant nucleic acids, and their
complements, up to about 43 percent of the bases may be so
changed.
[0372] The disclosed NOV14 protein of the invention includes the
Prostatin Precursor-like protein whose sequence is provided in
Table 14B, or 14D. The invention also includes a mutant or variant
protein any of whose residues may be changed from the corresponding
residue shown in Table 14B, or 14D while still encoding a protein
that maintains its Prostatin Precursor-like activities and
physiological functions, or a functional fragment thereof. In the
mutant or variant protein, up to about 43% percent of the residues
may be so changed.
[0373] The invention further encompasses antibodies and antibody
fragments, such as F.sub.ab or (F.sub.ab).sub.2, that bind
immunospecifically to any of the proteins of the invention.
[0374] The above defined information for this invention suggests
that this Prostatin Precursor-like protein (NOV14) may function as
a member of a "Prostatin Precursor family". Therefore, the NOV14
nucleic acids and proteins identified here may be useful in
potential therapeutic applications implicated in (but not limited
to) various pathologies and disorders as indicated below. The
potential therapeutic applications for this invention include, but
are not limited to: protein therapeutic, small molecule drug
target, antibody target (therapeutic, diagnostic, drug
targeting/cytotoxic antibody), diagnostic and/or prognostic marker,
gene therapy (gene delivery/gene ablation), research tools, tissue
regeneration in vivo and in vitro of all tissues and cell types
composing (but not limited to) those defined here.
[0375] The NOV14 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart
defects, Aortic stenosis, Atrial septal defect (ASD),
Atrioventricular (A-V) canal defect, Ductus arteribsus, Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD),
valve diseases, Tuberous sclerosis, Scleroderma, Obesity,
Transplantation, and/or other diseases and pathologies.
[0376] NOV14 nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOV14 substances for use in therapeutic or diagnostic
methods. These antibodies may be generated according to methods
known in the art, using prediction from hydrophobicity charts, as
described in the "Anti-NOVX Antibodies" section below. The
disclosed NOV14 protein has multiple hydrophilic regions, each of
which can be used as an immunogen. These novel proteins can be used
in assay systems for functional analysis of various human
disorders, which will help in understanding of pathology of the
disease and development of new drug targets for various
disorders.
[0377] NOVX Nucleic Acids and Polypeptides
[0378] One aspect of the invention pertains to isolated nucleic
acid molecules that encode NOVX polypeptides or biologically active
portions thereof. Also included in the invention are nucleic acid
fragments sufficient for use as hybridization probes to identify
NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for
use as PCR primers for the amplification and/or mutation of NOVX
nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA
generated using nucleotide analogs, and derivatives, fragments and
homologs thereof. The nucleic acid molecule may be single-stranded
or double-stranded, but preferably is comprised double-stranded
DNA.
[0379] An NOVX nucleic acid can encode a mature NOVX polypeptide.
As used herein, a "mature" form of a polypeptide or protein
disclosed in the present invention is the product of a naturally
occurring polypeptide or precursor form or proprotein. The
naturally occurring polypeptide, precursor or proprotein includes,
by way of nonlimiting example, the full-length gene product,
encoded by the corresponding gene. Alternatively, it may be defined
as the polypeptide, precursor or proprotein encoded by an ORF
described herein. The product "mature" form arises, again by way of
nonlimiting example, as a result of one or more naturally occurring
processing steps as they may take place within the cell, or host
cell, in which the gene product arises. Examples of such processing
steps leading to a "mature" form of a polypeptide or protein
include the cleavage of the N-terminal methionine residue encoded
by the initiation codon of an ORF, or the proteolytic cleavage of a
signal peptide or leader sequence. Thus a mature form arising from
a precursor polypeptide or protein that has residues 1 to N, where
residue 1 is the N-terminal methionine, would have residues 2
through N remaining after removal of the N-terminal methionine.
Alternatively, a mature form arising from a precursor polypeptide
or protein having residues 1 to N, in which an N-terminal signal
sequence from residue 1 to residue M is cleaved, would have the
residues from residue M+1 to residue N remaining. Further as used
herein, a "mature" form of a polypeptide or protein may arise from
a step of post-translational modification other than a proteolytic
cleavage event. Such additional processes include, by way of
non-limiting example, glycosylation, myristoylation or
phosphorylation. In general, a mature polypeptide or protein may
result from the operation of only one of these processes, or a
combination of any of them.
[0380] 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 may be
single- or double-stranded and designed to have specificity in PCR,
membrane-based hybridization technologies, or ELISA-like
technologies.
[0381] 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 NOVX nucleic acid molecules
can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or
0.1 kb of nucleotide sequences which naturally flank the nucleic
acid molecule in genomic DNA of the cell/tissue from which the
nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
Moreover, an "isolated" nucleic acid molecule, such as a
cDNA-molecule; can be substantially free of other cellular material
or culture medium when produced by recombinant techniques, or of
chemical precursors or other chemicals when chemically
synthesized.
[0382] A nucleic acid molecule of the invention, e.g., a nucleic
acid molecule having the nucleotide sequence SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, and 197, or a complement of this aforementioned nucleotide
sequence, can be isolated using standard molecular biology
techniques and the sequence information provided herein. Using all
or a portion of the nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, and 197 as a hybridization probe, NOVX molecules can be
isolated using standard hybridization and cloning techniques (e.g.,
as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A
LABORATORY MANUAL 2.sup.nd Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., (eds.),
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New
York, N.Y., 1993.)
[0383] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to NOVX nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0384] As used herein, the term "oligonucleotide" refers to a
series of linked nucleotide residues, which oligonucleotide has a
sufficient number of nucleotide bases to be used in a PCR reaction.
A short oligonucleotide sequence may be based on, or designed from,
a genomic or cDNA sequence and is used to amplify, confirm, or
reveal the presence of an identical, similar or complementary DNA
or RNA in a particular cell or tissue. Oligonucleotides comprise
portions of a nucleic acid sequence having about 10 nt, 50 nt, or
100 nt in length, preferably about 15 nt to 30 nt in length. In one
embodiment of the invention, an oligonucleotide comprising a
nucleic acid molecule less than 100 nt in length would further
comprise at least 6 contiguous nucleotides SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, and 197, or a complement thereof. Oligonucleotides may be
chemically synthesized and may also be used as probes.
[0385] 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 NOS:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, and 197, or a portion of this nucleotide sequence
(e.g., a fragment that can be used as a probe or primer or a
fragment encoding a biologically-active portion of an NOVX
polypeptide). A nucleic acid molecule that is complementary to the
nucleotide sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 197 is
one that is sufficiently complementary to the nucleotide sequence
shown SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 197 that it can hydrogen
bond with little or no mismatches to the nucleotide sequence shown
SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, and 197, thereby forming a stable
duplex.
[0386] As used herein, the term "complementary" refers to
Watson-Crick or Hoogsteen base pairing between nucleotides units of
a nucleic acid molecule, and the term "binding" means the physical
or chemical interaction between two polypeptides or compounds or
associated polypeptides or compounds or combinations thereof.
Binding includes ionic, non-ionic, van der Waals, hydrophobic
interactions, and the like. A physical interaction can be either
direct or indirect. Indirect interactions may be through or due to
the effects of another polypeptide or compound. Direct binding
refers to interactions that, do not take place through, or due to,
the effect of another polypeptide or compound, but instead are
without other substantial chemical intermediates.
[0387] Fragments provided herein are defined as sequences of at
least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino
acids, a length sufficient to allow for specific hybridization in
the case of nucleic acids or for specific recognition of an epitope
in the case of amino acids, respectively, and are at most some
portion less than a full length sequence. Fragments may be derived
from any contiguous portion of a nucleic acid or amino acid
sequence of choice. Derivatives are nucleic acid sequences or amino
acid sequences formed from the native compounds either directly or
by modification or partial substitution. Analogs are nucleic acid
sequences or amino acid sequences that have a structure similar to,
but not, identical to, the native compound but differs from it in
respect to certain components or side chains. Analogs may be
synthetic or from a different evolutionary origin and may have a
similar or opposite metabolic activity compared to wild type.
Homologs are nucleic acid sequences or amino acid sequences of a
particular gene that are derived from different species.
[0388] Derivatives and analogs may be full length or other than
full length, if the derivative or analog contains a modified
nucleic acid or amino acid, as described below. Derivatives or
analogs of the nucleic acids or proteins of the invention include,
but are not limited to, molecules comprising regions that are
substantially homologous to the nucleic acids or proteins of the
invention, in various embodiments, by at least about 70%, 80%, or
95% identity (with a preferred identity of 80-95%) over a nucleic
acid or amino acid sequence of identical size or when compared to
an aligned sequence in which the alignment is done by a computer
homology program known in the art, or whose encoding nucleic acid
is capable of hybridizing to the complement of a sequence encoding
the aforementioned proteins under stringent, moderately stringent,
or low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,
N.Y., 1993, and below.
[0389] 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 NOVX polypeptides. Isoforms
can be expressed in different tissues of the same organism as a
result of, for example, alternative splicing of RNA. Alternatively,
isoforms can be encoded by different genes. In the invention,
homologous nucleotide sequences include nucleotide sequences
encoding for an NOVX polypeptide of species other than humans,
including, but not limited to: vertebrates, and thus can include,
e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other
organisms. Homologous nucleotide sequences also include, but are
not limited to, naturally occurring allelic variations and
mutations of the nucleotide sequences set forth herein. A
homologous nucleotide sequence does not, however, include the exact
nucleotide sequence encoding human NOVX protein. Homologous nucleic
acid sequences include those nucleic acid sequences that encode
conservative amino acid substitutions (see below) in SEQ ID NOS:1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, and 197, as well as a polypeptide possessing NOVX
biological activity. Various biological activities of the NOVX
proteins are described below.
[0390] An NOVX polypeptide is encoded by the open reading frame
("ORF") of an NOVX nucleic acid. An ORF corresponds to a nucleotide
sequence that could potentially be translated into a polypeptide. A
stretch of nucleic acids comprising an ORF is uninterrupted by a
stop codon. An ORF that represents the coding sequence for a full
protein begins with an ATG "start" codon and terminates with one of
the three "stop" codons, namely, TAA, TAG, or TGA. For the purposes
of this invention, an ORF may be any part of a coding sequence,
with or without a start codon, a stop codon, or both. For an ORF to
be considered as a good candidate for coding for a bona 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.
[0391] The nucleotide sequences determined from the cloning of the
human NOVX genes allows for the generation of probes and primers
designed for use in identifying and/or cloning NOVX homologues in
other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/primer typically
comprises substantially purified oligonucleotide. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 12,
25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense
strand nucleotide sequence SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 197;
or an anti-sense strand nucleotide sequence of SEQ ID NOS:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, or 197; or of a naturally occurring mutant of SEQ ID
NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, and 197.
[0392] Probes based on the human NOVX nucleotide sequences can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins. In various embodiments, the probe further
comprises a label group attached thereto, e.g. the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissues which mis-express an NOVX
protein, such as by measuring a level of an NOVX-encoding nucleic
acid in a sample of cells from a subject e.g., detecting NOVX mRNA
levels or determining whether a genomic NOVX gene has been mutated
or deleted.
[0393] "A polypeptide having a biologically-active portion of an
NOVX polypeptide" refers to polypeptides exhibiting activity
similar, but not necessarily identical to, an activity of a
polypeptide of the invention, including mature forms, as measured
in a particular biological assay, with or without dose dependency.
A nucleic acid fragment encoding a "biologically-active portion of
NOVX" can be prepared by isolating a portion SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, or 197, that encodes a polypeptide having an NOVX
biological activity (the biological activities of the NOVX proteins
are described below), expressing the encoded portion of NOVX
protein (e.g., by recombinant expression in vitro) and assessing
the activity of the encoded portion of NOVX.
[0394] NOVX Nucleic Acid and Polypeptide Variants
[0395] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequences shown in SEQ ID NOS:1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, and 197 due to degeneracy of the genetic code and
thus encode the same NOVX proteins as that encoded by the
nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and
197. 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 NOS:2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or
198.
[0396] In addition to the human NOVX nucleotide sequences shown in
SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, and 197, it will be appreciated by
those skilled in the art that DNA sequence polymorphisms that lead
to changes in the amino acid sequences of the NOVX polypeptides may
exist within a population (e.g., the human population). Such
genetic polymorphism in the NOVX genes may exist among individuals
within a population due to natural allelic variation. As used
herein, the terms "gene" and "recombinant gene" refer to nucleic
acid molecules comprising an open reading frame (ORF) encoding an
NOVX protein, preferably a vertebrate NOVX protein. Such natural
allelic variations can typically result in 1-5% variance in the
nucleotide sequence of the NOVX genes. Any and all such nucleotide
variations and resulting amino acid polymorphisms in the NOVX
polypeptides, which are the result of natural allelic variation and
that do not alter the functional activity of the NOVX polypeptides,
are intended to be within the scope of the invention.
[0397] Moreover, nucleic acid molecules encoding NOVX proteins from
other species, and thus that have a nucleotide sequence that
differs from the human SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197 are
intended to be within the scope of the invention. Nucleic acid
molecules corresponding to natural allelic variants and homologues
of the NOVX cDNAs of the invention can be isolated based on their
homology to the human NOVX nucleic acids disclosed herein using the
human cDNAs, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions.
[0398] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 6 nucleotides in length and
hybridizes under stringent conditions to the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
and 197. 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.
[0399] Homologs (i.e., nucleic acids encoding NOVX proteins derived
from species other than human) or other related sequences (e.g.,
paralogs) can be obtained by low, moderate or high stringency
hybridization with all or a portion of the particular human
sequence as a probe using methods well known in the art for nucleic
acid hybridization and cloning.
[0400] 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.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present at excess, at Tm,
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
[0401] pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes, primers or oligonucleotides (e.g.,
10 nt to 50 nt) and at least about 60.degree. C. for longer probes,
primers and oligonucleotides. Stringent conditions may also be
achieved with the addition of destabilizing agents, such as
formamide.
[0402] 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 6.times.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.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.01% BSA at 50.degree. C. An isolated nucleic
acid molecule of the invention that hybridizes under stringent
conditions to the sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and
197, corresponds to a naturally-occurring nucleic acid molecule. As
used herein, a "naturally-occurring" nucleic acid molecule refers
to an RNA or DNA molecule having a nucleotide sequence that occurs
in nature (e.g., encodes a natural protein).
[0403] In a second embodiment, a nucleic acid sequence that is
hybridizable to the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197, 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
6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 mg/ml
denatured salmon sperm DNA at 55.degree. C., followed by one or
more washes in 1.times.SSC, 0.1% SDS at 37.degree. C. Other
conditions of moderate stringency that may be used are well-known
within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and
Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,
Stockton Press, NY.
[0404] In a third embodiment, a nucleic acid that is hybridizable
to the nucleic acid molecule comprising the nucleotide sequences
SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, and 197, 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, 5.times.SSC, 50 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA,
100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate
at 400C, followed by one or more washes in 2.times.SSC, 25 mM
Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50.degree. C. Other
conditions of low stringency that may be used are well known in the
art (e.g., as employed for cross-species hybridizations). See,
e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE
TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY;
Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.
[0405] Conservative Mutations
[0406] In addition to naturally-occurring allelic variants of NOVX
sequences that may exist in the population, the skilled artisan
will further appreciate that changes can be introduced by mutation
into the nucleotide sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and
197, thereby leading to changes in the amino acid sequences of the
encoded NOVX proteins, without altering the functional ability of
said NOVX proteins. For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or 198.
A "non-essential" amino acid residue is a residue that can be
altered from the wild-type sequences of the NOVX proteins without
altering their biological activity, whereas an "essential" amino
acid residue is required for such biological activity. For example,
amino acid residues that are conserved among the NOVX proteins of
the invention are predicted to be particularly non-amenable to
alteration. Amino acids for which conservative substitutions can be
made are well-known within the art.
[0407] Another aspect of the invention pertains to nucleic acid
molecules encoding NOVX proteins that contain changes in amino acid
residues that are not essential for activity. Such NOVX proteins
differ in amino acid sequence from SEQ ID NOS:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
and 197 yet retain biological activity. In one embodiment, the
isolated nucleic acid molecule comprises a nucleotide sequence
encoding a protein, wherein the protein comprises an amino acid
sequence at least about 45% homologous to the amino acid sequences
SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 26, 28, 40, 42, 44, 46, and 198. Preferably, the protein
encoded by the nucleic acid molecule is at least about 60%
homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, and 198; more
preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44,
46, or 198; still more preferably at least about 80% homologous to
SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 26, 28, 40, 42, 44, 46, or 198; even more preferably at
least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or
198; and most preferably at least about 95% homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
26, 28, 40, 42, 44, 46, or 198.
[0408] An isolated nucleic acid molecule encoding an NOVX protein
homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or 198
can be created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of SEQ ID
NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, and 197, such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein.
[0409] Mutations can be introduced into SEQ ID NOS:1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, and 197 by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Preferably, conservative
amino acid substitutions are made at one or more predicted,
non-essential amino acid residues. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined
within the art. These families include amino acids with basic side
chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted non-essential amino acid residue in
the NOVX protein is replaced with another amino acid residue from
the same side chain family. Alternatively, in another embodiment,
mutations can be introduced randomly along all or part of an NOVX
coding sequence, such as by saturation mutagenesis, and the
resultant mutants can be screened for NOVX biological activity to
identify mutants that retain activity. Following mutagenesis SEQ ID
NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, and 197, the encoded protein can be
expressed by any recombinant technology known in the art and the
activity of the protein can be determined.
[0410] The relatedness of amino acid families may also be
determined based on side chain interactions. Substituted amino
acids may be fully conserved "strong" residues or fully conserved
"weak" residues. The "strong" group of conserved amino acid
residues may be any one of the following groups: STA, NEQK, NHQK,
NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino
acid codes are grouped by those amino acids that may be substituted
for each other. Likewise, the "weak" group of conserved residues
may be any one of the following: CSA, ATV, SAG, SFNK, STPA, SGND,
SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within each
group represent the single letter amino acid code.
[0411] In one embodiment, a mutant NOVX protein can be assayed for
(i) the ability to form protein:protein interactions with other
NOVX proteins, other cell-surface proteins, or biologically-active
portions thereof, (ii) complex formation between a mutant NOVX
protein and an NOVX ligand; or (iii) the ability of a mutant NOVX
protein to bind to an intracellular target protein or
biologically-active portion thereof; (e.g. avidin proteins).
[0412] In yet another embodiment, a mutant NOVX protein can be
assayed for the ability to regulate a specific biological function
(e.g., regulation of insulin release).
[0413] Antisense Nucleic Acids
[0414] Another aspect of the invention pertains to isolated
antisense nucleic acid molecules that are hybridizable to or
complementary to the nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197, 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
NOVX coding strand, or to only a portion thereof. Nucleic acid
molecules encoding fragments, homologs, derivatives and analogs of
an NOVX protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or 198, or
antisense nucleic acids complementary to an NOVX nucleic acid
sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197, are
additionally provided.
[0415] In one embodiment, an antisense nucleic acid molecule is
antisense to a "coding region" of the coding strand of a nucleotide
sequence encoding an NOVX protein. The term "coding region" refers
to the region of the nucleotide sequence comprising codons which
are translated into amino acid residues. In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding the
NOVX protein. The term "noncoding region" refers to 5' and 3'
sequences which flank the coding region that are not translated
into amino acids (i.e., also referred to as 5' and 3' untranslated
regions).
[0416] Given the coding strand sequences encoding the NOVX protein
disclosed herein, antisense nucleic acids of the invention can be
designed according to the rules of Watson and Crick or Hoogsteen
base pairing. The antisense nucleic acid molecule can be
complementary to the entire coding region of NOVX mRNA, but more
preferably is an oligonucleotide that is antisense to only a
portion of the coding or noncoding region of NOVX mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of NOVX mRNA. An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20,
25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical
synthesis or enzymatic ligation reactions using procedures known in
the art. For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using
naturally-occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids (e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used).
[0417] 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-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 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-N-6-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).
[0418] 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 an NOVX protein to thereby inhibit expression of the
protein (e.g., by inhibiting transcription and/or translation). The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule that binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention includes direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface (e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies that
bind to cell surface receptors or antigens). The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient nucleic acid molecules,
vector constructs in which the antisense nucleic acid molecule is
placed under the control of a strong pol II or pol III promoter are
preferred.
[0419] 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 O-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., Inoue, et al. 1987. Nucl.
Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See,
e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330.
Ribozymes and PNA Moieties
[0420] Nucleic acid modifications include, by way of non-limiting
example, modified bases, and nucleic acids whose sugar phosphate
backbones are modified or derivatized. These modifications are
carried out at least in part to enhance the chemical stability of
the modified nucleic acid, such that they may be used, for example,
as antisense binding nucleic acids in therapeutic applications in a
subject.
[0421] In one embodiment, an antisense nucleic acid of the
invention is a ribozyme. Ribozymes are catalytic RNA molecules with
ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
as described in Haselhoff and Gerlach 1988. Nature 334: 585-591)
can be used to catalytically cleave NOVX mRNA transcripts to
thereby inhibit translation of NOVX mRNA. A ribozyme having
specificity for an NOVX-encoding nucleic acid can be designed based
upon the nucleotide sequence of an NOVX cDNA disclosed herein
(i.e., SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197). 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 an NOVX-encoding mRNA.
See, e.g., U.S. Pat. No. 4,987,071 to Cech, et al. and U.S. Pat.
No. 5,116,742 to Cech, et al. NOVX mRNA can also be used to select
a catalytic RNA having a specific ribonuclease activity from a pool
of RNA molecules. See, e.g., Bartel et al., (1993) Science
261:1411-1418.
[0422] Alternatively, NOVX gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the NOVX nucleic acid (e.g., the NOVX promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the NOVX gene in target cells. See, e.g., Helene,
1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann.
N.Y. Acad. Sci. 660:27-36; Maher, 1992. Bioassays 14:807-15.
[0423] In various embodiments, the NOVX nucleic acids can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve, e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids.
See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used
herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural nucleobases are retained. The neutral backbone of
PNAs has been shown to allow for specific hybridization to DNA and
RNA under conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using 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.
[0424] PNAs of NOVX can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or
antigene agents for sequence-specific modulation of gene expression
by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs of NOVX can also be used, for example,
in the analysis of single base pair mutations in a gene (e.g., PNA
directed PCR clamping; as artificial restriction enzymes when used
in combination with other enzymes, e.g., S.sub.1 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).
[0425] In another embodiment, PNAs of NOVX can be modified, e.g.,
to enhance their stability or cellular uptake, by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
NOVX can be generated that may combine the advantageous properties
of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g.,
RNase H and DNA 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 stacking, number of
bonds between the nucleobases, and orientation (see, Hyrup, et al.,
1996. supra). The synthesis of PNA-DNA chimeras can be performed as
described in Hyrup, et al., 1996. supra and Finn, et al., 1996.
Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be
synthesized on a solid support using standard phosphoramidite
coupling chemistry, and modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used between the PNA and the 5' end of DNA. See, e.g., Mag, et
al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra. Alternatively, chimeric molecules can be synthesized with a
5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al.,
1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
[0426] 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 barrier (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 may be conjugated to another molecule, e.g., a
peptide, a hybridization triggered cross-linking agent, a transport
agent, a hybridization-triggered cleavage agent, and the like.
[0427] NOVX Polypeptides
[0428] A polypeptide according to the invention includes a
polypeptide including the amino acid sequence of NOVX polypeptides
whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or
198. The invention also includes a mutant or variant protein any of
whose residues may be changed from the corresponding residues shown
in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 26, 28, 40, 42, 44, 46, or 198 while still encoding a
protein that maintains its NOVX activities and physiological
functions, or a functional fragment thereof.
[0429] In general, an NOVX variant that preserves NOVX-like
function includes any variant in which residues at a particular
position in the sequence have been substituted by other amino
acids, and further include the possibility of inserting an
additional residue or residues between two residues of the parent
protein as well as the possibility of deleting one or more residues
from the parent sequence. Any amino acid substitution, insertion,
or deletion is encompassed by the invention. In favorable
circumstances, the substitution is a conservative substitution as
defined above.
[0430] One aspect of the invention pertains to isolated NOVX
proteins, and biologically-active portions thereof, or derivatives,
fragments, analogs or homologs thereof. Also provided are
polypeptide fragments suitable for use as immunogens to raise
anti-NOVX antibodies. In one embodiment, native NOVX proteins can
be isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, NOVX proteins are produced by recombinant
DNA techniques. Alternative to recombinant expression, an NOVX
protein or polypeptide can be synthesized chemically using standard
peptide synthesis techniques.
[0431] 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 NOVX protein is derived, or
substantially free from chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of
cellular material" includes preparations of NOVX proteins in which
the protein is separated from cellular components of the cells from
which it is isolated or recombinantly-produced. In one embodiment,
the language "substantially free of cellular material" includes
preparations of NOVX proteins having less than about 30% (by dry
weight) of non-NOVX proteins (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-NOVX proteins, still more preferably less than about 10% of
non-NOVX proteins, and most preferably less than about 5% of
non-NOVX proteins. When the NOVX protein or biologically-active
portion thereof is recombinantly-produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
NOVX protein preparation.
[0432] The language "substantially free of chemical precursors or
other chemicals" includes preparations of NOVX proteins in which
the protein is separated from chemical precursors or other
chemicals that are involved in the synthesis of the protein. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of NOVX proteins having
less than about 30% (by dry weight) of chemical precursors or
non-NOVX chemicals, more preferably less than about 20% chemical
precursors or non-NOVX chemicals, still more preferably less than
about 10% chemical precursors or non-NOVX chemicals, and most
preferably less than about 5% chemical precursors or non-NOVX
chemicals.
[0433] Biologically-active portions of NOVX proteins include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequences of the NOVX proteins
(e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44,
46, or 198) that include fewer amino acids than the full-length
NOVX proteins, and exhibit at least one activity of an NOVX
protein. Typically, biologically-active portions comprise a domain
or motif with at least one activity of the NOVX protein. A
biologically-active portion of an NOVX protein can be a polypeptide
which is, for example, 10, 25, 50, 100 or more amino acid residues
in length.
[0434] Moreover, other biologically-active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native NOVX protein.
[0435] In an embodiment, the NOVX protein has an amino acid
sequence shown SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or 198. In other
embodiments, the NOVX protein is substantially homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
26, 28, 40, 42, 44, 46, or 198, and retains the functional activity
of the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or 198, yet
differs in amino acid sequence due to natural allelic variation or
mutagenesis, as described in detail, below. Accordingly, in another
embodiment, the NOVX protein is a protein that comprises an amino
acid sequence at least about 45% homologous to the amino acid
sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, 34, 26, 28, 40, 42, 44, 46, or 198, and retains the
functional activity of the NOVX proteins of SEQ ID NOS:2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42,
44, 46, or 198.
[0436] Determining Homology Between Two or More Sequences
[0437] 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 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").
[0438] The nucleic acid sequence homology may be determined as the
degree of identity between two sequences. The homology may be
determined using computer programs known in the art, such as GAP
software provided in the GCG program package. See, Needleman and
Wunsch, 1970. 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 NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and
197.
[0439] 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 number of positions in the region of comparison (i.e., the
window size), and multiplying the result by 100 to yield the
percentage of sequence 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.
[0440] Chimeric and Fusion Proteins
[0441] The invention also provides NOVX chimeric or fusion
proteins. As used herein, an NOVX "chimeric protein" or "fusion
protein" comprises an NOVX polypeptide operatively-linked to a
non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to an NOVX protein SEQ
ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 26, 28, 40, 42, 44, 46, or 198, whereas a "non-NOVX
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein that is not substantially homologous to
the NOVX protein, e.g., a protein that is different from the NOVX
protein and that is derived from the same or a different organism.
Within an NOVX fusion protein the NOVX polypeptide can correspond
to all or a portion of an NOVX protein. In one embodiment, an NOVX
fusion protein comprises at least one biologically-active portion
of an NOVX protein. In another embodiment, an NOVX fusion protein
comprises at least two biologically-active portions of an NOVX
protein. In yet another embodiment, an NOVX fusion protein
comprises at least three biologically-active portions of an NOVX
protein. Within the fusion protein, the term "operatively-linked"
is intended to indicate that the NOVX polypeptide and the non-NOVX
polypeptide are fused in-frame with one another. The non-NOVX
polypeptide can be fused to the N-terminus or C-terminus of the
NOVX polypeptide.
[0442] In one embodiment, the fusion protein is a GST-NOVX fusion
protein in which the NOVX sequences are fused to the C-terminus of
the GST (glutathione S-transferase) sequences. Such fusion proteins
can facilitate the purification of recombinant NOVX
polypeptides.
[0443] In another embodiment, the fusion protein is an NOVX protein
containing a heterologous signal sequence at its N-terminus. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of NOVX can be increased through use of a heterologous
signal sequence.
[0444] In yet another embodiment, the fusion protein is an
NOVX-immunoglobulin fusion protein in which the NOVX sequences are
fused to sequences derived from a member of the immunoglobulin
protein family. The NOVX-immunoglobulin fusion proteins of the
invention can be incorporated into pharmaceutical compositions and
administered to a subject to inhibit an interaction between an NOVX
ligand and an NOVX protein on the surface of a cell, to thereby
suppress NOVX-mediated signal transduction in vivo. The
NOVX-immunoglobulin fusion proteins can be used to affect the
bioavailability of an NOVX cognate ligand. Inhibition of the NOVX
ligand/NOVX interaction may be useful therapeutically for both the
treatment of proliferative and differentiative disorders, as well
as modulating (e.g. promoting or inhibiting) cell survival.
Moreover, the NOVX-immunoglobulin fusion proteins of the invention
can be used as immunogens to produce anti-NOVX antibodies in a
subject, to purify NOVX ligands, and in screening assays to
identify molecules that inhibit the interaction of NOVX with an
NOVX ligand.
[0445] An NOVX chimeric or fusion protein of the invention can be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
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 carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments that
can subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the NOVX protein.
[0446] NOVX Agonists and Antagonists
[0447] The invention also pertains to variants of the NOVX proteins
that function as either NOVX agonists (i.e., mimetics) or as NOVX
antagonists. Variants of the NOVX protein can be generated by
mutagenesis (e.g., discrete point mutation or truncation of the
NOVX protein). An agonist of the NOVX protein can retain
substantially the same, or a subset of, the biological activities
of the naturally occurring form of the NOVX protein. An antagonist
of the NOVX protein can inhibit one or more of the activities of
the naturally occurring form of the NOVX protein by, for example,
competitively binding to a downstream or upstream member of a
cellular signaling cascade which includes the NOVX protein. Thus,
specific biological effects can be elicited by treatment 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 NOVX proteins.
[0448] Variants of the NOVX proteins that function as either NOVX
agonists (i.e., mimetics) or as NOVX antagonists can be identified
by screening combinatorial libraries of mutants (e.g., truncation
mutants) of the NOVX proteins for NOVX protein agonist or
antagonist activity. In one embodiment, a variegated library of
NOVX variants is generated by combinatorial mutagenesis at the
nucleic acid level and is encoded by a variegated gene library. A
variegated library of NOVX variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential NOVX sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of NOVX sequences therein. There
are a variety of methods which can be used to produce libraries of
potential NOVX variants from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be performed
in an automatic DNA synthesizer, and the synthetic gene then
ligated into an appropriate expression vector. Use of a degenerate
set of genes allows for the provision, in one mixture, of all of
the sequences encoding the desired set of potential NOVX sequences.
Methods for synthesizing degenerate oligonucleotides are well-known
within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3;
Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et
al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res.
11: 477.
[0449] Polypeptide Libraries
[0450] In addition, libraries of fragments of the NOVX protein
coding sequences can be used to generate a variegated population of
NOVX fragments for screening and subsequent selection of variants
of an NOVX protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of an NOVX coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double-stranded DNA that can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S.sub.1 nuclease, and ligating
the resulting fragment library into an expression vector. By this
method, expression libraries can be derived which encodes
N-terminal and internal fragments of various sizes of the NOVX
proteins.
[0451] Various techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of NOVX proteins. The most widely used techniques,
which are amenable to high throughput analysis, for screening large
gene libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a new technique
that enhances the frequency of functional mutants in the libraries,
can be used in combination with the screening assays to identify
NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl.
Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein
Engineering 6:327-331.
[0452] NOVX Antibodies
[0453] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, single chain, F.sub.ab, F.sub.ab, and F.sub.(ab')2
fragments, and an F.sub.ab expression library. In general, antibody
molecules obtained from humans relates to any of the classes IgG,
IgM, IgA, IgE and IgD, which differ from one another by the nature
of the heavy chain present in the molecule. Certain classes have
subclasses as well, such as IgG.sub.1, IgG.sub.2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a
lambda chain. Reference herein to antibodies includes a reference
to all such classes, subclasses and types of human antibody
species.
[0454] An isolated protein of the invention intended to serve as an
antigen, or a portion or fragment thereof, can be used as an
immunogen to generate antibodies that immunospecifically bind the
antigen, using standard techniques for polyclonal and monoclonal
antibody preparation. The full-length protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of the antigen for use as immunogens. An antigenic peptide fragment
comprises at least 6 amino acid residues of the amino acid sequence
of the full length protein, such as an amino acid sequence shown in
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 26, 28, 40, 42, 44, 46, and 198, and encompasses an epitope
thereof such that an antibody raised against the peptide forms a
specific immune complex with the full length protein or with any
fragment that contains the epitope. Preferably, the antigenic
peptide comprises at least 10 amino acid residues, or at least 15
amino acid residues, or at least 20 amino acid residues, or at
least 30 amino acid residues. Preferred epitopes encompassed by the
antigenic peptide are regions of the protein that are located on
its surface; commonly these are hydrophilic regions.
[0455] In certain embodiments of the invention, at least one
epitope encompassed by the antigenic peptide is a region of NOVX
that is located on the surface of the protein, e.g., a hydrophilic
region. A hydrophobicity analysis of the human NOVX protein
sequence will indicate which regions of a NOVX polypeptide are
particularly hydrophilic and, therefore, are likely to encode
surface residues useful for targeting antibody production. As a
means for targeting antibody production, hydropathy plots showing
regions of hydrophilicity and hydrophobicity may be generated by
any method well known in the art, including, for example, the Kyte
Doolittle or the Hopp Woods methods, either with or without Fourier
transformation. See, e.g., Hopp and Woods, 1981, 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.
Antibodies that are specific for one or more domains within an
antigenic protein, or derivatives, fragments, analogs or homologs
thereof, are also provided herein.
[0456] A protein of the invention, or a derivative, fragment,
analog, homolog or ortholog thereof, may be utilized as an
immunogen in the generation of antibodies that immunospecifically
bind these protein components.
[0457] Various procedures known within the art may be used for the
production of polyclonal or monoclonal antibodies directed against
a protein of the invention, or against derivatives, fragments,
analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
incorporated herein by reference). Some of these antibodies are
discussed below.
Polyclonal Antibodies
[0458] For the production of polyclonal antibodies, various
suitable host animals (e.g., rabbit, goat, mouse or other mammal)
may be immunized by one or more injections with the native protein,
a synthetic variant thereof, or a derivative of the foregoing. An
appropriate immunogenic preparation can contain, for example, the
naturally occurring immunogenic protein, a chemically synthesized
polypeptide representing the immunogenic protein, or a
recombinantly expressed immunogenic protein. Furthermore, the
protein may be conjugated to a second protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. The preparation can further include an adjuvant.
Various adjuvants used to increase the immunological response
include, but are not limited to, Freund's (complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface
active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and
Corynebacterium parvum, or similar immunostimulatory agents.
Additional examples of adjuvants which can be employed include
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
[0459] The polyclonal antibody molecules directed against the
immunogenic protein can be isolated from the mammal (e.g., from the
blood) and further purified by well known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
Monodonal Antibodies
[0460] The term "monoclonal antibody" (MAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs thus contain an
antigen binding site capable of immunoreacting with a particular
epitope of the antigen characterized by a unique binding affinity
for it.
[0461] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes can be immunized in
vitro.
[0462] The immunizing agent will typically include the protein
antigen, a fragment thereof or a fusion protein thereof. Generally,
either peripheral blood lymphocytes are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells can be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0463] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies [Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0464] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the antigen. Preferably, the binding specificity
of monoclonal antibodies produced by the hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent
assay (ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980). It is an objective, especially important
in therapeutic applications of monoclonal antibodies, to identify
antibodies having a high degree of specificity and a high binding
affinity for the target antigen.
[0465] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods (Goding, 1986). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be
grown in vivo as ascites in a mammal.
[0466] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0467] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also can be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody.
Humanized Antibodies
[0468] The antibodies directed against the protein antigens of the
invention can further comprise humanized antibodies or human
antibodies. These antibodies are suitable for administration to
humans without engendering an immune response by the human against
the administered immunoglobulin. Humanized forms of antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab).sub.2 or other
antigen-binding subsequences of antibodies) that are principally
comprised of the sequence of a human immunoglobulin, and contain
minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. (See also U.S.
Pat. No. 5,225,539.) In some instances, Fv framework residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Humanized antibodies can also comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
Human Antibodies
[0469] Fully human antibodies essentially relate to antibody
molecules in which the entire sequence of both the light chain and
the heavy chain, including the CDRs, arise from human genes. Such
antibodies are termed "human antibodies", or "fully human
antibodies" herein. Human monoclonal antibodies can be prepared by
the trioma technique; the human B-cell hybridoma technique (see
Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma
technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,
Inc., pp. 77-96). Human monoclonal antibodies may be utilized in
the practice of the present invention and may be produced by using
human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr
Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES
AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
[0470] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies
can be made by introducing human immunoglobulin loci into
transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks
et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature
368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild
et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature
Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol. 13 65-93 (1995)).
[0471] Human antibodies may additionally be produced using
transgenic nonhuman animals which are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. (See PCT
publication WO94/02602). The endogenous genes encoding the heavy
and light immunoglobulin chains in the nonhuman host have been
incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. The preferred
embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells which secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv molecules.
[0472] An example of a method of producing a nonhuman host,
exemplified as a mouse, lacking expression of an endogenous
immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598.
It can be obtained by a method including deleting the J segment
genes from at least one endogenous heavy chain locus in an
embryonic stem cell to prevent rearrangement of the locus and to
prevent formation of a transcript of a rearranged immunoglobulin
heavy chain locus, the deletion being effected by a targeting
vector containing a gene encoding a selectable marker; and
producing from the embryonic stem cell a transgenic mouse whose
somatic and germ cells contain the gene encoding the selectable
marker.
[0473] A method for producing an antibody of interest, such as a
human antibody, is disclosed in U.S. Pat. No. 5,916,771. It
includes introducing an expression vector that contains a
nucleotide sequence encoding a heavy chain into one mammalian host
cell in culture, introducing an expression vector containing a
nucleotide sequence encoding a light chain into another mammalian
host cell, and fusing the two cells to form a hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and
the light chain.
[0474] In a further improvement on this procedure, a method for
identifying a clinically relevant epitope on an immunogen, and a
correlative method for selecting an antibody that binds
immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT publication WO 99/53049.
F.sub.ab Fragments and Single Chain Antibodies
[0475] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to an antigenic
protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of F.sub.ab
expression libraries (see e.g., Huse, et al., 1989 Science 246:
1275-1281) to allow rapid and effective identification of
monoclonal F.sub.ab fragments with the desired specificity for a
protein or derivatives, fragments, analogs or homologs thereof.
Antibody fragments that contain the idiotypes to a protein antigen
may be produced by techniques known in the art including, but not
limited to: (i) an F.sub.(ab')2 fragment produced by pepsin
digestion of an antibody molecule; (ii) an F.sub.ab fragment
generated by reducing the disulfide bridges of an F.sub.(ab')2
fragment; (iii) an F.sub.ab fragment generated by the treatment of
the antibody molecule with papain and a reducing agent and (iv)
F.sub.v fragments.
Bispecific Antibodies
[0476] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for an antigenic protein of the invention. The
second binding target is any other antigen, and advantageously is a
cell-surface protein or receptor or receptor subunit.
[0477] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305:537-539
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0478] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0479] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0480] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0481] Additionally, Fab' fragments can be directly recovered from
E. coli and chemically coupled to form bispecific antibodies.
Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized bispecific antibody F(ab').sub.2
molecule. Each Fab' fragment was separately secreted from E. coli
and subjected to directed chemical coupling in vitro to form the
bispecific antibody. The bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human
T cells, as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0482] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol. 148
(5):1547-1553 (1992). The leucine zipper peptides from the Fos and
Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0483] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0484] Exemplary bispecific antibodies can bind to two different
epitopes, at least one of which originates in the protein antigen
of the invention. Alternatively, an anti-antigenic arm of an
immunoglobulin molecule can be combined with an arm which binds to
a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the cell expressing the particular antigen. Bispecific antibodies
can also be used to direct cytotoxic agents to cells which express
a particular antigen. These antibodies possess an antigen-binding
arm and an arm which binds a cytotoxic agent or a radionuclide
chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific
antibody of interest binds the protein antigen described herein and
further binds tissue factor (TF).
Heteroconjugate Antibodies
[0485] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
Effector Function Engineering
[0486] It can be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) can be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity can also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design. 3: 219-230 (1989).
Immunoconjugates
[0487] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0488] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0489] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0490] In another embodiment, the antibody can be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is in turn
conjugated to a cytotoxic agent.
Immunoliposomes
[0491] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0492] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81 (19): 1484 (1989).
Diagnostic Applications of Antibodies Directed Against the Proteins
of the Invention
[0493] Antibodies directed against a protein of the invention may
be used in methods known within the art relating to the
localization and/or quantitation of the protein (e.g., for use in
measuring levels of the protein within appropriate physiological
samples, for use in diagnostic methods, for use in imaging the
protein, and the like). In a given embodiment, antibodies against
the proteins, or derivatives, fragments, analogs or homologs
thereof, that contain the antigen binding domain, are utilized as
pharmacologically-active compounds (see below).
[0494] An antibody specific for a protein of the invention can be
used to isolate the protein by standard techniques, such as
immunoaffinity chromatography or immunoprecipitation. Such an
antibody can facilitate the purification of the natural protein
antigen from cells and of recombinantly produced antigen expressed
in host cells. Moreover, such an antibody can be used to detect the
antigenic protein (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
antigenic protein. Antibodies directed against the protein 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, O-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include .sup.125I, 131I, .sup.35S
or .sup.3H.
Antibody Therapeutics
[0495] Antibodies of the invention, including polyclonal,
monoclonal, humanized and fully human antibodies, may used as
therapeutic agents. Such agents will generally be employed to treat
or prevent a disease or pathology in a subject. An antibody
preparation, preferably one having high specificity and high
affinity for its target antigen, is administered to the subject and
will generally have an effect due to its binding with the target.
Such an effect may be one of two kinds, depending on the specific
nature of the interaction between the given antibody molecule and
the target antigen in question. In the first instance,
administration of the antibody may abrogate or inhibit the binding
of the target with an endogenous ligand to which it naturally
binds. In this case, the antibody binds to the target and masks a
binding site of the naturally occurring ligand, wherein the ligand
serves as an effector molecule. Thus the receptor mediates a signal
transduction pathway for which ligand is responsible.
[0496] Alternatively, the effect may be one in which the antibody
elicits a physiological result by virtue of binding to an effector
binding site on the target molecule. In this case the target, a
receptor having an endogenous ligand which may be absent or
defective in the disease or pathology, binds the antibody as a
surrogate effector ligand, initiating a receptor-based signal
transduction event by the receptor.
[0497] A therapeutically effective amount of an antibody of the
invention relates generally to the amount needed to achieve a
therapeutic objective. As noted above, this may be a binding
interaction between the antibody and its target antigen that, in
certain cases, interferes with the functioning of the target, and
in other cases, promotes a physiological response. The amount
required to be administered will furthermore depend on the binding
affinity of the antibody for its specific antigen, and will also
depend on the rate at which an administered antibody is depleted
from the free volume other subject to which it is administered.
Common ranges for therapeutically effective dosing of an antibody
or antibody fragment of the invention may be, by way of nonlimiting
example, from about 0.1 mg/kg body weight to about 50 mg/kg body
weight. Common dosing frequencies may range, for example, from
twice daily to once a week.
Pharmaceutical Compositions of Antibodies
[0498] Antibodies specifically binding a protein of the invention,
as well as other molecules identified by the screening assays
disclosed herein, can be administered for the treatment of various
disorders in the form of pharmaceutical compositions. Principles
and considerations involved in preparing such compositions, as well
as guidance in the choice of components are provided, for example,
in Remington: The Science And Practice Of Pharmacy 19th ed.
(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.:
1995; Drug Absorption Enhancement: Concepts, Possibilities,
Limitations, And Trends, Harwood Academic Publishers, Langhorne,
Pa., 1994; and Peptide And Protein Drug Delivery (Advances In
Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
[0499] If the antigenic protein is intracellular and whole
antibodies are used as inhibitors, internalizing antibodies are
preferred. However, liposomes can also be used to deliver the
antibody, or an antibody fragment, into cells. Where antibody
fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is
preferred. For example, based upon the variable-region sequences of
an antibody, peptide molecules can be designed that retain the
ability to bind the target protein sequence. Such peptides can be
synthesized chemically and/or produced by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Nad. Acad. Sci. USA,
90: 7889-7893 (1993). The formulation herein can also contain more
than one active compound as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect each other. Alternatively, or in addition,
the composition can comprise an agent that enhances its function,
such as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0500] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions.
[0501] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0502] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ediyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrdgels release proteins
for shorter time periods.
ELISA Assay
[0503] An agent for detecting an analyte protein is an antibody
capable of binding to an analyte 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., F.sub.ab or F(ab).sub.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. Included within the usage of the term "biological
sample", therefore, is blood and a fraction or component of blood
including blood serum, blood plasma, or lymph. That is, the
detection method of the invention can be used to detect an analyte
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
an analyte mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of an analyte
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence. In
vitro techniques for detection of an analyte genomic DNA include
Southern hybridizations. Procedures for conducting immunoassays are
described, for example in "ELISA: Theory and Practice: Methods in
Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press,
Totowa, N.J., 1995; "Immunoassay", E. Diamandis and T.
Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and
"Practice and Thory of Enzyme Immunoassays", P. Tijssen, Elsevier
Science Publishers, Amsterdam, 1985. Furthermore, in vivo
techniques for detection of an analyte protein include introducing
into a subject a labeled anti-an analyte protein 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.
[0504] NOVX Recombinant Expression Vectors and Host Cells
[0505] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an NOVX protein, or derivatives, fragments, analogs or homologs
thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) 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-linked. Such
vectors are referred to herein as "expression vectors". In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" can be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0506] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively-linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector,
"operably-linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell).
[0507] The term "regulatory sequence" is intended to includes
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and
those that direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., NOVX proteins, mutant forms of NOVX
proteins, fusion proteins, etc.).
[0508] The recombinant expression vectors of the invention can be
designed for expression of NOVX proteins in prokaryotic or
eukaryotic cells. For example, NOVX proteins can be expressed in
bacterial cells such as 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.
[0509] 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: (1) to increase expression of recombinant protein; (it)
to increase the solubility of the recombinant protein; and (iii) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0510] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0511] 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.
[0512] In another embodiment, the NOVX expression vector is a yeast
expression vector. Examples of vectors for expression in yeast
Saccharomyces cerivisae include pYepSec1 (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
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[0513] Alternatively, NOVX can be expressed in insect cells using
baculovirus expression vectors. Baculovirus vectors available for
expression of proteins in cultured insect cells (e.g., SF9 cells)
include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology
170: 31-39).
[0514] 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 (Kaufman, et al., 1987.
EMBO J. 6: 187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0515] 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 (Baneiji, 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 mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the .alpha.-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
[0516] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively-linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to NOVX mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen that direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see, e.g., Weintraub, et al.,
"Antisense RNA as a molecular tool for genetic analysis,"
Reviews-Trends in Genetics, Vol. 1 (1) 1986.
[0517] 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.
[0518] A host cell can be any prokaryotic or eukaryotic cell. For
example, NOVX protein can be expressed in bacterial cells such as
E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0519] 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,
DEAE-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.
[0520] 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 G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding NOVX or can be introduced on a separate vector. Cells
stably transfected with the introduced nucleic acid can be
identified by drug selection (e.g., cells that have incorporated
the selectable marker gene will survive, while the other cells
die).
[0521] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) NOVX protein. Accordingly, the invention further provides
methods for producing NOVX protein using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of invention (into which a recombinant expression vector
encoding NOVX protein has been introduced) in a suitable medium
such that NOVX protein is produced. In another embodiment, the
method further comprises isolating NOVX protein from the medium or
the host cell.
[0522] Transgenic NOVX Animals
[0523] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which NOVX protein-coding sequences have been
introduced. Such host cells can then be used to create non-human
transgenic animals in which exogenous NOVX sequences have been
introduced into their genome or homologous recombinant animals in
which endogenous NOVX sequences have been altered. Such animals are
useful for studying the function and/or activity of NOVX protein
and for identifying and/or evaluating modulators of NOVX protein
activity. As used herein, a "transgenic animal" is a non-human
animal, preferably a mammal, more preferably a rodent such as a rat
or mouse, in which one or more of the cells of the animal includes
a transgene. Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A
transgene is exogenous DNA that is integrated into the genome of a
cell from which a transgenic animal develops and that remains in
the genome of the mature animal, thereby directing the expression
of an encoded gene product in one or more cell types or tissues of
the transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous NOVX gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0524] A transgenic animal of the invention can be created by
introducing NOVX-encoding nucleic acid into the male pronuclei of a
fertilized oocyte (e.g., by microinjection, retroviral infection)
and allowing the oocyte to develop in a pseudopregnant female
foster animal. The human NOVX cDNA sequences SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, and 197 can be introduced as a transgene into the genome of
a non-human animal. Alternatively, a non-human homologue of the
human NOVX gene, such as a mouse NOVX gene, can be isolated based
on hybridization to the human NOVX cDNA (described further supra)
and used as a transgene. Intronic sequences and polyadenylation
signals can also be included in the transgene to increase the
efficiency of expression of the transgene. A tissue-specific
regulatory sequence(s) can be operably-linked to the NOVX transgene
to direct expression of NOVX protein to particular cells. Methods
for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become
conventional in the art and are described, for example, in U.S.
Pat. 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 NOVX transgene in its
genome and/or expression of NOVX mRNA in tissues or cells of the
animals. A transgenic founder animal can then be used to breed
additional animals carrying the transgene. Moreover, transgenic
animals carrying a transgene-encoding NOVX protein can further be
bred to other transgenic animals carrying other transgenes.
[0525] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of an NOVX gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX
gene can be a human gene (e.g., the cDNA of SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, and 197), but more preferably, is a non-human homologue of
a human NOVX gene. For example, a mouse homologue of human NOVX
gene of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197 can be used to
construct a homologous recombination vector suitable for altering
an endogenous NOVX gene in the mouse genome. In one embodiment, the
vector is designed such that, upon homologous recombination, the
endogenous NOVX gene is functionally disrupted (i.e., no longer
encodes a functional protein; also referred to as a "knock out"
vector).
[0526] Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous NOVX gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous NOVX protein). In the homologous
recombination vector, the altered portion of the NOVX gene is
flanked at its 5'- and 3'-termini by additional nucleic acid of the
NOVX gene to allow for homologous recombination to occur between
the exogenous NOVX gene carried by the vector and an endogenous
NOVX gene in an embryonic stem cell. The additional flanking NOVX
nucleic acid is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several
kilobases of flanking DNA (both at the 5'- and 3'-termini) are
included in the vector. See, e.g., Thomas, et al., 1987. Cell 51:
503 for a description of homologous recombination vectors. The
vector is ten introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced NOVX gene has
homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
[0527] 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.
[0528] 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
acre/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.
[0529] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
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.
[0530] Pharmaceutical Compositions
[0531] The NOVX nucleic acid molecules, NOVX proteins, and
anti-NOVX antibodies (also referred to herein as "active
compounds") of the invention, and derivatives, fragments, analogs
and homologs thereof, can be incorporated into pharmaceutical
compositions suitable for administration. Such compositions
typically comprise the nucleic acid molecule, protein, or antibody
and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable 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.
[0532] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0533] 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 EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0534] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an NOVX protein or
anti-NOVX antibody) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, methods of preparation are vacuum drying and
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0535] 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 like 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.
[0536] 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.
[0537] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0538] 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.
[0539] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including 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. Pat. No.
4,522,811.
[0540] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0541] 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. Pat. 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.
[0542] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0543] Screening and Detection Methods
[0544] The isolated nucleic acid molecules of the invention can be
used to express NOVX protein (e.g., via a recombinant expression
vector in a host cell in gene therapy applications), to detect NOVX
mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX
gene, and to modulate NOVX activity, as described further, below.
In addition, the NOVX proteins can be used to screen drugs or
compounds that modulate the NOVX protein activity or expression as
well as to treat disorders characterized by insufficient or
excessive production of NOVX protein or production of NOVX protein
forms that have decreased or aberrant activity compared to NOVX
wild-type protein (e.g.; diabetes (regulates insulin release);
obesity (binds and transport lipids); metabolic disturbances
associated with obesity, the metabolic syndrome X as well as
anorexia and wasting disorders associated with chronic diseases and
various cancers, and infectious disease(possesses anti-microbial
activity) and the various dyslipidemias. In addition, the anti-NOVX
antibodies of the invention can be used to detect and isolate NOVX
proteins and modulate NOVX activity. In yet a further aspect, the
invention can be used in methods to influence appetite, absorption
of nutrients and the disposition of metabolic substrates in both a
positive and negative fashion.
[0545] The invention further pertains to novel agents identified by
the screening assays described herein and uses thereof for
treatments as described, supra.
[0546] Screening Assays
[0547] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) that bind to NOVX proteins or have a
stimulatory or inhibitory effect on, e.g., NOVX protein expression
or NOVX protein activity. The invention also includes compounds
identified in the screening assays described herein.
[0548] In one embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of the membrane-bound form of an NOVX protein or
polypeptide or biologically-active portion thereof. The test
compounds of the invention can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug
Design 12: 145.
[0549] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD and
most preferably less than about 4 kD. Small molecules can be, e.g.,
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules.
Libraries of chemical and/or biological mixtures, such as fungal,
bacterial, or algal extracts, are known in the art and can be
screened with any of the assays of the invention.
[0550] Examples of methods for the synthesis of molecular libraries
can 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: 2059; Carell, et al.,
1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al.,
1994. J. Med. Chem. 37: 1233.
[0551] Libraries of compounds may 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. Pat. No. 5,223,409), spores (Ladner, U.S.
Pat. No. 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. Pat. No.
5,233,409.).
[0552] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a membrane-bound form of NOVX protein, or a
biologically-active portion thereof, on the cell surface is
contacted with a test compound and the ability of the test compound
to bind to an NOVX protein determined. The cell, for example, can
of mammalian origin or a yeast cell. Determining the ability of the
test compound to bind to the NOVX protein can be accomplished, for
example, by coupling the test compound with a radioisotope or
enzymatic label such that binding of the test compound to the NOVX
protein or biologically-active portion thereof can be determined by
detecting the labeled compound in a complex. For example, test
compounds can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemission or by scintillation
counting. Alternatively, test compounds can be
enzymatically-labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product. In one embodiment, the assay comprises contacting a
cell which expresses a membrane-bound form of NOVX protein, or a
biologically-active portion thereof, on the cell surface with a
known compound which binds NOVX to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with an NOVX protein,
wherein determining the ability of the test compound to interact
with an NOVX protein comprises determining the ability of the test
compound to preferentially bind to NOVX protein or a
biologically-active portion thereof as compared to the known
compound.
[0553] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a membrane-bound form of
NOVX protein, or a biologically-active portion thereof, on the cell
surface with a test compound and determining the ability of the
test compound to modulate (e.g., stimulate or inhibit) the activity
of the NOVX protein or biologically-active portion thereof.
Determining the ability of the test compound to modulate the
activity of NOVX or a biologically-active portion thereof can be
accomplished, for example, by determining the ability of the NOVX
protein to bind to or interact with an NOVX target molecule. As
used herein, a "target molecule" is a molecule with which an NOVX
protein binds or interacts in nature, for example, a molecule on
the surface of a cell which expresses an NOVX interacting protein,
a molecule on the surface of a second cell, a molecule in the
extracellular milieu, a molecule associated with the internal
surface of a cell membrane or a cytoplasmic molecule. An NOVX
target molecule can be a non-NOVX molecule or an NOVX protein or
polypeptide of the invention. In one embodiment, an NOVX target
molecule is a component of a signal transduction pathway that
facilitates transduction of an extracellular signal (e.g. a signal
generated by binding of a compound to a membrane-bound NOVX
molecule) through the cell membrane and into the cell. The target,
for example, can be a second intercellular protein that has
catalytic activity or a protein that facilitates the association of
downstream signaling molecules with NOVX.
[0554] Determining the ability of the NOVX protein to bind to or
interact with an NOVX target molecule can be accomplished by one of
the methods described above for determining direct binding. In one
embodiment, determining the ability of the NOVX protein to bind to
or interact with an NOVX target molecule can be accomplished by
determining the activity of the target molecule. For example, the
activity of the target molecule can be determined by detecting
induction of a cellular second messenger of the target (i.e.
intracellular Ca.sup.2+, diacylglycerol, IP.sub.3, etc.), detecting
catalytic/enzymatic activity of the target an appropriate
substrate, detecting the induction of a reporter gene (comprising
an NOVX-responsive regulatory element operatively linked to a
nucleic acid encoding a detectable marker, e.g., luciferase), or
detecting a cellular response, for example, cell survival, cellular
differentiation, or cell proliferation.
[0555] In yet another embodiment, an assay of the invention is a
cell-free assay comprising contacting an NOVX protein or
biologically-active portion thereof with a test compound and
determining the ability of the test compound to bind to the NOVX
protein or biologically-active portion thereof. Binding of the test
compound to the NOVX protein can be determined either directly or
indirectly as described above. In one such embodiment, the assay
comprises contacting the NOVX protein or biologically-active
portion thereof with a known compound which binds NOVX to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
an NOVX protein, wherein determining the ability of the test
compound to interact with an NOVX protein comprises determining the
ability of the test compound to preferentially bind to NOVX or
biologically-active portion thereof as compared to the known
compound.
[0556] In still another embodiment, an assay is a cell-free assay
comprising contacting NOVX protein or biologically-active portion
thereof with a test compound and determining the ability of the
test compound to modulate (e.g. stimulate or inhibit) the activity
of the NOVX protein or biologically-active portion thereof.
Determining the ability of the test compound to modulate the
activity of NOVX can be accomplished, for example, by determining
the ability of the NOVX protein to bind to an NOVX target molecule
by one of the methods described above for determining direct
binding. In an alternative embodiment, determining the ability of
the test compound to modulate the activity of NOVX protein can be
accomplished by determining the ability of the NOVX protein further
modulate an NOVX target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as described, supra.
[0557] In yet another embodiment, the cell-free assay comprises
contacting the NOVX protein or biologically-active portion thereof
with a known compound which binds NOVX protein to form an assay
mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to interact with an
NOVX protein, wherein determining the ability of the test compound
to interact with an NOVX protein comprises determining the ability
of the NOVX protein to preferentially bind to or modulate the
activity of an NOVX target molecule.
[0558] The cell-free assays of the invention are amenable to use of
both the soluble form or the membrane-bound form of NOVX protein.
In the case of cell-free assays comprising the membrane-bound form
of NOVX protein, it may be desirable to utilize a solubilizing
agent such that the membrane-bound form of NOVX protein is
maintained in solution. Examples of such solubilizing agents
include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.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).
[0559] In more than one embodiment of the above assay methods of
the invention, it may be desirable to immobilize either NOVX
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 NOVX protein, or interaction of NOVX protein with a
target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided that adds a domain that allows one or both
of the proteins to be bound to a matrix. For example, GST-NOVX
fusion proteins or GST-target fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microfiter plates, that are then combined
with the test compound or the test compound and either the
non-adsorbed target protein or NOVX protein, and the mixture is
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described, supra. Alternatively, the complexes can be dissociated
from the matrix, and the level of NOVX protein binding or activity
determined using standard techniques.
[0560] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either the NOVX protein or its target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated NOVX
protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with NOVX
protein or target molecules, but which do not interfere with
binding of the NOVX protein to its target molecule, can be
derivatized to the wells of the plate, and unbound target or NOVX
protein trapped in the wells by antibody conjugation. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the NOVX protein or target molecule,
as well as enzyme-linked assays that rely on detecting an enzymatic
activity associated with the NOVX protein or target molecule.
[0561] In another embodiment, modulators of NOVX protein expression
are identified in a method wherein a cell is contacted with a
candidate compound and the expression of NOVX mRNA or protein in
the cell is determined. The level of expression of NOVX mRNA or
protein in the presence of the candidate compound is compared to
the level of expression of NOVX mRNA or protein in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of NOVX mRNA or protein expression based
upon this comparison. For example, when expression of NOVX mRNA or
protein is greater (i.e., statistically significantly greater) in
the presence of the candidate compound than in its absence, the
candidate compound is identified as a stimulator of NOVX mRNA or
protein expression. Alternatively, when expression of NOVX 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 NOVX mRNA or protein
expression. The level of NOVX mRNA or protein expression in the
cells can be determined by methods described herein for detecting
NOVX mRNA or protein.
[0562] In yet another aspect of the invention, the NOVX proteins
can be used as "bait proteins" in a two-hybrid assay or three
hybrid assay (see, e.g., U.S. Pat. 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
NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX
activity. Such NOVX-binding proteins are also likely to be involved
in the propagation of signals by the NOVX proteins as, for example,
upstream or downstream elements of the NOVX pathway.
[0563] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for NOVX is fused
to a gene encoding the DNA binding domain of a known transcription
factor (e.g., GAL-4). In the other construct, a DNA sequence, from
a library of DNA sequences, that encodes an unidentified protein
("prey" or "sample") is fused to a gene that codes for the
activation domain of the known transcription factor. If the "bait"
and the "prey" proteins are able to interact, in vivo, forming an
NOVX-dependent complex, the DNA-binding and activation domains of
the transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ) that
is operably linked to a transcriptional regulatory site responsive
to the transcription factor. Expression of the reporter gene can be
detected and cell colonies containing the functional transcription
factor can be isolated and used to obtain the cloned gene that
encodes the protein which interacts with NOVX.
[0564] The invention further pertains to novel agents identified by
the aforementioned screening assays and uses thereof for treatments
as described herein.
[0565] Detection Assays
[0566] 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
[0567] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the NOVX sequences,
SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, and 197, or fragments or
derivatives thereof, can be used to map the location of the NOVX
genes, respectively, on a chromosome. The mapping of the NOVX
sequences to chromosomes is an important first step in correlating
these sequences with genes associated with disease.
[0568] Briefly, NOVX genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the NOVX
sequences. Computer analysis of the NOVX, sequences can be used to
rapidly select primers that do not span more than one exon in the
genomic DNA, thus complicating the amplification process. These
primers can then be used for PCR screening of somatic cell hybrids
containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the NOVX sequences will
yield an amplified fragment.
[0569] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but in which human cells can, the one human
chromosome that contains the gene encoding the needed enzyme will
be retained. By using various media, panels of hybrid cell lines
can be established. Each cell line in a panel contains either a
single human chromosome or a small number of human chromosomes, and
a full set of mouse chromosomes, allowing easy mapping of
individual genes to specific human chromosomes. See, e.g.,
D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell
hybrids containing only fragments of human chromosomes can also be
produced by using human chromosomes with translocations and
deletions.
[0570] 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 NOVX sequences to design oligonucleotide primers,
sub-localization can be achieved with panels of fragments from
specific chromosomes.
[0571] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical like colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases, will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see, Verma, et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC
TECHNIQUES (Pergamon Press, New York 1988).
[0572] 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.
[0573] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, e.g.,
in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line
through Johns Hopkins University Welch Medical Library). The
relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g.,
Egeland, et al., 1987. Nature, 325: 783-787.
[0574] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the NOVX gene, can be determined. If a mutation is observed in some
or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
Tissue Typing
[0575] The NOVX sequences of the invention can also be used to
identify individuals from minute biological samples. In this
technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique
bands for identification. The sequences of the invention are useful
as additional DNA markers for RFLP ("restriction fragment length
polymorphisms," described in U.S. Pat. No. 5,272,057).
[0576] Furthermore, the sequences of the invention can be used to
provide an alternative technique that determines the actual
base-by-base DNA sequence of selected portions of an individual's
genome. Thus, the NOVX sequences described herein can be used to
prepare two PCR primers from the 5'- and 3'-termini of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0577] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
invention can be used to obtain such identification sequences from
individuals and from tissue. The NOVX sequences of the invention
uniquely represent portions of the human genome. Allelic variation
occurs to some degree in the coding regions of these sequences, and
to a greater degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a frequency
of about once per each 500 bases. Much of the allelic variation is
due to single nucleotide polymorphisms (SNPs), which include
restriction fragment length polymorphisms (RFLPs).
[0578] Each of the sequences described herein can, to some degree,
be used as a standard against which DNA from an individual can be
compared for identification purposes. Because greater numbers of
polymorphisms occur in the noncoding regions, fewer sequences are
necessary to differentiate individuals. The noncoding sequences can
comfortably provide positive individual identification with a panel
of perhaps 10 to 1,000 primers that each yield a noncoding
amplified sequence of 100 bases. If predicted coding sequences,
such as those in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 197 are used, a
more appropriate number of primers for positive individual
identification would be 500-2,000.
Predictive Medicine
[0579] The invention also pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trials are used for
prognostic (predictive) purposes to thereby treat an
individual-prophylactically. Accordingly, one aspect of the
invention relates to diagnostic assays for determining NOVX protein
and/or nucleic acid expression as well as NOVX activity, in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to thereby determine whether an individual is afflicted with a
disease or disorder, or is at risk of developing a disorder,
associated with aberrant NOVX expression or activity. The disorders
include metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-associated cachexia, cancer, neurodegenerative
disorders, Alzheimer's Disease, Parkinson's Disorder, immune
disorders, and hematopoietic disorders, and the various
dyslipidemias, metabolic disturbances associated with obesity, the
metabolic syndrome X and wasting disorders associated with chronic
diseases and various cancers. The invention also provides for
prognostic (or predictive) assays for determining whether an
individual is at risk of developing a disorder associated with NOVX
protein, nucleic acid expression or activity. For example,
mutations in an NOVX gene can be assayed in a biological sample.
Such assays can be used for prognostic or predictive purpose to
thereby prophylactically treat an individual prior to the onset of
a disorder characterized by or associated with NOVX protein,
nucleic acid expression, or biological activity.
[0580] Another aspect of the invention provides methods for
determining NOVX protein, nucleic acid expression or activity in an
individual to thereby select appropriate therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics"). Pharmacogenomics allows for the selection of
agents (e.g., drugs) for therapeutic or prophylactic treatment of
an individual based on the genotype of the individual (e.g., the
genotype of the individual examined to determine the ability of the
individual to respond to a particular agent.)
[0581] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs, compounds) on the expression
or activity of NOVX in clinical trials.
[0582] These and other agents are described in further detail in
the following sections.
[0583] Diagnostic Assays
[0584] An exemplary method for detecting the presence or absence of
NOVX in a biological sample involves obtaining a biological sample
from a test subject and contacting the biological sample with a
compound or an agent capable of detecting NOVX protein or nucleic
acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that
the presence of NOVX is detected in the biological sample. An agent
for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid
probe capable of hybridizing to NOVX mRNA or genomic DNA. The
nucleic acid probe can be, for example, a full-length NOVX nucleic
acid, such as the nucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and
197, or a portion thereof, such as an oligonucleotide of at least
15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to
specifically hybridize under stringent conditions to NOVX mRNA or
genomic DNA. Other suitable probes for use in the diagnostic assays
of the invention are described herein.
[0585] An agent for detecting NOVX protein is an antibody capable
of binding to NOVX protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g.,
F.sub.ab or F(ab).sub.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 NOVX mRNA, protein, or genomic DNA in a biological
sample in vitro as well as in vivo. For example, in vitro
techniques for detection of NOVX mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of NOVX protein include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of NOVX
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of NOVX protein include introducing into a
subject a labeled anti-NOVX antibody. For example, the antibody can
be labeled with a radioactive marker whose presence and location in
a subject can be detected by standard imaging techniques.
[0586] 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.
[0587] 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 NOVX
protein, mRNA, or genomic DNA, such that the presence of NOVX
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of NOVX protein, mRNA or genomic DNA in
the control sample with the presence of NOVX protein, mRNA or
genomic DNA in the test sample.
[0588] The invention also encompasses kits for detecting the
presence of NOVX in a biological sample. For example, the kit can
comprise: a labeled compound or agent capable of detecting NOVX
protein or mRNA in a biological sample; means for determining the
amount of NOVX in the sample; and means for comparing the amount of
NOVX in the sample with a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect NOVX protein or nucleic
acid.
[0589] Prognostic Assays
[0590] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
disease or disorder associated with aberrant NOVX expression or
activity. For example, the assays described herein, such as the
preceding diagnostic assays or the following assays, can be
utilized to identify a subject having or at risk of developing a
disorder associated with NOVX protein, nucleic acid expression or
activity. Alternatively, the prognostic assays can be utilized to
identify a subject having or at risk for developing a disease or
disorder. Thus, the invention provides a method for identifying a
disease or disorder associated with aberrant NOVX expression or
activity in which a test sample is obtained from a subject and NOVX
protein or nucleic acid (e.g., mRNA, genomic DNA) is detected,
wherein the presence of NOVX protein or nucleic acid is diagnostic
for a subject having or at risk of developing a disease or disorder
associated with aberrant NOVX expression or activity. As used
herein, a "test sample" refers to a biological sample obtained from
a subject of interest. For example, a test sample can be a
biological fluid (e.g., serum), cell sample, or tissue.
[0591] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant NOVX expression or
activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
disorder. Thus, the invention provides methods for determining
whether a subject can be effectively treated with an agent for a
disorder associated with aberrant NOVX expression or activity in
which a test sample is obtained and NOVX protein or nucleic acid is
detected (e.g., wherein the presence of NOVX protein or nucleic
acid is diagnostic for a subject that can be administered the agent
to treat a disorder associated with aberrant NOVX expression or
activity).
[0592] The methods of the invention can also be used to detect
genetic lesions in an NOVX gene, thereby determining if a subject
with the lesioned gene is at risk for a disorder characterized by
aberrant cell proliferation and/or differentiation. In various
embodiments, the methods include detecting, in a sample of cells
from the subject, the presence or absence of a genetic lesion
characterized by at least one of an alteration affecting the
integrity of a gene encoding an NOVX-protein, or the misexpression
of the NOVX gene. For example, such genetic lesions can be detected
by ascertaining the existence of at least one of: (i) a deletion of
one or more nucleotides from an NOVX gene; (ii) an addition of one
or more nucleotides to an NOVX gene; (iii) a substitution of one or
more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement
of an NOVX gene; (v) an alteration in the level of a messenger RNA
transcript of an NOVX gene, (vi) aberrant modification of an NOVX
gene, such as of the methylation pattern of the genomic DNA, (vii)
the presence of a non-wild-type splicing pattern of a messenger RNA
transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX
protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate
post-translational modification of an NOVX protein. As described
herein, there are a large number of assay techniques known in the
art which can be used for detecting lesions in an NOVX gene. A
preferred biological sample is a peripheral blood leukocyte sample
isolated by conventional means from a subject. However, any
biological sample containing nucleated cells may be used,
including, for example, buccal mucosal cells.
[0593] 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. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and
Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364),
the latter of which can be particularly useful for detecting point
mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl.
Acids Res. 23: 675-682). This method can include the steps of
collecting a sample of cells from a patient, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers that
specifically hybridize to an NOVX gene under conditions such that
hybridization and amplification of the NOVX gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[0594] 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.beta. 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.
[0595] In an alternative embodiment, mutations in an NOVX gene from
a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
e.g., U.S. Pat. No. 5,493,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0596] In other embodiments, genetic mutations in NOVX can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high-density arrays containing hundreds or thousands
of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For
example, genetic mutations in NOVX can be identified in two
dimensional arrays containing light-generated DNA probes as
described in Cronin, et al., supra. Briefly, a first hybridization
array of probes can be used to scan through long stretches of DNA
in a sample and control to identify base changes between the
sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This is
followed by a second hybridization array that allows the
characterization of specific mutations by using smaller,
specialized probe arrays complementary to all variants or mutations
detected. Each mutation array is composed of parallel probe sets,
one complementary to the wild-type gene and the other complementary
to the mutant gene.
[0597] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
NOVX gene and detect mutations by comparing the sequence of the
sample NOVX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA
74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is
also contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
(see, e.g., Naeve, et al., 1995. Biotechniques 19: 448), including
sequencing by mass spectrometry (see, e.g., PCT International
Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36: 127-162; and Griffin, et al., 1993. Appl.
Biochem. Biotechnol. 38: 147-159).
[0598] Other methods for detecting mutations in the NOVX gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See,
e.g., Myers, et al., 1985. Science 230: 1242. In general, the art
technique of "mismatch cleavage" starts by providing heteroduplexes
of formed by hybridizing (labeled) RNA or DNA containing the
wild-type NOVX sequence with potentially mutant RNA or DNA obtained
from a tissue sample. The double-stranded duplexes are treated with
an agent that cleaves single-stranded regions of the duplex such as
which will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S.sub.1 nuclease to
enzymatically digesting the mismatched regions. In other
embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to
digest mismatched regions. After digestion of the mismatched
regions, the resulting material is then separated by size on
denaturing polyacrylamide gels to determine the site of mutation.
See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an
embodiment, the control DNA or RNA can be labeled for
detection.
[0599] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in NOVX
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g.,
Hsu, et al., 1994. Carcinogenesis 15: 1657-1662. According to an
exemplary embodiment, a probe based on an NOVX sequence, e.g., a
wild-type NOVX sequence, is hybridized to a cDNA or other DNA
product from a test cell(s). The duplex is treated with a DNA
mismatch repair enzyme, and the cleavage products, if any, can be
detected from electrophoresis protocols or the like. See, e.g.,
U.S. Pat. No. 5,459,039.
[0600] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in NOVX genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc.
Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285:
125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79.
Single-stranded DNA fragments of sample and control NOVX nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In one embodiment, the subject method utilizes
heteroduplex analysis to separate double stranded heteroduplex
molecules on the basis of changes in electrophoretic mobility. See,
e.g., Keen, et al., 1991. Trends Genet., 7: 5.
[0601] 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 bp of high-melting GC-rich DNA by PCR. In
a further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987.
Biophys. Chem. 265: 12753.
[0602] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the 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.
[0603] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification may carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl.
Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one
primer where, under appropriate conditions, mismatch can prevent,
or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech.
11: 238). In addition it may be desirable to introduce a novel
restriction site in the region of the mutation to create
cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol.
Cell Probes 6: 1. It is anticipated that in certain embodiments
amplification may also be performed using Taq ligase for
amplification. See, e.g., Barany, 1991. Proc. Natl. 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.
[0604] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving an NOVX gene.
[0605] Furthermore, any cell type or tissue, preferably peripheral
blood leukocytes, in which NOVX is expressed may be utilized in the
prognostic assays described herein. However, any biological sample
containing nucleated cells may be used, including, for example,
buccal mucosal cells.
[0606] Pharmacogenomics
[0607] Agents, or modulators that have a stimulatory or inhibitory
effect on NOVX activity (e.g., NOVX gene expression), as identified
by a screening assay described herein can be administered to
individuals to treat (prophylactically or therapeutically)
disorders (The disorders include metabolic disorders, diabetes,
obesity, infectious disease, anorexia, cancer-associated cachexia,
cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's Disorder, immune disorders, and hematopoietic
disorders, and the various dyslipidemias, metabolic disturbances
associated with obesity, the metabolic syndrome X and wasting
disorders associated with chronic diseases and various cancers.) In
conjunction with such treatment, the pharmacogenomics (i.e., the
study of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) of the
individual may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the
individual permits the selection of effective agents (e.g., drugs)
for prophylactic or therapeutic treatments based on a consideration
of the individual's genotype. Such pharmacogenomics can further be
used to determine appropriate dosages and therapeutic regimens.
Accordingly, the activity of NOVX protein, expression of NOVX
nucleic acid, or mutation content of NOVX genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual.
[0608] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See e.g.,
Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985;
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.
[0609] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome PREGNANCY ZONE PROTEIN PRECURSOR 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 taking the standard and safe dose of a
drug. These polymorphisms are expressed in two phenotypes in the
population, the extensive metabolizer (EM) and poor metabolizer
(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.
[0610] Thus, the activity of NOVX protein, expression of NOVX
nucleic acid, or mutation content of NOVX genes in an individual
can be determined to thereby select appropriate agent(s) 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
an NOVX modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0611] Monitoring of Effects During Clinical Trials
[0612] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of NOVX (e.g., the ability to
modulate aberrant cell proliferation and/or differentiation) can be
applied not only in basic drug screening, but also in clinical
trials. For example, the effectiveness of an agent determined by a
screening assay as described herein to increase NOVX gene
expression, protein levels, or upregulate NOVX activity, can be
monitored in clinical trails of subjects exhibiting decreased NOVX
gene expression, protein levels, or downregulated NOVX activity.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease NOVX gene expression, protein levels,
or downregulate NOVX activity, can be monitored in clinical trails
of subjects exhibiting increased NOVX gene expression, protein
levels, or upregulated NOVX activity. In such clinical trials, the
expression or activity of NOVX and, preferably, other genes that
have been implicated in, for example, a cellular proliferation or
immune disorder can be used as a "read out" or markers of the
immune responsiveness of a particular cell.
[0613] By way of example, and not of limitation, genes, including
NOVX, that are modulated in cells by treatment with an agent (e.g.,
compound, drug or small molecule) that modulates NOVX activity
(e.g., identified in a screening assay as described herein) can be
identified. Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of NOVX and other genes implicated in the disorder. The
levels of gene expression (i.e., a gene expression pattern) can be
quantified by Northern blot analysis or RT-PCR, as described
herein, or alternatively by measuring the amount of protein
produced, by one of the methods as described herein, or by
measuring the levels of activity of NOVX or other genes. In this
manner, the gene expression pattern can serve as a marker,
indicative of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at
various points during, treatment of the individual with the
agent.
[0614] 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 (1) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of an NOVX 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 NOVX protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the NOVX protein, mRNA, or
genomic DNA in the pre-administration sample with the NOVX protein,
mRNA, or genomic DNA in the post administration sample or samples;
and (vi) altering the administration of the agent to the subject
accordingly. For example, increased administration of the agent may
be desirable to increase the expression or activity of NOVX to
higher levels than detected, i.e., to increase the effectiveness of
the agent. Alternatively, decreased administration of the agent may
be desirable to decrease expression or activity of NOVX to lower
levels than detected, i.e., to decrease the effectiveness of the
agent.
[0615] Methods of Treatment
[0616] The invention provides for both prophylactic and therapeutic
methods of treating a subject at risk of (or susceptible to) a
disorder or having a disorder associated with aberrant NOVX
expression or activity. The disorders include cardiomyopathy,
atherosclerosis, hypertension, congenital heart defects, aortic
stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal
defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis,
ventricular septal defect (VSD), valve diseases, tuberous
sclerosis, scleroderma, obesity, transplantation,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate
cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer,
fertility, hemophilia, hypercoagulation, idiopathic
thrombocytopenic purpura, immunodeficiencies, graft versus host
disease, AIDS, bronchial asthma, Crohn's disease; multiple
sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and
other diseases, disorders and conditions of the like.
[0617] These methods of treatment will be discussed more fully,
below.
[0618] Disease and Disorders
[0619] Diseases and disorders that are characterized by increased
(relative to a subject not suffering from the disease or disorder)
levels or biological activity may be treated with Therapeutics that
antagonize (i.e., reduce or inhibit) activity. Therapeutics that
antagonize activity may be administered in a therapeutic or
prophylactic manner. Therapeutics that may 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" endogenous 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.
[0620] Diseases and disorders that are characterized by decreased
(relative to a subject not suffering from the disease or disorder)
levels or biological activity may be treated with Therapeutics that
increase (i.e., are agonists to) activity. Therapeutics that
upregulate activity may be administered in a therapeutic or
prophylactic manner. Therapeutics that may be utilized include, but
are not limited to, an aforementioned peptide, or analogs,
derivatives, fragments or homologs thereof; or an agonist that
increases bioavailability.
[0621] 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 like).
[0622] Prophylactic Methods
[0623] In one aspect, the invention provides a method for
preventing, in a subject, a disease or condition associated with an
aberrant NOVX expression or activity, by administering to the
subject an agent that modulates NOVX expression or at least one
NOVX activity. Subjects at risk for a disease that is caused or
contributed to by aberrant NOVX 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 NOVX aberrancy, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression. Depending upon the type of NOVX aberrancy, for
example, an NOVX agonist or NOVX 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.
Therapeutic Methods
[0624] Another aspect of the invention pertains to methods of
modulating NOVX 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 NOVX
protein activity associated with the cell. An agent that modulates
NOVX protein activity can be an agent as described herein, such as
a nucleic acid or a protein, a naturally-occurring cognate ligand
of an NOVX protein, a peptide, an NOVX peptidomimetic, or other
small molecule. In one embodiment, the agent stimulates one or more
NOVX protein activity. Examples of such stimulatory agents include
active NOVX protein and a nucleic acid molecule encoding NOVX that
has been introduced into the cell. In another embodiment, the agent
inhibits one or more NOVX protein activity. Examples of such
inhibitory agents include antisense NOVX nucleic acid molecules and
anti-NOVX 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 an NOVX 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) NOVX expression or activity. In
another embodiment, the method involves administering an NOVX
protein or nucleic acid molecule as therapy to compensate for
reduced or aberrant NOVX expression or activity.
[0625] Stimulation of NOVX activity is desirable in situations in
which NOVX is abnormally downregulated and/or in which increased
NOVX 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).
[0626] Determination of the Biological Effect of the
Therapeutic
[0627] 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.
[0628] In various specific embodiments, in vitro assays may be
performed with representative cells of the type(s) 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
may 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 may be
used prior to administration to human subjects.
[0629] Prophylactic and Therapeutic Uses of the Compositions of the
Invention
[0630] The NOVX 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:
metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-associated cancer, neurodegenerative disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders,
hematopoietic disorders, and the various dyslipidemias, metabolic
disturbances associated with obesity, the metabolic syndrome X and
wasting disorders associated with chronic diseases and various
cancers.
[0631] As an example, a cDNA encoding the NOVX protein of the
invention may be useful in gene therapy, and the protein may 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: metabolic
disorders, diabetes, obesity, infectious disease, anorexia,
cancer-associated cachexia, cancer, neurodegenerative disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders,
hematopoietic disorders, and the various dyslipidemias.
[0632] Both the novel nucleic acid encoding the NOVX protein, and
the NOVX 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.
[0633] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Identification of NOVX Clones
[0634] The novel NOVX target sequences identified in the present
invention were subjected to the exon linking process to confirm the
sequence. PCR primers were designed by starting at the most
upstream sequence available, for the forward primer, and at the
most downstream sequence available for the reverse primer. Table
15A shows the sequences of the PCR primers used for obtaining
different clones. In each case, the sequence was examined, walking
inward from the respective termini toward the coding sequence,
until a suitable sequence that is either unique or highly selective
was encountered, or, in the case of the reverse primer, until the
stop codon was reached. Such primers were designed based on in
silico predictions for the full length cDNA, part (one or more
exons) of the DNA or protein sequence of the target sequence, or by
translated homology of the predicted exons to closely related human
sequences from other species. These primers were then employed in
PCR amplification based on the following pool of human cDNAs:
adrenal gland, bone marrow, 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, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. Usually the resulting amplicons were gel purified, cloned
and sequenced to high redundancy. The PCR product derived from exon
linking was cloned into the pCR2.1 vector from Invitrogen. The
resulting bacterial clone has an insert covering the entire open
reading frame cloned into the pCR2.1 vector. Table 15B shows a list
of these bacterial clones. The resulting sequences from all clones
were assembled with themselves, with other fragments in CuraGen
Corporation's database and with public ESTs. Fragments and ESTs
were included as components for an assembly when the extent of
their identity with another component of the assembly was at least
95% over 50 bp. In addition, sequence traces were evaluated
manually and edited for corrections if appropriate. These
procedures provide the sequence reported herein. TABLE-US-00097
TABLE 15A PCR Primers for Exon Linking SEQ SEQ NOVX ID ID Clone
Primer 1 (5'-3') NO Primer 2 (5'-3') NO NOV2a
TCAAATGTTCAGTTTTGATTGTTGTTCTTG 137 TTTTTGCTAAAAGCAGCAATGCCAT 138
and b NOV2c ATTGACTTATGCTTCCTAGTTCGTTGC 139
CAACATTTAAAAGAATGGACGATTTTCA 140 NOV2d CTGTATTCCGGATCGATGCAAGAAG
141 TCTTAAGGAGAAGAAAATCTGCCGAAG 142 NOV3a
TGGAAACTCTAAAAAGCAGAGCGCCTC 143 CCTCTAGGTGAGTCAGTGCGTCACTCT 144
NOV6 ATGGGGGGCCTGACAGC 145 TTATGTGGCACAGTCCATAGTCTGC 146 NOV8
ATGATATGTCTTCCACATTACTGACATTCA 147 TTAGAGCCACAAACTAACCAGCTCAT
148
[0635] Physical clone: Exons were predicted by homology and the
intron/exon boundaries were determined using standard genetic
rules. Exons were further selected and refined by means of
similarity determination using multiple BLAST (for example,
tBlastN, BlastX, and BlastN) searches, and, in some instances,
GeneScan and Grail. Expressed sequences from both public and
proprietary databases were also added when available to further
define and complete the gene sequence. The DNA sequence was then
manually corrected for apparent inconsistencies thereby obtaining
the sequences encoding the full-length protein. TABLE-US-00098
TABLE 15B Physical Clones for PCR products NOVX Clone Bacterial
Clone NOV1a Physical clone: 134912642 NOV1b Physical clone:
pc.253568.D11 Skin NOV2b Physical clone:
101349::AJ278717.698423.C24 FLC EL NOV2c Physical clone:
139266::Hs_S1638243.698892.A7 NOV2d Physical clone: 175223749
164837693 164830233 NOV5 Physical clone: AC007563 NOV7a Physical
clone: 151818950 151176749 87413691 148439395 146025263 NOV9
Physical clone: 135008015 NOV10 Physical clone: AC010175 NOV11a and
b Genomic clone: sggc_draft_ba58o1_20001005 NOV12 Genomic clone:
ba370b6 NOV13 Physical clone: 139720381 NOV14 Physical clone:
AC009088, 140129142
Example 2
Quantitative Expression Analysis of Clones in Various Cells and
Tissues
[0636] The quantitative expression of various clones was assessed
using microtiter plates containing RNA samples from a variety of
normal and pathology-derived cells, cell lines and tissues using
real time quantitative PCR (RTQ PCR). RTQ PCR was performed on an
Applied Biosystems ABI PRISM.RTM. 7700 or an ABI PRISM.RTM. 7900 HT
Sequence Detection System. Various collections of samples are
assembled on the plates, and referred to as Panel 1 (containing
normal tissues and cancer cell lines), Panel 2 (containing samples
derived from tissues from normal and cancer sources), Panel 3
(containing cancer cell lines), Panel 4 (containing cells and cell
lines from normal tissues and cells related to inflammatory
conditions), Panel 5D/51 (containing human tissues and cell lines
with an emphasis on metabolic diseases), AI_comprehensive_panel
(containing normal tissue and samples from autoimmune diseases),
Panel CNSD.01 (containing central nervous system samples from
normal and diseased brains) and CNS_neurodegeneration_panel
(containing samples from normal and Alzheimer's diseased
brains).
[0637] RNA integrity from all samples is controlled for quality by
visual assessment of agarose gel electropherograms using 28S and
18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1
28s:18s) and the absence of low molecular weight RNAs that would be
indicative of degradation products. Samples are controlled against
genomic DNA contamination by RTQ PCR reactions run in the absence
of reverse transcriptase using probe and primer sets designed to
amplify across the span of a single exon.
[0638] First, the RNA samples were normalized to reference nucleic
acids such as constitutively expressed genes (for example,
.beta.-actin and GAPDH). Normalized RNA (5 ul) was converted to
cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix
Reagents (Applied Biosystems; Catalog No. 4309169) and
gene-specific primers according to the manufacturer's
instructions.
[0639] In other cases, non-normalized RNA samples were converted to
single strand cDNA (sscDNA) using Superscript II (Invitrogen
Corporation; Catalog No. 18064-147) and random hexamers according
to the manufacturer's instructions. Reactions containing up to 10
.mu.g of total RNA were performed in a volume of 20 .mu.l and
incubated for 60 minutes at 42.degree. C. This reaction can be
scaled up to 50 .mu.g of total RNA in a final volume of 100 .mu.l.
sscDNA samples are then normalized to reference nucleic acids as
described previously, using 1.times. TaqMan.RTM. Universal Master
mix (Applied Biosystems; catalog No. 4324020), following the
manufacturer's instructions.
[0640] Probes and primers were designed for each assay according to
Applied Biosystems Primer 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 (Tm) range=58.degree.-60.degree. C., primer optimal
Tm=59.degree. C., maximum primer difference=2.degree. C., probe
does not have 5'G, probe Tm must be 10.degree. C. greater than
primer Tm, amplicon size 75 bp to 100 bp. The probes and primers
selected (see below) were synthesized by Synthegen (Houston, Tex.,
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, 200 nM.
[0641] PCR conditions: When working with RNA samples, normalized
RNA from each tissue and each cell line was spotted in each well of
either a 96 well or a 384-well PCR plate (Applied Biosystems). PCR
cocktails included either a single gene specific probe and primers
set, or two multiplexed probe and primers sets (a set specific for
the target clone and another gene-specific set multiplexed with the
target probe). PCR reactions were set up using TaqMan.RTM. One-Step
RT-PCR Master Mix (Applied Biosystems, Catalog No. 4313803)
following manufacturer's instructions. Reverse transcription was
performed at 48.degree. C. for 30 minutes followed by
amplification/PCR cycles as follows: 95.degree. C. 10 min, then 40
cycles of 95.degree. C. for 15 seconds, 60.degree. C. for 1 minute.
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.
[0642] When working with sscDNA samples, normalized sscDNA was used
as described previously for RNA samples. PCR reactions containing
one or two sets of probe and primers were set up as described
previously, using 1.times. TaqMan.RTM. Universal-Master mix
(Applied Biosystems; catalog No. 4324020), following the
manufacturer's instructions. PCR amplification was performed as
follows: 95.degree. C. 10 min, then 40 cycles of 95.degree. C. for
15 seconds, 60C for 1 minute. Results were analyzed and processed
as described previously.
[0643] Panels 1, 1.1, 1.2, and 13D
[0644] The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control
wells (genomic DNA control and chemistry control) and 94 wells
containing cDNA from various samples. The samples in these panels
are broken into 2 classes: samples derived from cultured cell lines
and samples derived from primary normal tissues. The cell lines are
derived from cancers of the following types: lung cancer, breast
cancer, melanoma, colon cancer, prostate cancer, CNS cancer,
squamous cell carcinoma, ovarian cancer, liver cancer, renal
cancer, gastric cancer and pancreatic cancer. Cell lines used in
these panels are widely available through the American Type Culture
Collection (ATCC), a repository for cultured cell lines, and were
cultured using the conditions recommended by the ATCC. The normal
tissues found on these panels are comprised of samples derived from
all major organ systems from single adult individuals or fetuses.
These samples are derived from the following organs: adult skeletal
muscle, fetal skeletal muscle, adult heart, fetal heart, adult
kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal
lung, various regions of the brain, the spleen, bone marrow, lymph
node, pancreas, salivary gland, pituitary gland, adrenal gland,
spinal cord, thymus, stomach, small intestine, colon, bladder,
trachea, breast, ovary, uterus, placenta, prostate, testis and
adipose.
[0645] In the results for Panels 1, 1.1, 1.2 and 1.3D, the
following abbreviations are used: [0646] ca.=carcinoma, [0647]
*=established from metastasis, [0648] met=metastasis, [0649] s cell
var=small cell variant, [0650] non-s=non-sm=non-small, [0651]
squam=squamous, [0652] pl. eff=pl effusion=pleural effusion, [0653]
glio=glioma, [0654] astro=astrocytoma, and [0655]
neuro=neuroblastoma.
[0656] General_screening_panel_v1.4
[0657] The plates for Panel 1.4 include 2 control wells (genomic
DNA control and chemistry control) and 94 wells containing cDNA
from various samples. The samples in Panel 1.4 are broken into 2
classes: samples derived from cultured cell lines and samples
derived from primary normal tissues. The cell lines are derived
from cancers of the following types: lung cancer, breast cancer,
melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell
carcinoma, ovarian cancer, liver cancer, renal cancer, gastric
cancer and pancreatic cancer. Cell lines used in Panel 1.4 are
widely available through the American Type Culture Collection
(ATCC), a repository for cultured cell lines, and were cultured
using the conditions recommended by the ATCC. The normal tissues
found on Panel 1.4 are comprised of pools of samples derived from
all major organ systems from 2 to 5 different adult individuals or
fetuses. These samples are derived from the following organs: adult
skeletal muscle, fetal skeletal muscle, adult heart, fetal heart,
adult kidney, fetal kidney, adult liver, fetal liver, adult lung,
fetal lung, various regions of the brain, the spleen, bone marrow,
lymph node, pancreas, salivary gland, pituitary gland, adrenal
gland, spinal cord, thymus, stomach, small intestine, colon,
bladder, trachea, breast, ovary, uterus, placenta, prostate, testis
and adipose. Abbreviations are as described for Panels 1, 1.1, 1.2,
and 1.3D.
[0658] Panels 2D and 2.2
[0659] The plates for Panels 2D and 2.2 generally include 2 control
wells and 94 test samples composed of RNA or cDNA isolated from
human tissue procured by surgeons working in close cooperation with
the National Cancer Institute's Cooperative Human Tissue Network
(CHTN) or the National Disease Research Initiative (NDR1). The
tissues are derived from human malignancies and in cases where
indicated many malignant tissues have "matched margins" obtained
from noncancerous tissue just adjacent to the tumor. These are
termed normal adjacent tissues and are denoted "NAT" in the results
below. The tumor tissue and the "matched margins" are evaluated by
two independent pathologists (the surgical pathologists and again
by a pathologist at NDR1 or CHTN). This analysis provides a gross
histopathological assessment of tumor differentiation grade.
Moreover, most samples include the original surgical pathology
report that provides information regarding the clinical stage of
the patient. These matched margins are taken from the tissue
surrounding (i.e. immediately proximal) to the zone of surgery
(designated "NAT", for normal adjacent tissue, in Table RR). In
addition, RNA and cDNA samples were obtained from various human
tissues derived from autopsies performed on elderly people or
sudden death victims (accidents, etc.). These tissues were
ascertained to be free of disease and were purchased from various
commercial sources such as Clontech (Palo Alto, Calif.), Research
Genetics, and Invitrogen.
[0660] Panel 3D
[0661] The plates of Panel 3D are comprised of 94 cDNA samples and
two control samples. Specifically, 92 of these samples are derived
from cultured human cancer cell lines, 2 samples of human primary
cerebellar tissue and 2 controls. The human cell lines are
generally obtained from ATCC (American Type Culture Collection),
NCI or the German tumor cell bank and fall into the following
tissue groups: Squamous cell carcinoma of the tongue, breast
cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas,
bladder carcinomas, pancreatic cancers, kidney cancers,
leukemiasilymphomas, ovarian/uterine/cervical, gastric, colon, lung
and CNS cancer cell lines. In addition, there are two independent
samples of cerebellum. These cells are all cultured under standard
recommended conditions and RNA extracted using the standard
procedures. The cell lines in panel 3D and 1.3D are of the most
common cell lines used in the scientific literature.
[0662] Panels 4D, 4R, and 4.1D
[0663] Panel 4 includes samples on a 96 well plate (2 control
wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels
4D/4.1 D) isolated from various human cell lines or tissues related
to inflammatory conditions. Total RNA from control normal tissues
such as colon and lung (Stratagene, La Jolla, Calif.) and thymus
and kidney (Clontech) was employed. Total RNA from liver tissue
from cirrhosis patients and kidney from lupus patients was obtained
from BioChain (Biochain Institute, Inc., Hayward, Calif.).
Intestinal tissue for RNA preparation from patients diagnosed as
having Crohn's disease and ulcerative colitis was obtained from the
National Disease Research Interchange (NDR1) (Philadelphia,
Pa.).
[0664] Astrocytes, lung fibroblasts, dermal fibroblasts, coronary
artery smooth muscle cells, small airway epithelium, bronchial
epithelium, microvascular dermal endothelial cells, microvascular
lung endothelial cells, human pulmonary aortic endothelial cells,
human umbilical vein endothelial cells were all purchased from
Clonetics (Walkersville, Md.) and grown in the media supplied for
these cell types by Clonetics. These primary cell types were
activated with various cytokines or combinations of cytokines for 6
and/or 12-14 hours, as indicated. The following cytokines were
used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at
approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml,
IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml,
IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes
starved for various times by culture in the basal media from
Clonetics with 0.1% serum.
[0665] Mononuclear cells were prepared from blood of employees at
CuraGen Corporation, using Ficoll. LAK cells were prepared from
these cells by culture in DMEM 5% FCS (Hyclone), 100 .mu.M non
essential amino acids (Gibco/Life Technologies, Rockville, Md.), 1
mM sodium pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M
(Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days.
Cells were then either activated with 10-20 ng/ml PMA and 1-2
.mu.g/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml
and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear
cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and 10 mM
Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed
mitogen) at approximately 5 .mu.g/ml. Samples were taken at 24, 48
and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction)
samples were obtained by taking blood from two donors, isolating
the mononuclear cells using Ficoll and mixing the isolated
mononuclear cells 1:1 at a final concentration of approximately
2.times.10.sup.6cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non
essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco),
mercaptoethanol (5.5.times.10.sup.-5M) (Gibco), and 110 mM Hepes
(Gibco). The MLR was cultured and samples taken at various time
points ranging from 1-7 days for RNA preparation.
[0666] Monocytes were isolated from mononuclear cells using CD14
Miltenyi Beads, +ve VS selection columns and a Vario Magnet
according to the manufacturer's instructions. Monocytes were
differentiated into dendritic cells by culture in DMEM 5% fetal
calf serum (FCS) (Hyclone, Logan, Utah), 1001M non essential amino
acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml
GMCSF and 5 ng/ml IL4 for 5-7 days. Macrophages were prepared by
culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), 10 mM Hepes
(Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml.
Monocytes, macrophages and dendritic cells were stimulated for 6
and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml.
Dendritic cells were also stimulated with anti-CD40 monoclonal
antibody (Pharmingen) at 10 .mu.g/ml for 6 and 12-14 hours.
[0667] CD4 lymphocytes, CD8 lymphocytes and NK cells were also
isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi
beads, positive VS selection columns and a Vario Magnet according
to the manufacturer's instructions. CD45RA and CD45RO CD4
lymphocytes were isolated by depleting mononuclear cells of CD8,
CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi
beads and positive selection. CD45RO beads were then used to
isolate the CD45RO CD4 lymphocytes with the remaining cells being
CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes
were placed in DMEM 5% FCS (Hyclone), 100 .mu.M non essential amino
acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), and 10 mM Hepes (Gibco) and plated at
10.sup.6cells/ml onto Falcon 6 well tissue culture plates that had
been coated overnight with 0.5 .mu.g/ml anti-CD28 (Pharmingen) and
3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the
cells were harvested for RNA preparation. To prepare chronically
activated CD8 lymphocytes, we activated the isolated CD8
lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and
then harvested the cells and expanded them in DMEM 5% FCS
(Hyclone), 100 .mu.M non essential amino acids (Gibco), 1 mM sodium
pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and
10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then
activated again with plate bound anti-CD3 and anti-CD28 for 4 days
and expanded as before. RNA was isolated 6 and 24 hours after the
second activation and after 4 days of the second expansion culture.
The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and 10 mM
Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
[0668] To obtain B cells, tonsils were procured from NDRI. The
tonsil was cut up with sterile dissecting scissors and then passed
through a sieve. Tonsil cells were then spun down and resupended at
10.sup.6cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non essential
amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.5M (Gibco), and 10 mM Hepes (Gibco). To activate
the cells, we used PWM at 5 .mu.g/ml or anti-CD40 (Pharmingen) at
approximately 10 .mu.g/ml and IL-4 at 5-10 ng/ml. Cells were
harvested for RNA preparation at 24, 48 and 72 hours.
[0669] To prepare the primary and secondary Th1/Th2 and Tr1 cells,
six-well Falcon plates were coated overnight with 10 .mu.g/ml
anti-CD28 (Pharmingen) and 2 .mu.g/ml OKT3 (ATCC), and then washed
twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic
Systems, German Town, Md.) were cultured at
10.sup.5-10.sup.6cells/ml in DMEM 5% FCS (Hyclone), 100M non
essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco),
mercaptoethanol 5.5.times.10.sup.-5M (Gibco), 10 mM Hepes (Gibco)
and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL4 (1 .mu.g/ml) were
used to direct to Th1, while IL-4 (5 ng/ml) and anti-IFN gamma (1
.mu.g/ml) were used to direct to Th2 and IL-10 at 5 ng/ml was used
to direct to Tr1. After 4-5 days, the activated Th1, Th2 and Tr1
lymphocytes were washed once in DMEM and expanded for 4-7 days in
DMEM 5% FCS (Hyclone), 100 .mu.M non essential amino acids (Gibco),
1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M
(Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this,
the activated Th1, Th2 and Tr1 lymphocytes were re-stimulated for 5
days with anti-CD28/OKT3 and cytokines as described above, but with
the addition of anti-CD95L (1 .mu.g/ml) to prevent apoptosis. After
4-5 days, the Th1, Th2 and Tr1 lymphocytes were washed and then
expanded again with IL-2 for 4-7 days. Activated Th1 and Th2
lymphocytes were maintained in this way for a maximum of three
cycles. RNA was prepared from primary and secondary Th1, Th2 and
Tr1 after 6 and 24 hours following the second and third activations
with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the
second and third expansion cultures in Interleukin 2.
[0670] The following leukocyte cells lines were obtained from the
ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated
by culture in 0.1 mM dbcAMP at 5.times.10.sup.5cells/ml for 8 days,
changing the media every 3 days and adjusting the cell
concentration to 5.times.10.sup.5cells/ml. For the culture of these
cells, we used DMEM or RPMI (as recommended by the ATCC), with the
addition of 5% FCS (Hyclone), 100 .mu.M non essential amino acids
(Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), 10 mM Hepes (Gibco). RNA was either
prepared from resting cells or cells activated with PMA at 10 ng/ml
and ionomycin at 1 .mu.g/ml for 6 and 14 hours. Keratinocyte line
CCD1106 and an airway epithelial tumor line NCI-H292 were also
obtained from the ATCC. Both were cultured in DMEM 5% FCS
(Hyclone), 100 .mu.M non essential amino acids (Gibco), 1 mM sodium
pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and
10 mM Hepes (Gibco). CCD1106 cells were activated for 6 and 14
hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta,
while NCI-H292 cells were activated for 6 and 14 hours with the
following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and
25 ng/ml IFN gamma.
[0671] For these cell lines and blood cells, RNA was prepared by
lysing approximately 10.sup.7cells/ml using Trizol (Gibco BRL).
Briefly, 1/10 volume of bromochloropropane (Molecular Research
Corporation) was added to the RNA sample, vortexed and after 10
minutes at room temperature, the tubes were spun at 14,000 rpm in a
Sorvall SS34 rotor. The aqueous phase was removed and placed in a
15 ml Falcon Tube. An equal volume of isopropanol was added and
left at -20.degree. C. overnight. The precipitated RNA was spun
down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and washed in
70% ethanol. The pellet was redissolved in 300 .mu.l of RNAse-free
water and 35 .mu.l buffer (Promega) 5.mu.l DTT, 7 .mu.l RNAsin and
8 .mu.l DNAse were added. The tube was incubated at 37.degree. C.
for 30 minutes to remove contaminating genomic DNA, extracted once
with phenol chloroform and re-precipitated with 1/10 volume of 3M
sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down
and placed in RNAse free water. RNA was stored at -80.degree.
C.
[0672] AI_comprehensive panel_v1.0
[0673] The plates for AI_comprehensive panel_v1.0 include two
control wells and 89 test samples comprised of cDNA isolated from
surgical and postmortem human tissues obtained from the Backus
Hospital and Clinomics (Frederick, Md.). Total RNA was extracted
from tissue samples from the Backus Hospital in the Facility at
CuraGen. Total RNA from other tissues was obtained from
Clinomics.
[0674] Joint tissues including synovial fluid, synovium, bone and
cartilage were obtained from patients undergoing total knee or hip
replacement surgery at the Backus Hospital. Tissue samples were
immediately snap frozen in liquid nitrogen to ensure that isolated
RNA was of optimal quality and not degraded. Additional samples of
osteoarthritis and rheumatoid arthritis joint tissues were obtained
from Clinomics. Normal control tissues were supplied by Clinomics
and were obtained during autopsy of trauma victims.
[0675] Surgical specimens of psoriatic tissues and adjacent matched
tissues were provided as total RNA by Clinomics. Two male and two
female patients were selected between the ages of 25 and 47. None
of the patients were taking prescription drugs at the time samples
were isolated.
[0676] Surgical specimens of diseased colon from patients with
ulcerative colitis and Crohns disease and adjacent matched tissues
were obtained from Clinomics. Bowel tissue from three female and
three male Crohn's patients between the ages of 41-69 were used.
Two patients were not on prescription medication while the others
were taking dexamethasone, phenobarbital, or tylenol. Ulcerative
colitis tissue was from three male and four female patients. Four
of the patients were taking lebvid and two were on
phenobarbital.
[0677] Total RNA from post mortem lung tissue from trauma victims
with no disease or with emphysema, asthma or COPD was purchased
from Clinomics. Emphysema patients ranged in age from 40-70 and all
were smokers, this age range was chosen to focus on patients with
cigarette-linked emphysema and to avoid those patients with
alpha-1anti-trypsin deficiencies. Asthma patients ranged in age
from 36-75, and excluded smokers to prevent those patients that
could also have COPD. COPD patients ranged in age from 35-80 and
included both smokers and non-smokers. Most patients were taking
corticosteroids, and bronchodilators.
[0678] In the labels employed to identify tissues in the
AI_comprehensive panel_v 1.0 panel, the following abbreviations are
used: [0679] AI=Autoimmunity [0680] Syn=Synovial [0681] Normal=No
apparent disease [0682] Rep22/Rep20=individual patients [0683]
RA=Rheumatoid arthritis [0684] Backus=From Backus Hospital [0685]
OA=Osteoarthritis [0686] (SS) (BA) (MF)=Individual patients [0687]
Adj=Adjacent tissue [0688] Match control=adjacent tissues [0689]
-M=Male [0690] -F=Female [0691] COPD=Chronic obstructive pulmonary
disease
[0692] Panels 5D and 5I
[0693] The plates for Panel 5D and 5I include two control wells and
a variety of cDNAs isolated from human tissues and cell lines with
an emphasis on metabolic diseases. Metabolic tissues were obtained
from patients enrolled in the Gestational Diabetes study. Cells
were obtained during different stages in the differentiation of
adipocytes from human mesenchymal stem cells. Human pancreatic
islets were also obtained.
[0694] In the Gestational Diabetes study subjects are young (18-40
years), otherwise healthy women with and without gestational
diabetes undergoing routine (elective) Caesarean section. After
delivery of the infant, when the surgical incisions were being
repaired/closed, the obstetrician removed a small sample (<1 cc)
of the exposed metabolic tissues during the closure of each
surgical level. The biopsy material was rinsed in sterile saline,
blotted and fast frozen within 5 minutes from the time of removal.
The tissue was then flash frozen in liquid nitrogen and stored,
individually, in sterile screw-top tubes and kept on dry ice for
shipment to or to be picked up by CuraGen. The metabolic tissues of
interest include uterine wall (smooth muscle), visceral adipose,
skeletal muscle (rectus) and subcutaneous adipose. Patient
descriptions are as follows: [0695] Patient 2 Diabetic Hispanic,
overweight, not on insulin [0696] Patient 7-9 Nondiabetic Caucasian
and obese (BMI>30) [0697] Patient 10 Diabetic Hispanic,
overweight, on insulin [0698] Patient 11 Nondiabetic African
American and overweight [0699] Patient 12 Diabetic Hispanic on
insulin
[0700] Adipocyte differentiation was induced in donor progenitor
cells obtained from Osirus (a division of Clonetics/BioWhittaker)
in triplicate, except for Donor 3U which had only two replicates.
Scientists at Clonetics isolated, grew and differentiated human
mesenchymal stem cells (HuMSCs) for CuraGen based on the published
protocol found in Mark F. Pittenger, et al., Multilineage Potential
of Adult Human Mesenchymal Stem Cells Science Apr. 2, 1999:
143-147. Clonetics provided Trizol lysates or frozen pellets
suitable for mRNA isolation and ds cDNA production. A general
description of each donor is as follows: [0701] Donor 2 and 3 U:
Mesenchymal Stem cells, Undifferentiated Adipose [0702] Donor 2 and
3 AM: Adipose, AdiposeMidway Differentiated [0703] Donor 2 and 3
AD: Adipose, Adipose Differentiated
[0704] Human cell lines were generally obtained from ATCC (American
Type Culture Collection), NCI or the German tumor cell bank and
fall into the following tissue groups: kidney proximal convoluted
tubule, uterine smooth muscle cells, small intestine, liver HepG2
cancer cells, heart primary stromal cells, and adrenal cortical
adenoma cells. These cells are all cultured under standard
recommended conditions and RNA extracted using the standard
procedures. All samples were processed at CuraGen to produce single
stranded cDNA.
[0705] Panel 5I contains all samples previously described with the
addition of pancreatic islets from a 58 year old female patient
obtained from the Diabetes Research Institute at the University of
Miami School of Medicine. Islet tissue was processed to total RNA
at an outside source and delivered to CuraGen for addition to panel
5I.
[0706] In the labels employed to identify tissues in the 5D and 5I
panels, the following abbreviations are used: [0707] GO
Adipose=Greater Omentum Adipose [0708] SK=Skeletal Muscle [0709]
UT=Uterus [0710] PL=Placenta [0711] AD=Adipose Differentiated
[0712] AM=Adipose Midway Differentiated [0713] U=Undifferentiated
Stem Cells
[0714] Panel CNSD.01
[0715] The plates for Panel CNSD.01 include two control wells and
94 test samples comprised of cDNA isolated from postmortem human
brain tissue obtained from the Harvard Brain Tissue Resource
Center. Brains are removed from calvaria of donors between 4 and 24
hours after death, sectioned by neuroanatomists, and frozen at
-80.degree. C. in liquid nitrogen vapor. All brains are sectioned
and examined by neuropathologists to confirm diagnoses with clear
associated neuropathology.
[0716] Disease diagnoses are taken from patient records. The panel
contains two brains from each of the following diagnoses:
Alzheimer's disease, Parkinson's disease, Huntington's disease,
Progressive Supernuclear Palsy, Depression, and "Normal controls".
Within each of these brains, the following regions are represented:
cingulate gyrus, temporal pole, globus palladus, substantia nigra,
Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal
cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17
(occipital cortex). Not all brain regions are represented in all
cases; e.g., Huntington's disease is characterized in part by
neurodegeneration in the globus palladus, thus this region is
impossible to obtain from confumed Huntington's cases. Likewise
Parkinson's disease is characterized by degeneration of the
substantia nigra making this region more difficult to obtain.
Normal control brains were examined for neuropathology and found to
be free of any pathology consistent with neurodegeneration.
[0717] In the labels employed to identify tissues in the CNS panel,
the following abbreviations are used: [0718] PSP=Progressive
supranuclear palsy [0719] Sub Nigra=Substantia nigra [0720] Glob
Palladus=Globus palladus [0721] Temp Pole=Temporal pole [0722] Cing
Gyr=Cingulate gyrus [0723] BA 4=Brodman Area 4
[0724] Panel CNS_Neurodegeneration_V1.0
[0725] The plates for Panel CNS_Neurodegeneration_V1.0 include two
control wells and 47 test samples comprised of cDNA isolated from
postmortem human brain tissue obtained from the Harvard Brain
Tissue Resource Center (McLean Hospital) and the Human Brain and
Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare
System). Brains are removed from calvaria of donors between 4 and
24 hours after death, sectioned by neuroanatomists, and frozen at
-80.degree. C. in liquid nitrogen vapor. All brains are sectioned
and examined by neuropathologists to confirm diagnoses with clear
associated neuropathology.
[0726] Disease diagnoses are taken from patient records. The panel
contains six brains from Alzheimer's disease (AD) patients, and
eight brains from "Normal controls" who showed no evidence of
dementia prior to death. The eight normal control brains are
divided into two categories: Controls with no dementia and no
Alzheimer's like pathology (Controls) and controls with no dementia
but evidence of severe Alzheimer's like pathology, (specifically
senile plaque load rated as level 3 on a scale of 0-3; 0=no
evidence of plaques, 3=severe AD senile plaque load). Within each
of these brains, the following regions are represented:
hippocampus, temporal cortex (Brodman Area 21), parietal cortex
(Brodman area 7), and occipital cortex (Brodman area 17). These
regions were chosen to encompass all levels of neurodegeneration in
AD. The hippocampus is a region of early and severe neuronal loss
in AD; the temporal cortex is known to show neurodegeneration in AD
after the hippocampus; the parietal cortex shows moderate neuronal
death in the late stages of the disease; the occipital cortex is
spared in AD and therefore acts as a "control" region within AD
patients. Not all brain regions are represented in all cases.
[0727] In the labels employed to identify tissues in the
CNS_Neurodegeneration-V 1.0 panel, the following abbreviations are
used: [0728] AD=Alzheimer's disease brain; patient was demented and
showed AD-like pathology upon autopsy [0729] Control=Control
brains; patient not demented, showing no neuropathology [0730]
Control (Path)=Control brains; pateint not demented but showing
sever AD-like pathology [0731] SupTemporal Ctx=Superior Temporal
Cortex [0732] Inf Temporal Ctx=Inferior Temporal Cortex NOV4
[0733] Expression of gene NOV4 was assessed using the primer-probe
set Ag3802, described in Table 16. TABLE-US-00099 TABLE 16 Probe
Name Ag3802 Primers Sequences Length Start Position SEQ ID NO:
Forward 5'-gtcgatgggacatctttcct-3' 20 108 149 Probe
TET-5'-cttcggatcactatcatccagtgcca-3'-TAMRA 26 134 150 Reverse
5'-atgaggaagtagcccacgtt-3' 20 171 151
[0734] General_screening_panel_v1.4 Summary: Ag3082 Expression of
the NOV4 gene is low/undetectable in all samples on this panel
(CTs>35). (Data not shown.) The data suggest that these
expression levels may be due to a probe failure.
NOV3a and NOV3b
[0735] Expression of gene NOV3a and variant NOV3b was assessed
using the primer-probe set Ag4849, described in Table 17. Results
of the RTQ-PCR runs are shown in Tables 18 and 19. TABLE-US-00100
TABLE 17 Probe Name Ag4849 Primers Sequences Length Start Position
SEQ ID NO: Forward 5'-gccagttctacctcaagttcct-3' 22 3895 152 Probe
TET-5'-ctaccaccatgtgtcccgccgttt-3'-TAMRA 24 3920 153 Reverse
5'-catagtcagagtcgagcaggaa-3' 22 3951 154
[0736] TABLE-US-00101 TABLE 18 General_screening_panel_v1.5 Rel.
Exp.(%) Ag4849, Rel. Exp.(%) Ag4849, Tissue Name Run 228887477
Tissue Name Run 228887477 Adipose 8.4 Renal ca. TK-10 31.2
Melanoma* 32.5 Bladder 18.6 Hs688(A).T Melanoma* 37.4 Gastric ca.
(liver met.) 76.8 Hs688(B).T NCI-N87 Melanoma* M14 58.2 Gastric ca.
KATO III 75.8 Melanoma* 15.5 Colon ca. SW-948 25.2 LOXIMVI
Melanoma* SK- 45.4 Colon ca. SW480 55.9 MEL-5 Squamous cell 17.0
Colon ca.* (SW480 25.5 carcinoma SCC-4 met) SW620 Testis Pool 30.1
Colon ca. HT29 17.2 Prostate ca.* (bone 40.3 Colon ca. HCT-116 36.1
met) PC-3 Prostate Pool 7.4 Colon ca. CaCo-2 29.5 Placenta 17.2
Colon cancer tissue 20.9 Uterus Pool 4.9 Colon ca. SW1116 11.3
Ovarian ca. 24.5 Colon ca. Colo-205 28.9 OVCAR-3 Ovarian ca.
SK-OV-3 71.2 Colon ca. SW-48 14.0 Ovarian ca. 30.1 Colon Pool 15.1
OVCAR-4 Ovarian ca. 32.8 Small Intestine Pool 13.1 OVCAR-5 Ovarian
ca. IGROV-1 19.9 Stomach Pool 7.4 Ovarian ca. 21.2 Bone Marrow Pool
7.9 OVCAR-8 Ovary 15.0 Fetal Heart 9.6 Breast ca. MCF-7 13.9 Heart
Pool 7.3 Breast ca. MDA- 38.4 Lymph Node Pool 16.5 MB-231 Breast
ca. BT 549 61.1 Fetal Skeletal Muscle 9.8 Breast ca. T47D 7.3
Skeletal Muscle Pool 29.1 Breast ca. MDA-N 17.0 Spleen Pool 9.0
Breast Pool 14.5 Thymus Pool 17.3 Trachea 13.2 CNS cancer
(glio/astro) 56.3 U87-MG Lung 2.6 CNS cancer (glio/astro) 67.4
U-118-MG Fetal Lung 25.5 CNS cancer 18.6 (neuro; met) SK-N-AS Lung
ca. NCI-N417 13.9 CNS cancer (astro) SF- 21.0 539 Lung ca. LX-1
37.9 CNS cancer (astro) 69.7 SNB-75 Lung ca. NCI-H146 9.6 CNS
cancer (glio) 14.3 SNB-19 Lung ca. SHP-77 34.9 CNS cancer (glio)
SF- 80.7 295 Lung ca. A549 19.3 Brain (Amygdala) Pool 32.1 Lung ca.
NCI-H526 16.6 Brain (cerebellum) 100.0 Lung ca. NCI-H23 25.2 Brain
(fetal) 61.1 Lung ca. NCI-H460 23.7 Brain (Hippocampus) 28.9 Pool
Lung ca. HOP-62 24.1 Cerebral Cortex Pool 32.1 Lung ca. NCI-H522
17.3 Brain (Substantia nigra) 46.3 Pool Liver 2.5 Brain (Thalamus)
Pool 47.3 Fetal Liver 17.4 Brain (whole) 41.8 Liver ca. HepG2 19.6
Spinal Cord Pool 18.2 Kidney Pool 27.2 Adrenal Gland 18.3 Fetal
Kidney 8.9 Pituitary gland Pool 4.1 Renal ca. 786-0 18.8 Salivary
Gland 7.9 Renal ca. A498 8.5 Thyroid (female) 15.7 Renal ca. ACHN
24.3 Pancreatic ca. CAPAN2 28.1 Renal ca. UO-31 19.2 Pancreas Pool
19.5
[0737] TABLE-US-00102 TABLE 19 Panel 4.1D Rel. Exp.(%) Rel. Exp.(%)
Ag4849, Run Ag4849, Run Tissue Name 223335772 Tissue Name 223335772
Secondary Th1 act 66.9 HUVEC IL-1beta 26.8 Secondary Th2 act 62.4
HUVEC IFN gamma 21.3 Secondary Tr1 act 65.5 HUVEC TNF alpha + IFN
29.3 gamma Secondary Th1 rest 36.9 HUVEC TNF alpha + IL4 24.7
Secondary Th2 rest 44.8 HUVEC IL-11 20.4 Secondary Tr1 rest 33.0
Lung Microvascular EC 34.2 none Primary Th1 act 56.6 Lung
Microvascular EC 39.2 TNF alpha + IL-1beta Primary Th2 act 51.8
Microvascular Dermal EC 18.3 none Primary Tr1 act 50.7
Microsvasular Dermal EC 22.8 TNF alpha + IL-1beta Primary Th1 rest
32.5 Bronchial epithelium 41.5 TNF alpha + IL1beta Primary Th2 rest
32.8 Small airway epithelium 23.5 none Primary Tr1 rest 49.3 Small
airway epithelium 36.6 TNF alpha + IL-1beta CD45RA CD4 55.5
Coronery artery SMC rest 19.3 lymphocyte act CD45RO CD4 57.0
Coronery artery SMC 26.1 lymphocyte act TNF alpha + IL-1beta CD8
lymphocyte act 73.7 Astrocytes rest 22.5 Secondary CD8 62.4
Astrocytes TNF alpha + IL- 13.5 lymphocyte rest 1beta Secondary CD8
48.6 KU-812 (Basophil) rest 19.3 lymphocyte act CD4 lymphocyte none
29.7 KU-812 (Basophil) 28.3 PMA/ionomycin 2ry Th1/Th2/Tr1_anti-
61.1 CCD1106 (Keratinocytes) 29.3 CD95 CH11 none LAK cells rest
47.3 CCD1106 (Keratinocytes) 31.6 TNF alpha + IL-1beta LAK cells
IL-2 55.9 Liver cirrhosis 4.6 LAK cells IL-2 + IL-12 43.5 NCI-H292
none 9.9 LAK cells IL-2 + IFN 53.6 NCI-H292 IL-4 16.6 gamma LAK
cells IL-2 + IL-18 48.6 NCI-H292 IL-9 20.0 LAK cells 35.6 NCI-H292
IL-13 19.9 PMA/ionomycin NK Cells IL-2 rest 100.0 NCI-H292 IFN
gamma 15.9 Two Way MLR 3 day 65.5 HPAEC none 16.6 Two Way MLR 5 day
47.6 HPAEC TNF alpha + IL- 38.4 1beta Two Way MLR 7 day 35.1 Lung
fibroblast none 43.2 PBMC rest 28.7 Lung fibroblast TNF alpha + IL-
34.4 1beta PBMC PWM 59.5 Lung fibroblast IL-4 53.2 PBMC PHA-L 58.6
Lung fibroblast IL-9 29.3 Ramos (B cell) none 80.1 Lung fibroblast
IL-13 33.4 Ramos (B cell) 79.6 Lung fibroblast IFN 49.0 ionomycin
gamma B lymphocytes PWM 31.9 Dermal fibroblast 45.7 CCD1070 rest B
lymphocytes CD40L 82.9 Dermal fibroblast 64.6 and IL-4 CCD1070 TNF
alpha EOL-1 dbcAMP 33.0 Dermal fibroblast 35.4 CCD1070 IL-1beta
EOL-1 dbcAMP 42.6 Dermal fibroblast IFN 34.6 PMA/ionomycin gamma
Dendritic cells none 31.6 Dermal fibroblast IL-4 51.1 Dendritic
cells LPS 20.2 Dermal Fibroblasts rest 39.2 Dendritic cells anti-
27.7 Neutrophils TNFa + LPS 7.7 CD40 Monocytes rest 15.5
Neutrophils rest 14.3 Monocytes LPS 52.1 Colon 15.9 Macrophages
rest 34.4 Lung 12.9 Macrophages LPS 14.8 Thymus 69.7 HUVEC none
23.5 Kidney 29.5 HUVEC starved 26.6
[0738] General_screening_panel_v1.5 Summary: Ag4849 The NOV3a gene
is a splice variant of SET-binding factor and is moderately
expressed in all tissues and cell lines in this panel. The
ubiquitous expression of this gene suggests a role in cell survival
and proliferation. As demonstrated in the abstract below, this gene
may regulate the activity of other genes by direct interaction. In
addition, highest expression of this gene in this panel is seen in
the brain, with high levels of expression detected in all regions
of the brain examined. Since SBF proteins are believed to play a
role in the cell cycle, this protein may be of use in neural stem
cell therapy, specifically in controlling the transition of stem
cells to post mitotic neurons.
REFERENCES
[0739] Firestein R, Cui X, Huie P, Cleary M L. Set domain-dependent
regulation of transcriptional silencing and growth control by
SUV39H1, a mammalian ortholog of Drosophila Su(var).sub.3-9. Mol
Cell Biol 2000 July; 20 (13):4900-9
[0740] Mammalian SET domain-containing proteins define a
distinctive class of chromatin-associated factors that are targets
for growth control signals and oncogenic activation. SUV39H1, a
mammalian ortholog of Drosophila Su(var)3-9, contains both SET and
chromo domains, signature motifs for proteins that contribute to
epigenetic control of gene expression through effects on the
regional organization of chromatin structure. In this report we
demonstrate that SUV39H1 represses transcription in a transient
transcriptional assay when tethered to DNA through the GALA DNA
binding domain. Under these conditions, SUV39H1 displays features
of a long-range repressor capable of acting over several kilobases
to silence basal promoters. A possible role in chromatin-mediated
gene silencing is supported by the localization of exogenously
expressed SUV39H1 to nuclear bodies with morphologic features
suggestive of heterochromatin in interphase cells. In addition, we
show that SUV39H1 is phosphorylated specifically at the G(1)/S cell
cycle transition and when forcibly expressed suppresses cell
growth. Growth suppression as well as the ability of SUV39H1 to
form nuclear bodies and silence transcription are antagonized by
the oncogenic antiphosphatase Sbf1 that when hyperexpressed
interacts with the SET domain and stabilizes the phosphorylated
form of SUV39H1. These studies suggest a phosphorylation-dependent
mechanism for regulating the chromatin organizing activity of a
mammalian su(var) protein and implicate the SET domain as a
gatekeeper motif that integrates upstream signaling pathways to
epigenetic regulation and growth control.
[0741] Cui X, De Vivo I, Slany R, Miyamoto A, Firestein R, Cleary M
L. Nat Genet 1998 April; 18 (4):331-7; Association of SET domain
and myotubularin-related proteins modulates growth control.
[0742] Several proteins that contribute to epigenetic mechanisms of
gene regulation contain a characteristic motif of unknown function
called the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain. We
have demonstrated that SET domains mediate highly conserved
interactions with a specific family of proteins that display
similarity with dual-specificity phosphatases (dsPTPases). These
include myotubularin, the gene of which is mutated in a subset of
patients with X-linked myotubular myopathy, and Sbf1, a newly
isolated homologue of myotubularin. In contrast with myotubularin,
Sbf1 lacks a functional catalytic domain which dephosphorylates
phospho-tyrosine and serine-containing peptides in vitro.
Competitive interference of endogenous SET domain-dsPTPase
interactions by forced expression of Sbf1 induced oncogenic
transformation of NIH 3T3 fibroblasts and impaired the in vitro
differentiation of C2 myoblast cells. We conclude that
myotubularin-type phosphatases link SET-domain containing
components of the epigenetic regulatory machinery with signalling
pathways involved in growth and differentiation.
[0743] Firestein R, Cleary M L. Pseudo-phosphatase Sbf1 contains an
N-terminal GEF homology domain that modulates its growth regulatory
properties. J Cell Sci 2001 August; 114 (Pt 16):2921-7
[0744] Sbf1 (SET binding factor 1) is a pseudo-phosphatase related
to the myotubularin family of dual specificity phosphatases, some
of which have been implicated in cellular growth and
differentiation by virtue of their mutation in human genetic
disorders. Sbf1 contains germline-encoded alterations of its
myotubularin homology domain that render it non-functional as a
phosphatase. We report here the complete structure of Sbf1 and
further characterization of its growth regulatory properties. In
addition to its similarity to myotubularin, the predicted
full-length Sbf1 protein contains pleckstrin (PH) and GEF homology
domains that are conserved in several-proteins implicated in
signaling and growth control. Forced expression of wild-type Sbf1
in NIH 3T3 cells inhibited their proliferation and altered their
morphology. These effects required intact PH, GEF and myotubularin
homology domains, implying that growth inhibition may be an
intrinsic property of wild-type Sbf1. Conversely, deletion of its
conserved N-terminal 44 amino acids alone was sufficient to convert
Sbf1 from an inhibitor of cellular growth to a transforming protein
in NIH 3T3 cells. Oncogenic forms of Sbf1 partially localized to
the nucleus, in contrast to the exclusively cytoplasmic subcellular
localization of endogenous Sbf1 in all cell lines and mammalian
tissues tested. These data show that the N-terminal GEF homology
domain serves to inhibit the transforming effects of Sbf1, possibly
sequestering the protein to the cytoplasm, and suggest that this
region may be a modulatory domain that relays growth control
signals.
[0745] Panel 4.1D Summary: Ag4849 The NOV3a gene is expressed at
high to moderate levels in a wide range of cell types of
significance in the immune response in health and disease. These
cells include members of the T-cell, B-cell, endothelial cell,
macrophage/monocyte, and peripheral blood mononuclear cell family,
as well as epithelial and fibroblast cell types from lung and skin,
and normal tissues represented by colon, lung, thymus and kidney.
This ubiquitous pattern of expression suggests that this gene
product may be involved in homeostatic processes for these and
other cell types and tissues. This pattern is in agreement with the
expression profile in General_screening-panel_v1.5 and also
suggests a role for the gene product in cell survival and
proliferation. Therefore, modulation of the gene product with a
functional therapeutic may lead to the alteration of functions
associated with these cell types and lead to improvement of the
symptoms of patients suffering from autoimmune and inflammatory
diseases such as asthma, allergies, inflammatory bowel disease,
lupus erythematosus, psoriasis, rheumatoid arthritis, and
osteoarthritis.
NOV1a and NOV1c
[0746] Expression of gene NOV1a and variant NOV1c was assessed
using the primer-probe sets Ag400, Ag2866 and Ag3077, described in
Tables 20, 21 and 22. Results of the RTQ-PCR runs are shown in
Tables 23, 24, 25, and 26. TABLE-US-00103 TABLE 20 Probe Name Ag400
Primers Sequences Length Start Position SEQ ID NO: Forward
5'-acgatcctgggctggacag-3' 19 2684 155 Probe
TET-5'-catctgcgcgtagcccctcca-3'-TAMRA 21 2659 156 Reverse
5'-gcttcaaccccctcgagttc-3' 20 2627 157
[0747] TABLE-US-00104 TABLE 21 Probe Name Ag2866 Primers Sequences
Length Start Position SEQ ID NO: Forward
5'-tatgtactcgtggtccctgaga-3' 22 4075 158 Probe
TET-5'-acgtctacagctttggctacctccgg-3'-TAMRA 26 4097 159 Reverse
5'-agtggctgatgaagtcatagga-3' 22 4141 160
[0748] TABLE-US-00105 TABLE 22 Probe Name Ag3077 Primers Sequences
Length Start Position SEQ ID NO: Forward 5'-aatgtggagctgtgcctgt-3'
19 5431 161 Probe TET-5'-gactcatgccaggaatgtgcccc-3'-TAMRA 23 5470
162 Reverse 5'-gaagagacctttgacgtccc-3' 20 5504 163
[0749] TABLE-US-00106 TABLE 23 Panel 1 Rel. Exp.(%) Rel. Exp.(%)
Rel. Exp.(%) Rel. Exp.(%) Ag400, Run Ag400, Run Ag400, Run Ag400,
Run Tissue Name 91010053 97802926 Tissue Name 91010053 97802926
Endothelial cells 4.5 2.3 Renal ca. 786-0 12.0 4.7 Endothelial
cells 4.0 3.1 Renal ca. A498 30.1 14.6 (treated) Pancreas 11.6 4.3
Renal ca. RXF393 13.2 5.1 Pancreatic ca. 18.8 11.3 Renal ca. 4.5
3.9 CAPAN 2 ACHN Adrenal gland 3.3 5.4 Renal ca. UO- 10.5 8.6 31
Thyroid 6.5 3.7 Renal ca. TK- 22.1 54.7 10 Salivary gland 3.3 2.3
Liver 4.8 5.1 Pituitary gland 5.8 8.4 Liver (fetal) 1.5 1.7 Brain
(fetal) 1.5 1.9 Liver ca. 13.8 23.0 (hepatoblast) HepG2 Brain
(whole) 8.7 4.2 Lung 24.3 8.4 Brain (amygdala) 1.1 2.6 Lung (fetal)
17.9 6.7 Brain 100.0 16.3 Lung ca. (small 15.2 18.7 (cerebellum)
cell) LX-1 Brain 2.3 2.5 Lung ca. (small 0.2 0.7 (hippocampus)
cell) NCI-H69 Brain (substantia 3.9 1.8 Lung ca. (s.cell 14.9 2.3
nigra) var.) SHP-77 Brain (thalamus) 3.0 5.0 Lung ca. (large 75.3
100.0 cell)NCI-H460 Brain 3.6 3.0 Lung ca. (non- 40.6 28.3
(hypothalamus) sm. cell) A549 Spinal cord 2.0 2.5 Lung ca. (non-
11.3 16.0 s.cell) NCI-H23 glio/astro U87- 0.2 0.6 Lung ca. (non-
8.4 10.9 MG s.cell) HOP-62 glio/astro U-118- 8.8 4.1 Lung ca. (non-
18.4 10.0 MG s.cl) NCI-H522 astrocytoma 4.9 2.1 Lung ca. 46.7 28.3
SW1783 (squam.) SW900 neuro*; met SK- 11.1 21.9 Lung ca. 0.2 1.3
N-AS (squam.) NCI- H596 astrocytoma SF- 4.8 4.2 Mammary 26.8 25.5
539 gland astrocytoma 3.7 2.5 Breast ca.* 23.0 13.3 SNB-75 (pl.ef)
MCF-7 glioma SNB-19 46.0 51.1 Breast ca.* 9.3 7.5 (pl.ef) MDA-
MB-231 glioma U251 19.6 27.0 Breast ca.* (pl. 96.6 56.6 ef) T47D
glioma SF-295 8.9 16.4 Breast ca. BT- 12.1 4.2 549 Heart 7.9 11.9
Breast ca. 0.0 0.1 MDA-N Skeletal muscle 0.8 7.2 Ovary 9.2 4.9 Bone
marrow 1.2 1.5 Ovarian ca. 30.6 43.5 OVCAR-3 Thymus 15.3 7.5
Ovarian ca. 50.3 22.5 OVCAR-4 Spleen 3.7 3.5 Ovarian ca. 39.8 34.2
OVCAR-5 Lymph node 6.7 2.1 Ovarian ca. 9.9 19.6 OVCAR-8 Colon
(ascending) 5.9 1.2 Ovarian ca. 63.3 70.2 IGROV-1 Stomach 22.2 5.2
Ovarian ca. 12.8 10.1 (ascites) SK- OV-3 Small intestine 8.6 4.3
Uterus 13.8 13.8 Colon ca. SW480 7.2 9.5 Placenta 38.4 28.9 Colon
ca.* 6.0 3.7 Prostate 16.5 10.4 SW620 (SW480 met) Colon ca. HT29
15.0 13.9 Prostate ca.* 71.7 87.7 (bone met) PC-3 Colon ca. HCT-
11.3 7.9 Testis 15.1 2.2 116 Colon ca. CaCo-2 65.1 29.5 Melanoma
0.0 0.0 Hs688(A).T Colon ca. HCT- 12.1 9.2 Melanoma* 0.3 0.3 15
(met) Hs688(B).T Colon ca. HCC- 12.1 18.6 Melanoma 0.5 4.7 2998
UACC-62 Gastric ca.* 92.0 52.5 Melanoma M14 3.8 1.3 (liver met)
NCI- N87 Bladder 10.0 17.4 Melanoma 0.0 0.0 LOX IMVI Trachea 13.4
6.2 Melanoma* 2.3 2.9 (met) SK-MEL-5 Kidney 11.3 14.8 Melanoma SK-
2.4 0.0 MEL-28 Kidney (fetal) 19.1 16.6
[0750] TABLE-US-00107 TABLE 24 Panel 1.3D Rel. Exp.(%) Rel. Exp.(%)
Rel. Exp.(%) Rel. Exp.(%) Ag2866, Run Ag3077, Run Ag2866, Run
Ag3077, Run Tissue Name 161974612 165724514 Tissue Name 161974612
165724514 Liver 100.0 100.0 Kidney (fetal) 13.3 13.0 adenocarcinoma
Pancreas 1.1 4.2 Renal ca. 786-0 2.2 2.6 Pancreatic ca. 5.1 8.5
Renal ca. 12.3 25.3 CAPAN 2 A498 Adrenal gland 3.2 1.6 Renal ca.
RXF393 7.2 25.9 Thyroid 4.7 6.5 Renal ca. 2.3 7.7 ACHN Salivary
gland 0.7 3.3 Renal ca. UO- 2.4 4.4 31 Pituitary gland 1.5 4.2
Renal ca. TK- 5.9 8.6 10 Brain (fetal) 1.1 2.3 Liver 0.3 0.5 Brain
(whole) 2.4 3.1 Liver (fetal) 0.7 1.9 Brain (amygdala) 1.3 3.3
Liver ca. 4.9 9.4 (hepatoblast) HepG2 Brain (cerebellum) 3.6 13.1
Lung 10.6 14.3 Brain 2.5 3.4 Lung (fetal) 11.0 6.0 (hippocampus)
Brain (substantia 0.4 1.2 Lung ca. 5.6 9.2 nigra) (small cell) LX-1
Brain (thalamus) 2.0 4.2 Lung ca. 0.2 0.0 (small cell) NCI-H69
Cerebral Cortex 3.6 0.4 Lung ca. 3.9 1.8 (s.cell var.) SHP-77
Spinal cord 2.2 2.3 Lung ca. (large 19.9 89.5 cell)NCI-H460
glio/astro U87-MG 0.6 0.5 Lung ca. (non- 8.4 10.0 sm. cell) A549
glio/astro U-118- 4.5 22.8 Lung ca. (non- 6.4 3.8 MG s.cell) NCI-
H23 astrocytoma 10.7 4.5 Lung ca. (non- 5.1 7.0 SW1783 s.cell)
HOP-62 neuro*; met SK-N- 7.0 8.3 Lung ca. (non- 2.9 1.5 AS s.cl)
NCI- H522 astrocytoma SF- 5.0 3.6 Lung ca. 7.0 18.9 539 (squam.)
SW900 astrocytoma SNB- 2.6 10.1 Lung ca. 0.3 0.1 75 (squam.) NCI-
H596 glioma SNB-19 27.4 20.6 Mammary 3.4 7.1 gland glioma U251 14.6
46.0 Breast ca.* 5.9 5.5 (pl.ef) MCF-7 glioma SF-295 6.7 6.0 Breast
ca.* 6.5 20.4 (pl.ef) MDA- MB-231 Heart (fetal) 10.4 2.6 Breast
ca.* 14.6 28.3 (pl.ef) T47D Heart 3.7 8.5 Breast ca. BT- 2.2 15.4
549 Skeletal muscle 27.9 0.9 Breast ca. 0.1 0.0 (fetal) MDA-N
Skeletal muscle 1.7 1.0 Ovary 5.6 0.6 Bone marrow 0.5 0.5 Ovarian
ca. 18.2 33.0 OVCAR-3 Thymus 5.1 2.7 Ovarian ca. 10.5 45.7 OVCAR-4
Spleen 4.6 4.0 Ovarian ca. 13.4 14.3 OVCAR-5 Lymph node 2.3 8.0
Ovarian ca. 5.2 3.0 OVCAR-8 Colorectal 5.4 0.6 Ovarian ca. 17.4
22.1 IGROV-1 Stomach 2.9 6.4 Ovarian ca.* 6.6 3.3 (ascites) SK-
OV-3 Small intestine 5.1 22.7 Uterus 3.4 16.2 Colon ca. SW480 7.2
10.7 Placenta 12.5 44.4 Colon ca.* 2.5 3.0 Prostate 3.8 11.4
SW620(SW480 met) Colon ca. HT29 8.5 2.9 Prostate ca.* 14.6 19.2
(bone met)PC-3 Colon ca. HCT- 3.6 3.5 Testis 1.2 3.6 116 Colon ca.
CaCo-2 27.5 22.5 Melanoma 0.0 0.0 Hs688(A).T Colon ca. 5.5 6.0
Melanoma* 1.1 1.2 tissue(ODO3866) (met) Hs688(B).T Colon ca. HCC-
6.3 3.9 Melanoma 0.7 1.3 2998 UACC-62 Gastric ca.* (liver 28.5 47.3
Melanoma 0.6 1.6 met) NCI-N87 M14 Bladder 9.3 2.9 Melanoma 0.0 0.0
LOX IMVI Trachea 8.2 11.7 Melanoma* 0.9 1.0 (met) SK- MEL-5 Kidney
7.2 7.2 Adipose 3.1 2.8
[0751] TABLE-US-00108 TABLE 25 Panel 2D Rel. Exp.(%) Rel. Exp.(%)
Ag2866, Run Ag2866, Tissue Name 162011074 Tissue Name Run 162011074
Normal Colon 41.5 Kidney Margin 25.0 8120608 CC Well to Mod Diff
13.9 Kidney Cancer 28.7 (ODO3866) 8120613 CC Margin (ODO3866) 10.3
Kidney Margin 27.5 8120614 CC Gr.2 rectosigmoid 4.3 Kidney Cancer
33.0 (ODO3868) 9010320 CC Margin (ODO3868) 11.6 Kidney Margin 55.1
9010321 CC Mod Diff (ODO3920) 7.4 Normal Uterus 12.0 CC Margin
(ODO3920) 26.2 Uterus Cancer 064011 26.6 CC Gr.2 ascend colon 14.3
Normal Thyroid 13.5 (ODO3921) CC Margin (ODO3921) 11.4 Thyroid
Cancer 22.5 064010 CC from Partial 21.9 Thyroid Cancer 22.2
Hepatectomy (ODO4309) A302152 Mets Liver Margin (ODO4309) 8.2
Thyroid Margin 17.0 A302153 Colon mets to lung 8.8 Normal Breast
24.5 (OD04451-01) Lung Margin (OD04451- 17.2 Breast Cancer 15.9 02)
(OD04566) Normal Prostate 6546-1 11.9 Breast Cancer 87.1
(OD04590-01) Prostate Cancer 22.4 Breast Cancer Mets 100.0
(OD04410) (OD04590-03) Prostate Margin 26.4 Breast Cancer 48.6
(OD04410) Metastasis (OD04655-05) Prostate Cancer 32.3 Breast
Cancer 064006 28.9 (OD04720-01) Prostate Margin 30.6 Breast Cancer
1024 42.9 (OD04720-02) Normal Lung 061010 32.8 Breast Cancer 27.9
9100266 Lung Met to Muscle 23.2 Breast Margin 18.4 (ODO4286)
9100265 Muscle Margin 25.3 Breast Cancer 20.0 (ODO4286) A209073
Lung Malignant Cancer 22.8 Breast Margin 18.2 (OD03126) A2090734
Lung Margin (OD03126) 28.1 Normal Liver 3.8 Lung Cancer (OD04404)
23.8 Liver Cancer 064003 1.9 Lung Margin (OD04404) 25.7 Liver
Cancer 1025 6.3 Lung Cancer (OD04565) 16.6 Liver Cancer 1026 10.5
Lung Margin (OD04565) 16.3 Liver Cancer 6004-T 15.3 Lung Cancer
(OD04237- 27.5 Liver Tissue 6004-N 9.0 01) Lung Margin (OD04237-
16.8 Liver Cancer 6005-T 13.4 02) Ocular Mel Met to Liver 6.3 Liver
Tissue 6005-N 1.7 (ODO4310) Liver Margin (ODO4310) 3.6 Normal
Bladder 26.1 Melanoma Mets to Lung 3.0 Bladder Cancer 1023 6.6
(OD04321) Lung Margin (OD04321) 24.8 Bladder Cancer 12.2 A302173
Normal Kidney 32.1 Bladder Cancer 32.5 (OD04718-01) Kidney Ca,
Nuclear grade 46.7 Bladder Normal 19.9 2 (OD04338) Adjacent
(OD04718- 03) Kidney Margin 28.9 Normal Ovary 6.7 (OD04338) Kidney
Ca Nuclear grade 28.7 Ovarian Cancer 28.7 1/2 (OD04339) 064008
Kidney Margin 31.6 Ovarian Cancer 91.4 (OD04339) (OD04768-07)
Kidney Ca, Clear cell 50.7 Ovary Margin 13.6 type (OD04340)
(OD04768-08) Kidney Margin 47.6 Normal Stomach 25.0 (OD04340)
Kidney Ca, Nuclear grade 15.7 Gastric Cancer 7.1 3 (OD04348)
9060358 Kidney Margin 27.7 Stomach Margin 6.3 (OD04348) 9060359
Kidney Cancer 29.7 Gastric Cancer 29.5 (OD04622-01) 9060395 Kidney
Margin 7.4 Stomach Margin 14.6 (OD04622-03) 9060394 Kidney Cancer
41.5 Gastric Cancer 51.8 (OD04450-01) 9060397 Kidney Margin 17.6
Stomach Margin 3.6 (OD04450-03) 9060396 Kidney Cancer 8120607 23.5
Gastric Cancer 22.1 064005
[0752] TABLE-US-00109 TABLE 26 Panel 4D Rel. Rel. Rel. Rel. Exp.(%)
Exp.(%) Exp.(%) Exp.(%) Ag2866, Ag3077, Ag2866, Ag3077, Run Run Run
Run Tissue Name 159616591 164681476 Tissue Name 159616591 164681476
Secondary Th1 act 0.0 0.0 HUVEC IL-1beta 2.9 2.6 Secondary Th2 act
0.1 0.0 HUVEC IFN 4.4 9.7 gamma Secondary Tr1 act 0.1 0.3 HUVEC TNF
7.1 9.3 alpha + IFN gamma Secondary Th1 rest 0.0 0.0 HUVEC TNF 4.3
11.3 alpha + IL4 Secondary Th2 rest 0.0 0.0 HUVEC IL-11 3.7 3.8
Secondary Tr1 rest 0.1 0.0 Lung 10.2 15.5 Microvascular EC none
Primary Th1 act 0.0 0.0 Lung 9.5 15.8 Microvascular EC TNF alpha +
IL- 1beta Primary Th2 act 0.1 0.0 Microvascular 30.4 29.7 Dermal EC
none Primary Tr1 act 0.0 0.0 Microsvasular 13.8 18.2 Dermal EC TNF
alpha + IL- 1beta Primary Th1 rest 0.0 0.0 Bronchial 8.4 83.5
epithelium TNF alpha + IL 1beta Primary Th2 rest 0.0 0.0 Small
airway 9.5 13.9 epithelium none Primary Tr1 rest 0.1 0.0 Small
airway 33.9 21.3 epithelium TNF alpha + IL- 1beta CD45RA CD4 0.2
0.4 Coronery artery 0.0 7.2 SMC rest CD45RO CD4 0.0 0.0 Coronery
artery 5.9 9.3 lymphocyte act SMC TNF alpha + IL- 1beta CD8
lymphocyte 0.0 0.0 Astrocytes rest 28.1 38.4 act Secondary CD8 0.0
0.0 Astrocytes 19.6 32.8 lymphocyte rest TNF alpha + IL- 1beta
Secondary CD8 0.0 0.0 KU-812 0.9 0.0 lymphocyte act (Basophil) rest
CD4 lymphocyte 0.1 0.0 KU-812 1.3 1.8 none (Basophil) PMA/ionomycin
2ry 0.0 0.0 CCD1106 10.6 21.5 Th1/Th2/Tr1_anti- (Keratinocytes)
CD95 CH11 none LAK cells rest 0.2 0.0 CCD1106 4.4 18.3
(Keratinocytes) TNF alpha + IL- 1beta LAK cells IL-2 0.0 0.2 Liver
cirrhosis 2.9 2.2 LAK cells IL-2 + IL- 0.0 0.1 Lupus kidney 4.2 2.9
12 LAK cells IL- 0.1 0.0 NCI-H292 none 71.2 55.1 2 + IFN gamma LAK
cells IL-2 + IL- 0.0 0.0 NCI-H292 IL-4 91.4 67.8 18 LAK cells 0.1
0.0 NCI-H292 IL-9 100.0 75.8 PMA/ionomycin NK Cells IL-2 rest 0.1
0.0 NCI-H292 IL-13 80.7 100.0 Two Way MLR 3 0.1 0.3 NCI-H292 IFN
68.8 95.9 day gamma Two Way MLR 5 0.1 0.0 HPAEC none 11.1 11.4 day
Two Way MLR 7 0.0 0.0 HPAEC TNF 9.2 15.6 day alpha + IL-1 beta PBMC
rest 0.1 0.2 Lung fibroblast 10.9 7.8 none PBMC PWM 0.3 0.2 Lung
fibroblast 5.9 7.0 TNF alpha + IL-1 beta PBMC PHA-L 0.1 0.0 Lung
fibroblast 7.9 4.5 IL-4 Ramos (B cell) 1.8 1.5 Lung fibroblast 9.2
5.3 none IL-9 Ramos (B cell) 3.7 3.0 Lung fibroblast 6.6 5.1
ionomycin IL-13 B lymphocytes 0.3 0.2 Lung fibroblast 5.2 4.2 PWM
IFN gamma B lymphocytes 1.9 0.9 Dermal fibroblast 1.9 0.5 CD40L and
IL-4 CCD1070 rest EOL-1 dbcAMP 0.0 0.0 Dermal fibroblast 1.5 0.4
CCD1070 TNF alpha EOL-1 dbcAMP 0.0 0.0 Dermal fibroblast 0.8 1.2
PMA/ionomycin CCD1070 IL-1 beta Dendritic cells 0.1 0.0 Dermal
fibroblast 1.5 1.4 none IFN gamma Dendritic cells LPS 0.1 0.0
Dermal fibroblast 17.1 1.8 IL-4 Dendritic cells anti- 0.1 0.0 IBD
Colitis 2 1.0 0.5 CD40 Monocytes rest 0.0 0.2 IBD Crohn's 0.8 0.9
Monocytes LPS 0.1 0.0 Colon 9.3 6.7 Macrophages rest 0.3 0.0 Lung
10.7 7.7 Macrophages LPS 0.3 0.5 Thymus 21.6 8.3 HUVEC none 8.9 7.2
Kidney 8.9 4.5 HUVEC starved 9.9 6.0
[0753] Panel 1 Summary: Ag400 Two experiments with the same probe
and primer produce results that are in very good agreement, with
highest expression of the NOV1a gene in a lung cancer cell line and
the brain. There are also significant levels of expression in
clusters of cell lines derived from prostate, renal, ovarian,
brain, and colon cancers. This suggests that expression of this
gene may be associated with these cancers. Therefore, therapeutic
modulation of this gene might be of use in the treatment of these
cancers.
[0754] In addition, this gene, a laminin alpha 5 homolog, is
expressed in several metabolic tissues including liver, pancreas
and skeletal muscle. The gene also shows moderate to high levels of
expression in several endocrine tissues including, pituitary,
thyroid and testes, indicating an importance in general endocrine
physiology. Thus, these levels of expression indicate that laminin
alpha 5 may be involved in both endocrine and metabolic processes.
Therapeutic modulation of this gene and/or gene product may
therefore aid in the treatment of a number of endocrine disorders
including metabolic disease.
[0755] This panel confirms the expression of this gene at moderate
levels in all regions of the CNS examined. For a discussion of
utility of this gene in the central nervous system, please see
panel 1.3D.
[0756] Panel 1.3D Summary: Ag2866/3077 The expression of the NOV1 a
gene was assessed in two independent runs on this panel using two
independent probes. There is reasonably good concordance between
the runs with the highest expression in liver adenocarcinoma
(CTs=24-28). There is also significant expression of this gene in
prostate, renal, ovarian, lung, brain and colon cancer cell lines.
This pattern is in agreement with the expression seen in Panel 1,
with these data indicating that the expression of this gene might
be associated with cancer of these tissues. Therefore, therapeutic
modulation of this gene might be of use in the treatment of these
cancers.
[0757] The gene, a laminin alpha 5 homolog, is also expressed in
several metabolic tissues including adipose, liver, pancreas and
skeletal muscle. The gene also has moderate to high levels of
expression in several endocrine tissues as well, including
pituitary, thyroid, and ovaries and testes. This expression profile
suggests that this gene product may play a role in general
endocrine physiology and be involved in both endocrine and
metabolic processes. Therefore, therapeutic modulation of this gene
and/or gene product may aid in the treatment of a number of
endocrine disorders including metabolic disease.
[0758] In addition, this gene is expressed at moderate levels in
all regions of the CNS examined. Laminin has been implicated in
muscular dystrophy. Laminin alpha2 chain deficiency causes
merosin-deficient congenital muscular dystrophy. Furthermore,
laminin alpha 5 may be a functional component of the neuromuscular
synapse. Therefore, therapeutic modulation of this gene may be of
use in the treatment of muscular dystrophy.
REFERENCES
[0759] Nakagawa M, Miyagoe-Suzuki Y, Ikezoe K, Miyata Y, Nonaka I,
Harii K, Takeda S. Schwann cell myelination occurred without basal
lamina formation in laminin alpha2 chain-null mutant (dy3K/dy3K)
mice. Glia 2001 August; 35 (2):101-10
[0760] The laminin alpha2 chain is a major component of basal
lamina in both skeletal muscle and the peripheral nervous system.
Laminin alpha2 chain deficiency causes merosin-deficient congenital
muscular dystrophy, which affects not only skeletal muscles, but
also the peripheral and central nervous systems. It has been
reported that the formation of basal lamina is required for
myelination in the peripheral nervous system. In fact, the spinal
root of dystrophic mice (dy/dy mice), whose laminin alpha2 chain
expression is greatly reduced, shows lack of basal lamina and
clusters of naked axons. To investigate the role of laminin alpha2
chain and basal lamina in vivo, we examined the peripheral nervous
system of dy3K/dy3K mice, which are null mutants of laminin alpha2
chain. The results indicate the presence of myelination although
Schwann cells lacked basal lamina in the spinal roots of dy3K/dy3K
mice, suggesting that basal lamina is not an absolute requirement
for myelination in vivo. Immunohistochemically, the expression of
laminin alpha4 chain was increased and laminin alpha5 chain was
preserved in the endoneurium of the spinal root. Laminin alpha4 and
alpha5 chains may play the critical role in myelination instead of
laminin alpha2 chain in dy3K/dy3K mice. In addition, the motor
conduction velocity of the sciatic nerve was significantly reduced
compared with that of wild-type littermate. This reduction in
conduction velocity may be due to small axon diameter, thin myelin
sheath and the patchy disruption of the basal lamina of the nodes
of Ranvier in dy3K/dy3K mice.
[0761] Son Y J, Scranton T W, Sunderland W J, Baek S J, Miner J H,
Sanes J R, Carlson S S. The synaptic vesicle protein SV2 is
complexed with an alpha5-containing laminin on the nerve terminal
surface. J Biol Chem 2000 Jan. 7; 275 (1):451-60
[0762] Interactions between growing axons and synaptic basal lamina
components direct the formation of neuromuscular junctions during
nerve regeneration. Isoforms of laminin containing alpha5 or beta2
chains are potential basal lamina ligands for these interactions.
The nerve terminal receptors are unknown. Here we show that SV2, a
synaptic vesicle transmembrane proteoglycan, is complexed with a
900-kDa laminin on synaptosomes from the electric organ synapse
that is similar to the neuromuscular junctions. Although two
laminins are present on synaptosomes, only the 900-kDa laminin is
associated with SV2. Other nerve terminal components are absent
from this complex. The 900-kDa laminin contains an alpha5, a beta1,
and a novel gamma chain. To test whether SV2 directly binds the
900-kDa laminin, we looked for interaction between purified SV2 and
laminin-1, a laminin isoform with a similar structure. We find SV2
binds with high affinity to purified laminin-1. Our results suggest
that a synaptic vesicle component may act as a laminin receptor on
the presynaptic plasma membrane; they also suggest a mechanism for
activity-dependent adhesion at the synapse.
[0763] Panel 2.2 Summary: Ag3077 Data from one run with the NOV1a
gene is not included because the amp plot indicates that there were
experimental difficulties with this run.
[0764] Panel 2D Summary: Ag2866 The NOV1a gene is moderately
expressed in all the tissues used in this panel. There is increased
expression in stomach, breast and ovarian cancer samples compared
to normal adjacent tissues from the same sample. Therefore,
expression of this gene could potentially be used as a diagnostic
marker for the presence of these cancers. Furthermore, therapeutic
modulation of the activity of the gene product through the use of
antibodies, small molecule inhibitors and chimeric molecules may be
effective in the treatment of these cancers.
[0765] Panel 4D Summary: Ag2866/Ag3077 Two experiments with two
different probe and primer sets produce results that are in very
good agreement, with highest expression of the NOVIA gene in
activated-NC1--H292 mucoepidermoid cells (CTs=24-28). Significant
levels of expression are seen in IL-4, IL-9, IL-13 and IFN gamma
activated-NC1--H292 mucoepidermoid cells as well as untreated
NC1--H292 cells. Moderate/low expression is also detected in IL-4,
IL-9, IL-13 and IFN gamma activated lung fibroblasts, human
pulmonary aortic endothelial cells (treated and untreated), small
airway epithelium (treated and untreated), treated bronchial
epithelium and lung microvascular endothelial cells (treated and
untreated). The expression of this gene in cells derived from or
within the lung suggests that this gene may be involved in normal
conditions as well as pathological and inflammatory lung disorders
that include chronic obstructive pulmonary disease, asthma, allergy
and emphysema. Moderate/low expression of NOVIA is also detected in
treated and untreated HUVECs (endothelial cells), coronary artery
smooth muscle cells (treated and untreated), treated and untreated
astrocytes, treated KU-812 basophils, treated and untreated CCD1106
keratinocytes, IL-4 treated dermal fibroblasts, and normal tissues
that include lung, colon, thymus and kidney. Low level expression
is also detected in treated and untreated Ramos (B cell) cells as
well as liver cirrhosis and lupus kidney samples. Expression in the
various immune cell types (as well as in diseased tissue samples)
suggests that therapeutic modulation of NOV1a may ameliorate
symptoms associated with infectious conditions as well as
inflammatory and autoimmune disorders that include psoriasis,
allergy, asthma, inflammatory bowel disease, rheumatoid arthritis
and osteoarthritis. Also, owing to the importance of immune
cells/lymphoid cells (eg. T and B cells) in lupus and cirrhosis,
therapeutic modulation of NOVIA may ameliorate symptoms associated
with lupus and other autoimmune diseases as well as liver
cirrhosis. NOV1a may also serve as a marker for these diseases.
NOV14b
[0766] Expression of gene NOV14b was assessed using the
primer-probe set Ag2908, described in Table 27. Results of the
RTQ-PCR runs are shown in Table 28. TABLE-US-00110 TABLE 27 Probe
Name Ag2908 Primers Sequences Length Start Position SEQ ID NO:
Forward 5'-attgtttacatcaaacggcatt-3' 22 944 164 Probe
TET-5'-aatccttttgaggcccttgtcccata-3'-TAMRA 26 981 165 Reverse
5'-tcccagttgagactcctactga-3' 22 1009 166
[0767] TABLE-US-00111 TABLE 28 Panel 4D Rel. Exp.(%) Rel. Exp.(%)
Ag2908, Run Ag2908, Run Tissue Name 164403110 Tissue Name 164403110
Secondary Th1 act 11.7 HUVEC IL-1beta 0.0 Secondary Th2 act 2.4
HUVEC IFN gamma 0.0 Secondary Tr1 act 11.5 HUVEC TNF alpha + IFN
0.0 gamma Secondary Th1 rest 1.7 HUVEC TNF alpha + IL4 0.0
Secondary Th2 rest 7.4 HUVEC IL-11 0.0 Secondary Tr1 rest 6.0 Lung
Microvascular EC 0.0 none Primary Th1 act 2.0 Lung Microvascular EC
0.8 TNF alpha + IL-1beta Primary Th2 act 5.6 Microvascular Dermal
EC 0.0 none Primary Tr1 act 1.7 Microsvasular Dermal EC 0.0 TNF
alpha + IL-1beta Primary Th1 rest 20.0 Bronchial epithelium 0.0 TNF
alpha + IL1beta Primary Th2 rest 13.8 Small airway epithelium 0.0
none Primary Tr1 rest 8.0 Small airway epithelium 0.7 TNF alpha +
IL-1beta CD45RA CD4 0.8 Coronery artery SMC rest 0.0 lymphocyte act
CD45RO CD4 4.9 Coronery artery SMC 0.0 lymphocyte act TNF alpha +
IL-1beta CD8 lymphocyte act 0.0 Astrocytes rest 0.0 Secondary CD8
0.8 Astrocytes TNF alpha + IL- 0.0 lymphocyte rest 1beta Secondary
CD8 4.2 KU-812 (Basophil) rest 0.0 lymphocyte act CD4 lymphocyte
none 0.0 KU-812 (Basophil) 1.8 PMA/ionomycin 2ry Th1/Th2/Tr1_anti-
13.1 CCD1106 (Keratinocytes) 0.0 CD95 CH11 none LAK cells rest 46.3
CCD1106 (Keratinocytes) 0.0 TNF alpha + IL-1beta LAK cells IL-2 7.3
Liver cirrhosis 3.9 LAK cells IL-2 + IL-12 3.3 Lupus kidney 1.3 LAK
cells IL-2 + IFN 11.3 NCI-H292 none 0.0 gamma LAK cells IL-2 +
IL-18 6.2 NCI-H292 IL-4 1.6 LAK cells 11.5 NCI-H292 IL-9 0.6
PMA/ionomycin NK Cells IL-2 rest 15.1 NCI-H292 IL-13 0.7 Two Way
MLR 3 day 8.2 NCI-H292 IFN gamma 0.0 Two Way MLR 5 day 4.0 HPAEC
none 0.0 Two Way MLR 7 day 2.5 HPAEC TNF alpha + IL- 0.0 1beta PBMC
rest 0.0 Lung fibroblast none 0.0 PBMC PWM 3.8 Lung fibroblast TNF
alpha + IL- 0.0 1beta PBMC PHA-L 5.5 Lung fibroblast IL-4 0.0 Ramos
(B cell) none 0.0 Lung fibroblast IL-9 0.0 Ramos (B cell) 0.0 Lung
fibroblast IL-13 0.0 ionomycin B lymphocytes PWM 3.4 Lung
fibroblast IFN 0.0 gamma B lymphocytes CD40L 1.5 Dermal fibroblast
0.0 and IL-4 CCD1070 rest EOL-1 dbcAMP 0.0 Dermal fibroblast 52.5
CCD1070 TNF alpha EOL-1 dbcAMP 9.7 Dermal fibroblast 0.0
PMA/ionomycin CCD1070 IL-1beta Dendritic cells none 94.0 Dermal
fibroblast IFN 0.0 gamma Dendritic cells LPS 20.3 Dermal fibroblast
IL-4 0.0 Dendritic cells anti- 100.0 IBD Colitis 2 0.9 CD40
Monocytes rest 23.2 IBD Crohn's 0.0 Monocytes LPS 0.0 Colon 0.0
Macrophages rest 54.7 Lung 0.7 Macrophages LPS 7.7 Thymus 0.9 HUVEC
none 0.0 Kidney 0.0 HUVEC starved 0.0
[0768] CNS_neurodegeneration_v1.0 Summary: Ag2908 Expression of the
NOV14b gene is low/undetectable in all samples on this panel.
(CTs>35). (Data not shown.)
[0769] Panel 1.3D Summary: Ag2908 Expression of the NOV14b gene is
restricted to bone marrow (CT=34.78). Thus, expression of this gene
could be used as a marker for this tissue.
[0770] Panel 4D Summary: Ag2908 The NOV14b gene is expressed at low
levels in resting lymphokine activated killer cells (LAK), resting
macrophages and monocytes., and CCD1070 dermal fibroblasts treated
with TNF alpha. Low level expression is also detected in both
stimulated and resting dendritic cells. The expression of this gene
in resting cells of these lineages suggests that the protein
encoded by this transcript may be involved in normal immunological
processes associated with immune homeostasis. Expression in
TNFalpha treated dermal fibroblasts also suggests that this gene
product may be involved in skin disorders, including psoriasis.
[0771] In addition, low level expression of this transcript is
detected in stimulated, lymphokine-activated killer cells (LAK).
Since these cells are involved in tumor immunology and tumor cell
clearance, as well as virally and bacterial infected cells,
therapeutic modulation of this gene product may alter the functions
of these cells and lead to improvement in cancer cell killing as
well as host immunity to microbial and viral infections. This
expression in immune cells suggests that therapeutic modulation of
this gene product may ameliorate symptoms associated with
inflammatory and autoimmune disorders that include psoriasis,
allergy, asthma, inflammatory bowel disease, rheumatoid arthritis
and osteoarthritis.
NOV11
[0772] Expression of gene NOV11 was assessed using the primer-probe
sets Ag1522, Ag1848, Ag2263 and Ag2422, described in Tables 29, 30,
31 and 32. Results of the RTQ-PCR runs are shown in Tables 33, 34,
35, 36, 37, 38, 39 and 40. TABLE-US-00112 TABLE 29 Probe Name
Ag1522 Primers Sequences Length Start Position SEQ ID NO: Forward
5'-tgacttcgacacagacatcact-3' 22 1242 167 Probe
TET-5'-actcatctgctgccctgactggtg-3'-TAMRA 24 1265 168 Reverse
5'-ccttgccgtcttaaagttgac-3' 21 1300 169
[0773] TABLE-US-00113 TABLE 30 Probe Name Ag1848 Primers Sequences
Length Start Position SEQ ID NO: Forward
5'-tgacttcgacacagacatcact-3' 22 1242 170 Probe
TET-5'-actcatctgctgccctgactggtg-3'-TAMRA 24 1265 171 Reverse
5'-ccttgccgtcttaaagttgac-3' 21 1300 172
[0774] TABLE-US-00114 TABLE 31 Probe Name Ag2263 Primers Sequences
Length Start Position SEQ ID NO: Forward
5'-tgacttcgacacagacatcact-3' 22 1242 173 Probe
TET-5'-actcatctgctgccctgactggtg-3'-TAMRA 24 1265 174 Reverse
5'-ccttgccgtcttaaagttgac-3' 21 1300 175
[0775] TABLE-US-00115 TABLE 32 Probe Name Ag2422 Primers Sequences
Length Start Position SEQ ID NO: Forward 5'-ggctccctggacactctct-3'
19 2530 176 Probe TET-5'-ctgtcaccacccagctgggaccttat-3'-TAMRA 26
2567 177 Reverse 5'-tggacagtgggatcttgaag-3' 20 2595 178
[0776] TABLE-US-00116 TABLE 33 AI_comprehensive panel_v1.0 Rel.
Exp.(%) Rel. Exp.(%) Rel. Exp.(%) Rel. Exp.(%) Ag1522, Run Ag1848,
Run Ag1522, Run Ag1848, Run Tissue Name 229393906 229440541 Tissue
Name 229393906 229440541 110967 COPD-F 2.5 1.5 112427 Match 13.0
6.9 Control Psoriasis-F 110980 COPD-F 5.6 5.4 12418 2.5 1.9
Psoriasis-F 110968 COPD-M 2.3 1.7 112723 Match 3.3 3.0 Control
Psoriasis-M 110977 COPD-M 12.2 10.0 112419 3.0 2.8 Psoriasis-M
110989 6.4 4.5 112424 Match 2.5 1.7 Emphysema-F Control Psoriasis-M
110992 4.2 2.6 112420 7.2 7.1 Emphysema-F Psoriasis-M 110993 3.5
3.1 112425 Match 5.5 6.7 Emphysema-F Control Psoriasis-M 110994 2.8
1.3 104689 (MF) 100.0 92.7 Emphysema-F OA Bone- Backus 110995 10.0
3.8 104690 (MF) 32.1 35.1 Emphysema-F Adj "Normal" Bone-Backus
110996 2.1 0.9 104691 (MF) 3.1 3.0 Emphysema-F OA Synovium- Backus
110997 3.5 1.1 104692 (BA) 27.9 22.1 Asthma-M OA Cartilage- Backus
111001 5.6 1.2 104694 (BA) 81.2 100.0 Asthma-F OA Bone- Backus
111002 6.7 3.4 104695 (BA) 57.0 54.7 Asthma-F Adj "Normal"
Bone-Backus 111003 Atopic 5.8 4.0 104696 (BA) 14.4 11.9 Asthma-F OA
Synovium- Backus 111004 Atopic 13.1 6.8 104700 (SS) 34.4 27.0
Asthma-F OA Bone- Backus 111005 Atopic 5.2 3.3 104701 (SS) 44.1
45.7 Asthma-F Adj "Normal" Bone-Backus 111006 Atopic 1.8 1.0 104702
(SS) 5.3 4.9 Asthma-F OA Synovium- Backus 111417 4.3 3.3 117093 OA
3.4 3.0 Allergy-M Cartilage Rep7 112347 0.4 0.1 112672 OA 3.6 3.8
Allergy-M Bone5 112349 Normal 0.3 0.1 112673 OA 1.5 2.2 Lung-F
Synovium5 112357 Normal 2.6 3.0 112674 OA 1.5 1.4 Lung-F Synovial
Fluid cells5 112354 Normal 1.1 1.3 117100 OA 1.6 1.9 Lung-M
Cartilage Rep14 112374 Crohns-F 9.7 5.7 112756 OA 7.3 5.7 Bone9
112389 Match 16.5 8.4 112757 OA 0.8 0.5 Control Synovium9 Crohns-F
112375 Crohns-F 9.2 6.3 112758 OA 3.3 2.3 Synovial Fluid Cells9
112732 Match 2.7 2.5 117125 RA 4.6 3.0 Control Cartilage Rep2
Crohns-F 112725 Crohns-M 0.8 0.6 113492 Bone2 6.5 5.3 RA 112387
Match 6.3 4.5 113493 3.1 1.8 Control Synovium2 Crohns-M RA 112378
Crohns-M 0.5 0.2 113494 Syn 5.9 4.7 Fluid Cells RA 112390 Match
23.7 16.7 113499 5.2 3.4 Control Cartilage4 RA Crohns-M 112726
Crohns-M 2.0 3.3 113500 Bone4 4.6 3.2 RA 112731 Match 4.8 3.4
113501 3.1 2.1 Control Synovium4 Crohns-M RA 112380 Ulcer 4.8 3.3
113502 Syn 2.7 2.2 Col-F Fluid Cells4 RA 112734 Match 7.4 3.8
113495 4.4 3.1 Control Ulcer Cartilage3 RA Col-F 112384 Ulcer 6.7
5.6 113496 Bone3 5.0 3.0 Col-F RA 112737 Match 1.7 0.9 113497 3.3
2.4 Control Ulcer Synovium3 Col-F RA 112386 Ulcer 1.6 3.7 113498
Syn 6.2 4.0 Col-F Fluid Cells3 RA 112738 Match 1.4 1.0 117106 3.1
3.0 Control Ulcer Normal Col-F Cartilage Rep20 112381 Ulcer 0.2 0.1
113663 Bone3 0.3 0.1 Col-M Normal 112735 Match 1.1 1.5 113664 0.1
0.0 Control Ulcer Synovium3 Col-M Normal 112382 Ulcer 11.5 8.7
113665 Syn 0.2 0.0 Col-M Fluid Cells3 Normal 112394 Match 2.0 1.7
117107 0.6 0.4 Control Ulcer Normal Col-M Cartilage Rep22 112383
Ulcer 4.8 2.9 113667 Bone4 0.7 0.7 Col-M Normal 112736 Match 22.2
6.3 113668 0.8 0.7 Control Ulcer Synovium4 Col-M Normal 112423 1.3
0.9 113669 Syn 0.8 0.9 Psoriasis-F Fluid Cells4 Normal
[0777] TABLE-US-00117 TABLE 34 CNS_neurodegeneration_v1.0 Rel.
Exp.(%) Rel. Exp.(%) Rel. Exp.(%) Rel. Exp.(%) Rel. Exp.(%) Ag1848,
Run Ag2263, Run Ag2263, Run Ag2422, Run Ag2422, Run Tissue Name
207776125 219933384 224115886 206262709 230512499 AD 1 Hippo 28.3
39.0 19.3 21.3 16.6 AD 2 Hippo 37.9 45.1 23.5 38.7 40.1 AD 3 Hippo
12.0 20.6 13.9 14.9 13.0 AD 4 Hippo 17.7 27.2 9.0 13.3 16.4 AD 5
hippo 45.4 60.3 8.1 57.8 59.0 AD 6 Hippo 66.9 96.6 70.2 95.9 66.0
Control 2 43.2 81.2 67.8 46.0 48.3 Hippo Control 4 34.2 36.6 38.7
30.4 27.5 Hippo Control (Path) 3.9 11.0 4.6 12.7 12.1 3 Hippo AD 1
Temporal 47.0 79.0 69.7 40.6 27.2 Ctx AD 2 Temporal 49.3 61.6 70.7
39.8 50.7 Ctx AD 3 Temporal 14.5 20.7 15.3 15.7 14.5 Ctx AD 4
Temporal 41.5 53.6 31.9 36.3 39.0 Ctx AD 5 Inf 77.9 95.9 72.2 88.9
100.0 Temporal Ctx AD 5 40.9 57.4 3.7 57.0 69.3 SupTemporal Ctx AD
6 Inf 84.1 99.3 100.0 74.2 83.5 Temporal Ctx AD 6 Sup 58.2 64.6
81.8 71.7 61.1 Temporal Ctx Control 1 17.9 18.0 21.5 11.3 16.5
Temporal Ctx Control 2 45.7 39.8 66.4 44.8 55.1 Temporal Ctx
Control 3 14.7 21.8 22.7 15.6 13.5 Temporal Ctx Control 4 23.2 21.5
23.8 19.1 24.1 Temporal Ctx Control (Path) 46.0 39.8 19.3 40.3 51.1
1 Temporal Ctx Control (Path) 24.7 40.6 23.7 21.8 24.0 2 Temporal
Ctx Control (Path) 6.0 8.2 8.0 7.7 7.3 3 Temporal Ctx Control
(Path) 32.1 29.5 31.0 24.0 18.6 4 Temporal Ctx AD 1 Occipital 24.1
48.0 5.5 26.4 13.7 Ctx AD 2 Occipital 0.0 0.0 0.0 0.0 0.0 Ctx
(Missing) AD 3 Occipital 19.2 25.3 20.4 18.2 18.8 Ctx AD 4
Occipital 30.1 58.2 30.6 23.3 30.8 Ctx AD 5 Occipital 6.0 51.8 53.6
26.8 23.0 Ctx AD 6 Occipital 43.2 39.0 8.5 50.3 47.6 Ctx Control 1
14.6 22.2 19.1 12.8 13.4 Occipital Ctx Control 2 66.9 85.9 94.6
76.3 70.2 Occipital Ctx Control 3 17.8 37.1 8.0 17.4 13.1 Occipital
Ctx Control 4 23.3 22.2 2.7 15.7 19.1 Occipital Ctx Control (Path)
100.0 100.0 63.7 100.0 90.1 1 Occipital Ctx Control (Path) 18.7
20.9 11.0 12.3 11.7 2 Occipital Ctx Control (Path) 7.9 6.1 9.4 7.1
5.8 3 Occipital Ctx Control (Path) 24.5 21.5 11.1 14.0 13.1 4
Occipital Ctx Control 1 23.2 26.8 7.4 22.2 17.6 Parietal Ctx
Control 2 46.0 65.1 71.2 64.6 50.0 Parietal Ctx Control 3 26.1 27.2
16.5 17.3 19.5 Parietal Ctx Control (Path) 51.1 66.0 80.1 54.3 55.1
1 Parietal Ctx Control (Path) 36.3 16.5 34.2 27.9 27.9 2 Parietal
Ctx Control (Path) 6.1 10.5 1.4 5.1 4.6 3 Parietal Ctx Control
(Path) 46.0 52.5 10.7 36.6 12.2 4 Parietal Ctx
[0778] TABLE-US-00118 TABLE 35 Panel 1.2 Rel. Exp.(%) Ag1522, Rel.
Exp.(%) Ag1522, Tissue Name Run 142131145 Tissue Name Run 142131145
Endothelial cells 1.2 Renal ca. 786-0 0.0 Heart (Fetal) 17.9 Renal
ca. A498 0.3 Pancreas 0.7 Renal ca. RXF 393 0.2 Pancreatic ca.
CAPAN2 4.9 Renal ca. ACHN 0.1 Adrenal Gland 7.9 Renal ca. UO-31 0.5
Thyroid 0.1 Renal ca. TK-10 0.3 Salivary gland 2.5 Liver 2.4
Pituitary gland 0.1 Liver (fetal) 0.5 Brain (fetal) 0.2 Liver ca.
0.3 (hepatoblast) HepG2 Brain (whole) 3.2 Lung 0.3 Brain (amygdala)
4.4 Lung (fetal) 0.4 Brain (cerebellum) 9.0 Lung ca. (small cell)
25.3 LX-1 Brain (hippocampus) 18.9 Lung ca. (small cell) 43.8
NCI-H69 Brain (thalamus) 15.7 Lung ca. (s. cell var.) 0.3 SHP-77
Cerebral Cortex 35.4 Lung ca. (large 54.7 cell)NCI-H460 Spinal cord
1.6 Lung ca. (non-sm. 0.3 cell) A549 glio/astro U87-MG 72.2 Lung
ca. (non-s. cell) 2.4 NCI-H23 glio/astro U-118-MG 3.1 Lung ca.
(non-s. cell) 1.7 HOP-62 astrocytoma SW1783 0.3 Lung ca. (non-s.
cl) 9.3 NCI-H522 neuro*; met SK-N-AS 36.3 Lung ca. (squam.) 1.5 SW
900 astrocytoma SF-539 5.8 Lung ca. (squam.) 22.4 NCI-H596
astrocytoma SNB-75 1.7 Mammary gland 1.4 glioma SNB-19 23.8 Breast
ca.* (pl. ef) 0.8 MCF-7 glioma U251 2.9 Breast ca.* (pl. ef) 0.1
MDA-MB-231 glioma SF-295 100.0 Breast ca.* (pl. ef) 18.4 T47D Heart
31.6 Breast ca. BT-549 0.1 Skeletal Muscle 3.4 Breast ca. MDA-N 0.0
Bone marrow 0.2 Ovary 6.9 Thymus 0.2 Ovarian ca. OVCAR-3 1.7 Spleen
2.1 Ovarian ca. OVCAR-4 12.9 Lymph node 0.5 Ovarian ca. OVCAR-5 5.7
Colorectal Tissue 1.4 Ovarian ca. OVCAR-8 5.3 Stomach 1.3 Ovarian
ca. IGROV-1 0.8 Small intestine 3.3 Ovarian ca. (ascites) 5.4
SK-OV-3 Colon ca. SW480 0.8 Uterus 0.9 Colon ca.* SW620 2.2
Placenta 0.9 (SW480 met) Colon ca. HT29 0.1 Prostate 10.0 Colon ca.
HCT-116 7.5 Prostate ca.* (bone 0.1 met) PC-3 Colon ca. CaCo-2 6.3
Testis 0.3 Colon ca. Tissue 3.0 Melanoma 21.2 (ODO3866) Hs688(A).T
Colon ca. HCC-2998 1.2 Melanoma* (met) 28.5 Hs688(B).T Gastric ca.*
(liver 24.7 Melanoma UACC-62 2.4 met) NCI-N87 Bladder 12.8 Melanoma
M14 0.1 Trachea 0.3 Melanoma LOX 0.1 IMVI Kidney 19.2 Melanoma*
(met) 1.2 SK-MEL-5 Kidney (fetal) 6.6
[0779] TABLE-US-00119 TABLE 36 Panel 1.3D Rel. Exp.(%) Rel. Exp.(%)
Rel. Exp.(%) Rel. Exp.(%) Ag1522, Run Ag1848, Run Ag2263, Run
Ag2422, Run Tissue Name 159601761 160201402 166011650 159319549
Liver 15.8 12.3 31.4 18.3 adenocarcinoma Pancreas 1.7 1.4 2.8 2.9
Pancreatic ca. 6.7 4.6 21.6 5.5 CAPAN 2 Adrenal gland 3.9 2.0 3.5
3.0 Thyroid 1.7 1.5 0.0 2.5 Salivary gland 0.6 0.2 2.3 0.3
Pituitary gland 2.1 1.4 2.9 4.3 Brain (fetal) 1.4 1.1 3.5 1.1 Brain
(whole) 28.7 13.5 43.2 10.4 Brain (amygdala) 16.8 13.0 31.2 18.6
Brain (cerebellum) 8.2 6.5 42.3 9.2 Brain (hippocampus) 60.7 47.6
16.8 51.8 Brain (substantia 8.9 5.2 32.3 6.8 nigra) Brain
(thalamus) 40.1 22.2 62.0 19.8 Cerebral Cortex 25.9 18.4 36.6 14.3
Spinal cord 10.2 5.4 37.9 7.9 glio/astro U87-MG 43.2 34.6 100.0
48.6 glio/astro U-118-MG 10.2 8.0 6.4 7.5 astrocytoma 0.9 0.8 2.8
1.1 SW1783 neuro*; met SK-N- 100.0 100.0 59.0 100.0 AS astrocytoma
SF-539 9.7 8.3 17.7 9.0 astrocytoma SNB-75 12.9 12.1 8.4 12.1
glioma SNB-19 19.5 17.6 46.3 17.2 glioma U251 13.4 10.6 24.5 10.9
glioma SF-295 66.9 62.4 64.2 62.0 Heart (fetal) 15.6 12.5 20.0 18.7
Heart 2.2 1.1 3.4 3.3 Skeletal muscle 22.2 14.0 6.7 19.3 (fetal)
Skeletal muscle 0.3 0.2 1.4 0.7 Bone marrow 0.7 0.3 0.4 0.8 Thymus
2.0 1.6 3.6 3.4 Spleen 7.9 5.6 4.5 5.9 Lymph node 2.6 1.9 2.7 2.1
Colorectal 4.7 9.2 12.8 10.3 Stomach 6.1 2.4 3.6 4.5 Small
intestine 2.9 2.9 4.5 4.9 Colon ca. SW480 2.0 1.0 1.9 1.5 Colon
ca.* 1.0 1.2 2.0 2.1 SW620(SW480 met) Colon ca. HT29 0.1 0.1 0.0
0.1 Colon ca. HCT-116 4.2 2.9 4.7 5.6 Colon ca. CaCo-2 5.3 3.9 12.5
7.2 Colen ca. 14.8 17.3 19.8 23.5 tissue(ODO3866) Colon ca.
HCC-2998 0.7 1.6 0.0 0.5 Gastric ca.* (liver 21.9 22.8 19.1 25.7
met) NCI-N87 Bladder 2.1 1.7 3.4 1.5 Trachea 12.2 6.8 1.6 13.8
Kidney 1.4 0.6 3.9 3.0 Kidney (fetal) 5.3 5.8 5.2 6.3 Renal ca.
786-0 0.1 0.0 0.0 0.0 Renal ca. A498 7.7 7.9 6.8 9.7 Renal ca. RXF
393 0.1 3.6 0.8 0.1 Renal ca. ACHN 0.0 0.0 0.0 0.0 Renal ca. UO-31
0.2 0.3 0.5 0.3 Renal ca. TK-10 0.1 0.0 0.0 0.0 Liver 0.3 0.1 0.0
0.6 Liver (fetal) 1.1 1.0 0.3 1.2 Liver ca. 0.2 0.0 0.8 0.3
(hepatoblast) HepG2 Lung 8.2 9.4 4.1 10.3 Lung (fetal) 4.3 4.2 7.3
4.5 Lung ca. (small cell) 8.4 6.9 31.6 9.9 LX-1 Lung ca. (small
cell) 44.4 48.6 90.8 54.3 NCI-H69 Lung ca. (s. cell var.) 0.7 0.8
0.5 1.1 SHP-77 Lung ca. (large 16.2 11.9 22.4 12.1 cell)NCI-H460
Lung ca. (non-sm. 0.4 0.3 0.2 0.4 cell) A549 Lung ca. (non-s. cell)
2.0 0.9 3.3 1.2 NCI-H23 Lung ca. (non-s. cell) 0.4 0.9 1.6 0.7
HOP-62 Lung ca. (non-s. cl) 1.7 0.8 1.7 1.1 NCI-H522 Lung ca.
(squam.) 0.5 0.3 1.9 0.2 SW 900 Lung ca. (squam.) 4.0 4.1 26.4 2.4
NCI-H596 Mammary gland 6.3 4.4 3.0 2.8 Breast ca.* (pl.ef) 1.1 0.4
1.5 0.9 MCF-7 Breast ca.* (pl.ef) 0.8 1.2 0.7 1.4 MDA-MB-231 Breast
ca.* (pl.ef) 9.6 5.7 14.0 4.5 T47D Breast ca. BT-549 0.2 0.3 0.2
0.3 Breast ca. MDA-N 0.0 0.0 0.0 0.0 Ovary 6.4 4.9 6.2 9.5 Ovarian
ca. 1.1 0.6 1.1 0.8 OVCAR-3 Ovarian ca. 1.0 1.4 11.4 1.5 OVCAR-4
Ovarian ca. 2.4 2.6 5.7 3.3 OVCAR-5 Ovarian ca. 3.6 1.6 2.6 5.4
OVCAR-8 Ovarian ca. IGROV-1 0.6 0.2 0.7 0.2 Ovarian ca.* 2.0 2.6
2.1 1.1 (ascites) SK-OV-3 Uterus 2.7 1.3 3.9 4.2 Placenta 2.0 2.0
5.8 4.8 Prostate 4.4 2.5 3.4 5.4 Prostate ca.* (bone 0.1 0.1 0.2
0.0 met)PC-3 Testis 8.1 5.5 3.5 6.4 Melanoma 31.6 25.0 59.5 27.4
Hs688(A).T Melanoma* (met) 46.0 17.1 87.1 28.5 Hs688(B).T Melanoma
UACC- 0.1 0.2 2.0 0.5 62 Melanoma M14 0.0 0.0 0.0 0.0 Melanoma LOX
0.1 0.2 0.0 0.1 IMVI Melanoma* (met) 0.9 0.9 1.7 0.6 SK-MEL-5
Adipose 3.6 2.3 5.1 2.9
[0780] TABLE-US-00120 TABLE 37 Panel 2D Rel. Exp.(%) Rel. Exp.(%)
Rel. Exp.(%) Rel. Exp.(%) Rel. Exp.(%) Ag1522, Run Ag1522, Run
Ag1848, Run Ag2263, Run Ag2422, Run Tissue Name 145049854 145492337
160202834 165725935 159317774 Normal Colon 20.2 46.0 35.1 59.0 36.9
CC Well to Mod 15.3 45.1 22.5 21.8 21.3 Diff (ODO3866) CC Margin
6.1 15.2 7.4 7.7 5.5 (ODO3866) CC Gr.2 7.0 8.2 5.8 5.9 13.2
rectosigmoid (ODO3868) CC Margin 0.3 0.5 0.5 9.3 0.8 (ODO3868) CC
Mod Diff 1.2 4.0 2.5 5.6 5.8 (ODO3920) CC Margin 3.0 4.7 4.1 5.4
7.2 (ODO3920) CC Gr.2 ascend 10.7 22.5 24.1 19.9 25.5 colon
(ODO3921) CC Margin 3.6 4.3 7.3 5.6 5.8 (ODO3921) CC from Partial
12.1 19.9 20.7 19.3 27.0 Hepatectomy (ODO4309) Mets Liver Margin
0.4 3.6 2.4 2.6 3.3 (ODO4309) Colon mets to 5.8 11.9 6.1 8.5 10.7
lung (OD04451- 01) Lung Margin 9.3 17.7 7.7 10.0 15.4 (OD04451-02)
Normal Prostate 10.5 51.1 7.3 21.6 7.0 6546-1 Prostate Cancer 12.2
14.9 14.9 9.0 17.4 (OD04410) Prostate Margin 14.6 13.8 25.3 19.2
29.7 (OD04410) Prostate Cancer 12.2 18.0 22.7 31.6 30.6
(OD04720-01) Prostate Margin 11.8 11.8 17.7 16.7 25.0 (OD04720-02)
Normal Lung 7.3 17.8 17.6 12.8 22.4 061010 Lung Met to 12.7 27.4
25.0 31.0 22.1 Muscle (ODO4286) Muscle Margin 7.4 8.7 6.2 7.3 9.5
(ODO4286) Lung Malignant 22.7 27.4 26.1 28.3 20.4 Cancer (OD03126)
Lung Margin 12.7 21.9 21.9 13.9 31.9 (OD03126) Lung Cancer 17.9
41.5 41.5 30.4 48.0 (OD04404) Lung Margin 16.4 28.7 10.0 11.8 12.4
(OD04404) Lung Cancer 22.5 38.2 28.5 27.9 40.6 (OD04565) Lung
Margin 8.1 11.7 8.5 8.6 16.3 (OD04565) Lung Cancer 9.8 7.1 10.9 8.8
9.6 (OD04237-01) Lung Margin 12.9 23.0 14.3 14.0 16.0 (OD04237-02)
Ocular Mel Met 0.6 0.5 0.7 0.5 1.1 to Liver (ODO4310) Liver Margin
3.5 2.6 1.8 3.3 3.0 (ODO4310) Melanoma Mets 1.4 2.0 3.6 4.3 2.9 to
Lung (OD04321) Lung Margin 20.4 14.4 25.2 24.0 18.6 (OD04321)
Normal Kidney 20.2 19.9 18.0 17.4 26.1 Kidney Ca, 1.7 4.2 2.9 2.7
4.9 Nuclear grade 2 (OD04338) Kidney Margin 6.2 11.7 17.2 11.3 22.8
(OD04338) Kidney Ca 3.6 10.0 3.7 4.6 6.6 Nuclear grade 1/2
(OD04339) Kidney Margin 11.7 12.2 11.4 12.1 11.0 (OD04339) Kidney
Ca, Clear 46.7 50.7 66.0 65.1 70.7 cell type (OD04340) Kidney
Margin 15.3 19.1 14.8 12.9 16.8 (OD04340) Kidney Ca, 21.0 9.5 16.3
16.8 17.0 Nuclear grade 3 (OD04348) Kidney Margin 8.2 5.8 8.8 11.5
9.3 (OD04348) Kidney Cancer 24.0 25.3 27.7 24.8 41.5 (OD04622-01)
Kidney Margin 2.1 4.6 3.4 3.1 5.9 (OD04622-03) Kidney Cancer 0.2
0.0 0.2 0.5 0.5 (OD04450-01) Kidney Margin 5.9 6.3 9.3 9.9 12.9
(OD04450-03) Kidney Cancer 7.3 9.1 11.9 12.8 13.4 8120607 Kidney
Margin 12.2 6.2 7.9 5.6 8.0 8120608 Kidney Cancer 3.6 8.0 5.2 8.8
10.1 8120613 Kidney Margin 6.3 6.7 8.9 7.5 9.3 8120614 Kidney
Cancer 18.7 61.1 25.0 21.9 22.1 9010320 Kidney Margin 14.0 20.3
16.4 12.9 17.9 9010321 Normal Uterus 4.1 5.6 3.3 8.4 6.0 Uterus
Cancer 9.6 10.7 17.1 11.7 15.6 064011 Normal Thyroid 2.6 9.2 2.6
1.5 3.6 Thyroid Cancer 100.0 72.7 100.0 82.9 100.0 064010 Thyroid
Cancer 7.6 4.5 12.5 8.0 11.7 A302152 Thyroid Margin 3.0 2.4 2.8 3.2
6.0 A302153 Normal Breast 10.3 5.7 9.9 12.9 7.2 Breast Cancer 11.7
15.9 12.8 12.9 12.8 (OD04566) Breast Cancer 17.9 39.0 27.2 16.5
25.3 (OD04590-01) Breast Cancer 26.1 66.0 35.4 42.0 27.9 Mets
(OD04590- 03) Breast Cancer 4.5 5.4 6.0 5.2 3.5 Metastasis
(OD04655-05) Breast Cancer 30.8 32.1 28.1 21.6 36.3 064006 Breast
Cancer 20.7 46.7 19.8 16.7 14.8 1024 Breast Cancer 13.1 15.9 13.9
11.0 22.1 9100266 Breast Margin 10.4 14.4 15.6 16.4 20.9 9100265
Breast Cancer 22.2 26.8 34.2 25.5 50.0 A209073 Breast Margin 6.7
9.7 7.1 4.3 11.3 A2090734 Normal Liver 1.4 4.2 1.6 1.7 2.3 Liver
Cancer 1.0 2.8 1.7 1.3 1.3 064003 Liver Cancer 1.4 1.1 3.3 2.3 3.2
1025 Liver Cancer 7.8 6.5 4.9 6.4 10.7 1026 Liver Cancer 5.0 9.9
4.2 3.0 5.2 6004-T Liver Tissue 4.7 7.9 3.5 4.2 3.7 6004-N Liver
Cancer 7.9 11.5 8.2 10.3 6.7 6005-T Liver Tissue 2.0 3.2 2.7 1.6
2.3 6005-N Normal Bladder 6.8 17.9 13.6 11.5 15.2 Bladder Cancer
10.7 22.8 14.5 14.2 14.2 1023 Bladder Cancer 18.0 29.3 22.7 17.7
23.5 A302173 Bladder Cancer 14.5 29.3 26.1 21.0 28.3 (OD04718-01)
Bladder Normal 2.9 5.0 3.1 3.2 4.2 Adjacent (OD04718-03) Normal
Ovary 1.4 4.7 3.6 4.6 5.4 Ovarian Cancer 40.9 100.0 89.5 100.0 76.3
064008 Ovarian Cancer 9.7 43.2 16.7 15.6 19.5 (OD04768-07) Ovary
Margin 6.5 7.9 10.8 6.7 8.3 (OD04768-08) Normal Stomach 11.8 39.5
14.7 14.8 13.1 Gastric Cancer 1.4 6.0 2.9 2.8 2.9 9060358 Stomach
Margin 6.4 19.9 7.4 10.8 8.7 9060359 Gastric Cancer 11.1 58.6 21.6
21.2 32.3 9060395 Stomach Margin 6.8 34.6 23.7 13.8 22.2 9060394
Gastric Cancer 15.4 78.5 24.8 25.2 31.9 9060397 Stomach Margin 3.9
14.5 6.1 7.5 7.9 9060396 Gastric Cancer 2.5 14.8 7.0 7.3 13.0
064005
[0781] TABLE-US-00121 TABLE 38 Panel 3D Rel. Exp.(%) Rel. Exp.(%)
Ag2263, Run Ag2263, Run Tissue Name 170189128 Tissue Name 170189128
Daoy-Medulloblastoma 19.1 Ca Ski-Cervical epidermoid 0.4 carcinoma
(metastasis) TE671-Medulloblastoma 8.4 ES-2-Ovarian clear cell 0.0
carcinoma D283 Med- 39.2 Ramos-Stimulated with 0.0 Medulloblastoma
PMA/ionomycin 6 h PFSK-1-Primitive 59.5 Ramos-Stimulated with 0.0
Neuroectodermal PMA/ionomycin 14 h XF-498-CNS 0.9 MEG-01-Chronic
3.8 myelogenous leukemia (megokaryoblast) SNB-78-Glioma 35.4
Raji-Burkitt's lymphoma 0.0 SF-268-Glioblastoma 0.0 Daudi-Burkitt's
lymphoma 0.0 T98G-Glioblastoma 1.2 U266-B-cell plasmacytoma 0.0
SK-N-SH- 94.6 CA46-Burkitt's lymphoma 0.0 Neuroblastoma
(metastasis) SF-295-Glioblastoma 0.3 RL-non-Hodgkin's B-cell 0.0
lymphoma Cerebellum 37.4 JM1-pre-B-cell lymphoma 0.0 Cerebellum
35.1 Jurkat-T cell leukemia 0.5 NCI-H292- 4.3 TF-1-Erythroleukemia
73.2 Mucoepidermoid lung carcinoma DMS-114-Small cell 6.6 HUT
78-T-cell lymphoma 0.0 lung cancer DMS-79-Small cell lung 100.0
U937-Histiocytic lymphoma 0.0 cancer NCI-H146-Small cell 37.4
KU-812-Myelogenous 0.6 lung cancer leukemia NCI-H526-Small cell
17.2 769-P-Clear cell renal 0.0 lung cancer carcinoma
NCI-N417-Small cell 88.9 Caki-2-Clear cell renal 0.0 lung cancer
carcinoma NCI-H82-Small cell 95.3 SW 839-Clear cell renal 0.0 lung
cancer carcinoma NCI-H157-Squamous 0.8 G401-Wilms'tumor 2.8 cell
lung cancer (metastasis) NCI-H1155-Large cell 55.5
Hs766T-Pancreatic 0.6 lung cancer carcinoma (LN metastasis)
NCI-H1299-Large cell 0.0 CAPAN-1-Pancreatic 3.1 lung cancer
adenocarcinoma (liver metastasis) NCI-H727-Lung 0.7
SU86.86-Pancreatic 0.4 carcinoid carcinoma (liver metastasis)
NCI-UMC-11-Lung 7.9 BxPC-3-Pancreatic 22.8 carcinoid adenocarcinoma
LX-1-Small cell lung 1.8 HPAC-Pancreatic 35.6 cancer adenocarcinoma
Colo-205-Colon cancer 0.3 MIA PaCa-2-Pancreatic 0.6 carcinoma
KM12-Colon cancer 0.1 CFPAC-1-Pancreatic ductal 1.1 adenocarcinoma
KM20L2-Colon cancer 0.6 PANC-1-Pancreatic 0.3 epithelioid ductal
carcinoma NCI-H716-Colon cancer 70.2 T24-Bladder carcinma 0.0
(transitional cell) SW-48-Colon 0.0 5637-Bladder carcinoma 2.2
adenocarcinoma SW1116-Colon 16.6 HT-1197-Bladder carcinoma 0.4
adenocarcinoma LS 174T-Colon 4.2 UM-UC-3-Bladder carcinma 0.2
adenocarcinoma (transitional cell) SW-948-Colon 0.4
A204-Rhabdomyosarcoma 0.0 adenocarcinoma SW-480-Colon 0.0
HT-1080-Fibrosarcoma 7.9 adenocarcinoma NCI-SNU-5-Gastric 1.7
MG-63-Osteosarcoma 16.3 carcinoma KATO III-Gastric 17.4
SK-LMS-1-Leiomyosarcoma 0.0 carcinoma (vulva) NCI-SNU-16-Gastric
0.7 SJRH30-Rhabdomyosarcoma 3.9 carcinoma (met to bone marrow)
NCI-SNU-1-Gastric 23.0 A431-Epidermoid carcinoma 34.9 carcinoma
RF-1-Gastric 0.0 WM266-4-Melanoma 0.0 adenocarcinoma RF-48-Gastric
0.0 DU 145-Prostate carcinoma 0.0 adenocarcinoma (brain metastasis)
MKN-45-Gastric 11.5 MDA-MB-468-Breast 16.4 carcinoma adenocarcinoma
NCI-N87-Gastric 24.0 SCC-4-Squamous cell 0.0 carcinoma carcinoma of
tongue OVCAR-5-Ovarian 3.7 SCC-9-Squamous cell 0.0 carcinoma
carcinoma of tongue RL95-2-Uterine 4.6 SCC-15-Squamous cell 0.0
carcinoma carcinoma of tongue HelaS3-Cervical 5.9 CAL 27-Squamous
cell 7.1 adenocarcinoma carcinoma of tongue
[0782] TABLE-US-00122 TABLE 39 Panel 4D Rel. Exp.(%) Rel. Exp.(%)
Rel. Exp.(%) Rel. Exp.(%) Ag1522, Run Ag1848, Run Ag2263, Run
Ag2422, Run Tissue Name 145789191 160202841 151562852 159318890
Secondary Th1 act 0.0 0.1 0.0 0.2 Secondary Th2 act 0.0 0.0 0.0 0.0
Secondary Tr1 act 0.0 0.0 0.0 4.6 Secondary Th1 rest 0.1 0.0 0.1
0.0 Secondary Th2 rest 0.0 0.0 0.0 0.0 Secondary Th1 rest 0.0 0.0
0.0 0.2 Primary Th1 act 0.1 0.2 0.2 1.0 Primary Th2 act 0.1 0.2 0.1
0.3 Primary Th1 act 0.2 0.5 0.0 0.6 Primary Th1 rest 0.0 0.0 0.0
0.0 Primary Th2 rest 0.0 0.0 0.0 0.0 Primary Tr1 rest 0.0 0.0 0.0
0.0 CD45RA CD4 4.9 6.3 8.5 10.6 lymphocyte act CD45RO CD4 0.0 0.0
0.0 0.0 lymphocyte act CD8 lymphocyte act 0.0 0.0 0.0 0.0 Secondary
CD8 0.0 0.0 0.0 0.0 lymphocyte rest Secondary CD8 0.0 0.0 0.0 0.0
lymphocyte act CD4 lymphocyte none 0.0 0.0 0.0 0.0 2ry
Th1/Th2/Tr1_anti- 0.0 0.0 0.0 0.0 CD95 CH11 LAK cells rest 1.8 2.7
2.0 5.8 LAK cells IL-2 0.0 0.0 0.0 0.0 LAK cells IL-2 + IL-12 0.0
0.1 0.0 0.2 LAK cells IL-2 + IFN 0.0 0.1 0.0 0.2 gamma LAK cells
IL-2 + IL-18 0.0 0.4 0.0 0.1 LAK cells 1.1 1.0 1.7 2.5
PMA/ionomycin NK Cells IL-2 rest 0.0 0.1 0.0 0.0 Two Way MLR 3 day
0.0 0.1 0.2 0.2 Two Way MLR 5 day 0.2 0.3 0.8 0.6 Two Way MLR 7 day
0.5 0.2 0.1 0.3 PBMC rest 0.0 0.0 0.1 0.0 PBMC PWM 0.0 0.1 0.0 0.0
PBMC PHA-L 0.0 0.1 0.0 0.0 Ramos (B cell) none 0.0 0.0 0.0 0.0
Ramos (B cell) 0.0 0.0 0.0 0.0 ionomycin B lymphocytes PWM 0.2 0.0
0.0 0.0 B lymphocytes CD40L 0.0 0.1 0.1 0.3 and IL-4 EOL-1 dbcAMP
0.2 0.2 0.4 0.0 EOL-1 dbcAMP 0.1 0.4 0.2 0.6 PMA/ionomycin
Dendritic cells none 1.4 1.1 1.0 2.8 Dendritic cells LPS 0.3 0.4
0.3 0.4 Dendritic cells anti- 2.4 3.0 3.5 6.7 CD40 Monocytes rest
0.8 0.8 0.6 1.3 Monocytes LPS 0.0 0.0 0.3 0.0 Macrophages rest 1.3
1.0 0.0 2.0 Macrophages LPS 0.0 0.2 0.1 0.4 HUVEC none 1.1 1.4 0.6
2.5 HUVEC starved 4.4 4.7 2.9 6.0 HUVEC IL-1beta 1.7 2.8 1.0 2.3
HUVEC IFN gamma 1.6 1.4 2.5 1.9 HUVEC TNF alpha + IFN 0.3 0.3 0.5
0.5 gamma HUVEC TNF alpha + IL4 0.2 0.3 0.3 1.3 HUVEC IL-11 0.9 1.2
2.2 0.5 Lung Microvascular EC 2.2 6.5 2.8 6.7 none Lung
Microvascular EC 12.7 11.9 8.5 15.5 TNF alpha + IL-1beta
Microvascular Dermal 32.1 30.8 22.4 22.4 EC none Microsvasular
Dermal 16.3 16.2 8.8 14.4 EC TNF alpha + IL- 1beta Bronchial
epithelium 24.0 31.2 15.1 50.7 TNF alpha + IL-1beta Small airway
epithelium 8.8 5.9 6.7 12.8 none Small airway epithelium 31.9 43.5
21.0 44.8 TNF alpha + IL-1beta Coronery artery SMC 27.4 28.7 8.5
35.8 rest Coronery artery SMC 12.9 21.6 27.4 17.8 TNF alpha +
IL-1beta Astrocytes rest 17.1 14.9 23.8 24.3 Astrocytes TNF alpha +
IL- 32.8 29.5 28.1 35.1 1beta KU-812 (Basophil) rest 1.0 1.8 1.3
0.7 KU-812 (Basophil) 1.4 3.3 2.0 3.7 PMA/ionomycin CCD1106 1.4 0.2
0.7 2.7 (Keratinocytes) none CCD1106 0.9 0.3 0.8 1.3
(Keratinocytes) TNF alpha + IL-1beta Liver cirrhosis 2.9 3.0 2.4
4.8 Lupus kidney 3.0 2.9 0.9 4.4 NCI-H292 none 10.4 13.7 6.8 18.8
NCI-H292 IL-4 14.2 14.9 6.8 17.1 NCI-H292 IL-9 13.2 16.7 9.3 12.8
NCI-H292 IL-13 9.4 8.6 15.9 9.0 NCI-H292 IFN gamma 3.8 4.7 4.7 5.3
HPAEC none 1.2 1.0 1.6 2.8 HPAEC TNF alpha + IL- 5.8 2.6 4.7 6.0
1beta Lung fibroblast none 100.0 100.0 100.0 100.0 Lung fibroblast
TNF 8.5 12.2 15.9 15.2 alpha + IL-1beta Lung fibroblast IL-4 74.2
79.6 45.7 97.3 Lung fibroblast IL-9 27.7 48.6 30.6 50.3 Lung
fibroblast IL-13 48.0 39.5 27.4 55.9 Lung fibroblast IFN 76.3 82.9
42.6 98.6 gamma Dermal fibroblast 52.9 56.3 27.2 65.5 CCD1070 rest
Dermal fibroblast 33.9 42.6 19.8 46.7 CCD1070 TNF alpha Dermal
fibroblast 29.1 27.9 70.2 28.9 CCD1070 IL-1beta Dermal fibroblast
IFN 6.1 3.6 8.9 7.9 gamma Dermal fibroblast IL-4 14.5 16.2 17.3
18.9 IBD Colitis 2 0.1 0.1 0.2 0.5 IBD Crohn's 0.6 0.4 0.0 0.8
Colon 7.6 6.4 8.0 11.3 Lung 59.5 75.8 47.6 74.7 Thymus 16.5 17.3
10.2 19.6 Kidney 6.8 9.0 3.0 6.5
[0783] TABLE-US-00123 TABLE 40 Panel CNS_1 Rel. Exp.(%) Rel.
Exp.(%) Ag2263, Ag2263, Run Run Tissue Name 171669090 Tissue Name
171669090 BA4 Control 22.8 BA17 PSP 11.2 BA4 Control2 38.2 BA17
PSP2 7.1 BA4 3.7 Sub Nigra Control 100.0 Alzheimer's2 BA4
Parkinson's 45.7 Sub Nigra Control2 51.8 BA4 31.2 Sub Nigra 30.8
Parkinson's2 Alzheimer's2 BA4 12.3 Sub Nigra 89.5 Huntington's
Parkinson's2 BA4 12.2 Sub Nigra 59.0 Huntington's Huntington's BA4
PSP 13.6 Sub Nigra 16.2 Huntington's2 BA4 PSP2 42.6 Sub Nigra PSP2
22.5 BA4 Depression 27.9 Sub Nigra 40.6 Depression BA4 10.9 Sub
Nigra 12.8 Depression2 Depression2 BA7 Control 28.3 Glob Palladus
36.1 Control BA7 Control2 27.2 Glob Palladus 21.3 Control2 BA7 5.5
Glob Palladus 26.1 Alzheimer's2 Alzheimer's BA7 Parkinson's 13.2
Glob Palladus 11.2 Alzheimer's2 BA7 12.8 Glob Palladus 73.2
Parkinson's2 Parkinson's BA7 14.8 Glob Palladus 15.7 Huntington's
Parkinson's2 BA7 22.2 Glob Palladus PSP 15.0 Huntington's2 BA7 PSP
29.1 Glob Palladus PSP2 10.4 BA7 PSP2 8.9 Glob Palladus 28.3
Depression BA7 Depression 5.4 Temp Pole Control 5.4 BA9 Control
14.3 Temp Pole Control2 25.2 BA9 Control2 57.0 Temp Pole 10.0
Alzheimer's BA9 Alzheimer's 5.5 Temp Pole 2.5 Alzheimer's2 BA9 13.8
Temp Pole 15.5 Alzheimer's2 Parkinson's BA9 Parkinson's 16.2 Temp
Pole 27.9 Parkinson's2 BA9 21.0 Temp Pole 22.4 Parkinson's2
Huntington's BA9 21.5 Temp Pole PSP 1.3 Huntington's BA9 11.9 Temp
Pole PSP2 6.4 Huntington's2 BA9 PSP 27.7 Temp Pole 12.3 Depression2
BA9 PSP2 5.9 Cing Gyr Control 48.3 BA9 Depression 11.0 Cing Gyr
Control2 28.1 BA9 9.5 Cing Gyr 27.2 Depression2 Alzheimer's BA17
Control 25.0 Cing Gyr 13.1 Alzheimer's2 BA17 Control2 45.7 Cing Gyr
Parkinson's 29.7 BA17 6.5 Cing Gyr 37.4 Alzheimer's2 Parkinson's2
BA17 35.4 Cing Gyr 70.7 Parkinson's Huntington's BA17 15.3 Cing Gyr
32.1 Parkinson's2 Huntington's2 BA17 15.5 Cing Gyr PSP 42.6
Huntington's BA17 8.1 Cing Gyr PSP2 8.3 Huntington's2 BA17 26.2
Cing Gyr Depression 20.6 Depression BA17 59.9 Cing Gyr 36.3
Depression2 Depression2
[0784] AI_comprehensive panel_v1.0 Summary: Ag1522/1848: The
results of two runs with the same probe and primer set are in good
agreement. Low to moderate levels of expression of the NOV11 gene
are detected in samples derived from osteoarthritic (OA) bone and
adjacent bone as well as OA cartilage, OA synovium and OA synovial
fluid samples. Low level expression is also detected in cartilage,
bone, synovium and synovial fluid samples from rheumatoid arthritis
patients. With the exception of the cartilage Rep20 sample, no
significant expression is detected in normal samples of cartilage,
synovium, bone or synovial fluid cells. Low level expression is
also detected in samples derived from normal lung samples, COPD
lung, emphysema, atopic asthma, asthma, allergy, Crohn's disease
(normal matched control and diseased), ulcerative colitis(normal
matched control and diseased), and psoriasis (normal matched
control and diseased). Therefore, therapeutic modulation of this
gene product may ameliorate symptoms/conditions associated with
autoimmune and inflammatory disorders including psoriasis, allergy,
asthma, inflammatory bowel disease, rheumatoid arthritis and
osteoarthritis.
[0785] CNS_neurodegeneration_v1.0 Summary: Ag1848/Ag2263/Ag2422
Multiple experiments using different probe/primer sets produce
results that are in good agreement. Highest expression of the NOV11
gene is detected in the occipital cortex of a control patient.
Significant levels of expression are also detected in the
hippocampus, inferior temporal cortex, and the superior temporal
cortex of brain tissue from an Alzheimer's patient.
[0786] Based on its homology, the NOV11 gene product is most
similar to an UNC5H receptor, which as a class are known to act
both in axon guidance and neuronal migration during development, as
well as inducers of apoptosis (except when stimulated by the ligand
netrin-1). Panel CNS_Neurodegeneration_V1.0 shows a moderate
increase (1.5 to 2-fold) in the temporal cortex of the Alzheimer's
disease brain when compared to non-demented elderly either with or
without a high amyloid plaque load [this difference is apparent
after scaling the RTQ-PCR data based upon overall RNA
amount/quality, and is most apparent on Aq2263]. Thus, the NOV11
gene represents a protein that differentiates demented and
non-demented elderly who have a severe amyloid plaque load, making
it an excellent drug target in Alzheimer's disease. The modulation
and/or selective stimulation of this receptor may be of use in
enhancing or directing compensatory synaptogenesis and
axon/dendritic outgrowth in response to neuronal death (stroke,
head trauma) neurodegeneration (Alzheimer's, Parkinson's,
Huntington's, spinocerebellar ataxia, progressive supranuclear
palsy) or spinal cord injury. Furthermore, antagonism of this
receptor may decrease apoptosis in Alzheimer's disease.
REFERENCES
[0787] Ellezam B, Selles-Navarro I, Manitt C, Kennedy T E,
McKerracher L. Expression of netrin-1 and its receptors DCC and
UNC-5H2 after axotomy and during regeneration of adult rat retinal
ganglion cells. Exp Neurol 2001 March; 168 (1):105-15
[0788] Netrins are a family of chemotropic factors that guide axon
outgrowth during development; however, their function in the adult
CNS remains to be established. We examined the expression of the
netrin receptors DCC and UNC5H2 in adult rat retinal ganglion cells
(RGCs) after grafting a peripheral nerve (PN) to the transected
optic nerve and following optic nerve transection alone. In situ
hybridization revealed that both Dcc and Unc5h2 mRNAs are expressed
by normal adult RGCs. In addition, netrin-1 was found to be
constitutively expressed by RGCs. Quantitative analysis using in
situ hybridization demonstrated that both Dcc and Unc5h2 were
down-regulated by RGCs following axotomy. In the presence of an
attached PN graft, Dcc and Unc5h2 were similarly down-regulated in
surviving RGCs regardless of their success in regenerating an axon.
Northern blot analysis demonstrated expression of netrin-1 in both
optic and sciatic nerve, and Western blot analysis revealed the
presence of netrin protein in both nerves. Immunohistochemical
analysis indicated that netrin protein was closely associated with
glial cells in the optic nerve. These results suggest that
netrin-1, DCC, and UNC5H2 may contribute to regulating the
regenerative capacity of adult RGCs.
[0789] Braisted J E, Catalano S M, Stimac R, Kennedy T E,
Tessier-Lavigne M, Shatz C J, Oleary D D Netrin-1 promotes thalamic
axon growth and is required for proper development of the
thalamocortical projection. J Neurosci 2000 Aug. 1; 20
(15):5792-801
[0790] The thalamocortical axon (TCA) projection originates in
dorsal thalamus, conveys sensory input to the neocortex, and has a
critical role in cortical development. We show that the secreted
axon guidance molecule netrin-1 acts in vitro as an attractant and
growth promoter for dorsal thalamic axons and is required for the
proper development of the TCA projection in vivo. As TCAs approach
the hypothalamus, they turn laterally into the ventral
telencephalon and extend toward the cortex through a population of
netrin-1-expressing cells. DCC and neogenin, receptors implicated
in mediating the attractant effects of netrin-1, are expressed in
dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors
implicated in repulsion, are not. In vitro, dorsal thalamic axons
show biased growth toward a source of netrin-1, which can be
abolished by netrin-1-blocking antibodies. Netrin-1 also enhances
overall axon outgrowth from explants of dorsal thalamus. The biased
growth of dorsal thalamic axons toward the internal capsule zone of
ventral telencephalic explants is attenuated, but not
significantly, by netrin-1-blocking antibodies, suggesting that it
releases another attractant activity for TCAs in addition to
netrin-1. Analyses of netrin-1-/- mice reveal that the TCA
projection through the ventral telencephalon is disorganized, their
pathway is abnormally restricted, and fewer dorsal thalamic axons
reach cortex. These findings demonstrate that netrin-1 promotes the
growth of TCAs through the ventral telencephalon and cooperates
with other guidance cues to control their pathfinding from dorsal
thalamus to cortex.
[0791] Panel 1.2 Summary: Ag1522 Expression of the NOV11 gene is
highest in CNS cancer cell lines (CT=26.1). Of nine tissue samples
derived from CNS cancer cell lines, expression of the NOV11 gene
occurs in all samples, with expression high (CT=26.1, 26.6, 27.6)
in three samples, moderate in five samples and low in one sample.
High expression is also detectable in melanoma cell lines
(CT=27.9). Significant expression of the NOV11 gene is seen in
gastric cancer (28.1) and all ten samples of lung cancer cell lines
in this sample. Thus, expression of the NOV11 gene could be used to
distinguish those cancer cell lines from normal tissues. In
addition, therapeutic modulation of the expression, or activity of
the NOV11 gene product, might be of use in the treatment of
melanoma, gastric cancer, lung cancer and brain cancer.
[0792] Panel 1.3D Summary: Ag1522/Ag1848/Ag2263/Ag2422 Four
experiments using different probe/primer sets on the same tissue
panel produce results that are in excellent agreement. In all four
experiments, highest expression of the NOV11 gene is detected in
CNS cancer cell lines. Expression is also significant in lung
cancer and melanoma cell lines and in healthy brain tissue from the
hippocampus and thalamus regions. Thus, the expression of the NOV11
gene could be used to distinguish these tissue samples from other
samples. Moreover, therapeutic modulation of the expression, or
function, of the NOV11 gene, through the use of small molecule
drugs or antibodies, might be beneficial in the treatment of
melanoma, lung cancer and brain cancer.
[0793] Among metabolic tissues, there is high expression of the
NOV11 gene in adult heart tissue (CT=27.8) and moderate expression
in fetal heart, adult and fetal liver, pancreas, adrenal gland,
thyroid and pituitary. The NOV11 gene appears to be differentially
expressed in fetal (CT value=31) and adult skeletal muscle (CT
value=37) using the probe and primer set Ag1848 and may be useful
for the differentiation of the adult from the fetal phenotype in
this tissue.
[0794] Panel 2D Summary: Ag1522/Ag1848/Ag2263/Ag2422 Results from
multiple experiments with four different probe and primer sets are
in very good agreement. In all experiments, highest expression of
the NOV11 gene is detected in thyroid and ovarian cancers
(CTs=27-30), with lower expression also seen in most of the other
tissues on this panel. Thus, the expression of the NOV11 gene could
be used to distinguish ovarian and thyroid cancer cell lines from
other tissues. Moreover, therapeutic modulation of the expression
this gene, or its function, through the use of small molecule drugs
or antibodies, might be of benefit in the treatment of ovarian and
thyroid cancer. In addition, experiments with Ag2263 show
differential expression between samples derived from lung cancer
and their adjacent normal tissues. Thus, expression of the NOV11
gene could be used to distinguish cancerous lung tissue from normal
lung tissue. Moreover, therapeutic modulation of the expression or
function of this gene or its protein product, through the use of
antibodies or small molecule drugs, might be of benefit in the
treatment of lung cancer.
[0795] Panel 3D Summary: Ag2263 Expression of the NOV11 gene occurs
at moderate levels across all the tissues in this panel. Highest
expression is detected in a small cell lung cancer (CT=30.6) and
neuroblastoma (CT=30.7). In addition, significant expression is
detected in a cluster of small cell lung cancer lines. Thus, this
gene could be used to distinguish lung cancer cell lines from other
samples. Moreover, therapeutic modulation of the NOV11 gene or its
protein product, through the use of small molecule drugs or
antibodies might be of benefit in the treatment of small cell lung
cancer.
[0796] Panel 4D Summary: Ag522/Ag1848/Ag2263/Ag2422 Experiments
using each of the four probe and primer sets that correspond to the
NOV11 gene produce results that are in excellent agreement. In all
the experiments, expression of the NOV11 gene occurs at moderate to
low levels in many of the tissues in the sample. Highest expression
in each experiment occurs in lung fibroblasts (CT=29). Moderate
expression in lung fibroblasts treated with IL-4 is also consistent
among all four experiments (CT=30). Lower expression is also
detected in a variety of fibroblasts, endothelial and smooth muscle
cells. The expression of the NOV11 gene produces a complex profile;
it is upregulated by TNF-alpha in small airway epithelium, but
clearly downregulated by the same stimulus in lung fibroblasts. The
gene most probably encodes a netrin receptor that may be important
in understanding cell migration. Regulation of the protein encoded
for by the NOV11 gene could potentially control the progression of
keloid formation, emphysema and other conditions in which TNF-alpha
and IL-1 beta are present and tissue remodeling may occur.
[0797] Panel CNS.sub.--1 Summary: Ag2263 Expression of the NOV11
gene is moderate to low across many of the tissues in this panel.
Highest expression is detected in the substantia nigra (CT=31.4).
Although no disease-specific expression is seen in this panel, the
expression profile confirms the expression of this gene in the
central nervous system. Please see panel-CNS_neurodegeneration for
potential utility of the NOV11 gene regarding the CNS.
[0798] NOV10
[0799] Expression of gene NOV10 was assessed using the primer-probe
set Ag2421, described in Table 41. Results of the RTQ-PCR runs are
shown in Tables 42 and 43. TABLE-US-00124 TABLE 41 Probe Name
Ag2421 Primers Sequences Length Start Position SEQ ID NO: Forward
5'-tgaggctgagctctctgtgt-3' 20 1952 179 Probe
TET-5'-tctgctaactgtgaaggatctcacca-3'-TAMRA 26 1985 180 Reverse
5'-ctggtccacattgtcaggaa-3' 20 2014 181
[0800] TABLE-US-00125 TABLE 42 Panel 1.3D Rel. Exp.(%) Ag2421, Rel.
Exp.(%) Ag2421, Tissue Name Run 159299536 Tissue Name Run 159299536
Liver adenocarcinoma 0.0 Kidney (fetal) 0.6 Pancreas 0.0 Renal ca.
786-0 0.0 Pancreatic ca. CAPAN 2 0.0 Renal ca. A498 0.0 Adrenal
gland 0.0 Renal ca. RXF 393 0.0 Thyroid 0.0 Renal ca. ACHN 0.0
Salivary gland 0.0 Renal ca. UO-31 0.0 Pituitary gland 0.0 Renal
ca. TK-10 0.0 Brain (fetal) 0.0 Liver 33.4 Brain (whole) 0.0 Liver
(fetal) 0.0 Brain (amygdala) 0.0 Liver ca. 0.0 (hepatoblast) HepG2
Brain (cerebellum) 0.0 Lung 26.2 Brain (hippocampus) 8.4 Lung
(fetal) 8.7 Brain (substantia nigra) 0.0 Lung ca. (small cell) 0.0
LX-1 Brain (thalamus) 10.4 Lung ca. (small cell) 0.0 NCI-H69
Cerebral Cortex 0.0 Lung ca. (s.cell var.) 0.0 SHP-77 Spinal cord
1.1 Lung ca. (large 0.0 cell)NCI-H460 glio/astro U87-MG 0.0 Lung
ca. (non-sm. 0.0 cell) A549 glio/astro U-118-MG 0.0 Lung ca.
(non-s.cell) 0.0 NCI-H23 astrocytoma SW1783 0.0 Lung ca.
(non-s.cell) 0.0 HOP-62 neuro*; met SK-N-AS 0.0 Lung ca. (non-s.cl)
0.0 NCI-H522 astrocytoma SF-539 0.0 Lung ca. (squam.) 0.0 SW 900
astrocytoma SNB-75 1.4 Lung ca. (squam.) 0.0 NCI-H596 glioma SNB-19
0.0 Mammary gland 0.0 glioma U251 0.0 Breast ca.* (pl.ef) 0.0 MCF-7
glioma SF-295 0.0 Breast ca.* (pl.ef) 0.0 MDA-MB-231 Heart (fetal)
0.0 Breast ca.* (pl.ef) 0.0 T47D Heart 0.0 Breast ca. BT-549 0.0
Skeletal muscle (fetal) 0.0 Breast ca. MDA-N 0.0 Skeletal muscle
0.0 Ovary 1.5 Bone marrow 0.0 Ovarian ca. OVCAR-3 0.0 Thymus 0.0
Ovarian ca. OVCAR-4 0.0 Spleen 0.0 Ovarian ca. OVCAR-5 0.0 Lymph
node 0.0 Ovarian ca. OVCAR-8 0.0 Colorectal 0.0 Ovarian ca. IGROV-1
0.0 Stomach 0.0 Ovarian ca.* (ascites) 0.0 SK-OV-3 Small intestine
0.0 Uterus 0.0 Colon ca. SW480 0.0 Placenta 0.0 Colon ca.* 0.0
Prostate 0.0 SW620(SW480 met) Colon ca. HT29 0.0 Prostate ca.*
(bone 0.0 met)PC-3 Colon ca. HCT-116 0.0 Testis 0.0 Colon ca.
CaCo-2 4.2 Melanoma 0.0 Hs688(A).T Colon ca. 0.0 Melanoma* (met)
0.0 tissue(ODO3866) Hs688(B).T Colon ca. HCC-2998 0.0 Melanoma
UACC-62 0.0 Gastric ca.* (liver met) 0.0 Melanoma M14 0.0 NCI-N87
Bladder 0.0 Melanoma LOX 0.0 IMVI Trachea 100.0 Melanoma* (met) 0.0
SK-MEL-5 Kidney 0.0 Adipose 0.0
[0801] TABLE-US-00126 TABLE 43 Panel 2D Rel. Exp.(%) Rel. Exp.(%)
Ag2421, Ag2421, Tissue Name Run 159298043 Tissue Name Run 159298043
Normal Colon 0.0 Kidney Margin 0.0 8120608 CC Well to Mod Diff 0.0
Kidney Cancer 0.0 (ODO3866) 8120613 CC Margin (ODO3866) 0.0 Kidney
Margin 0.4 8120614 CC Gr.2 rectosigmoid 0.0 Kidney Cancer 0.0
(ODO3868) 9010320 CC Margin (ODO3868) 0.0 Kidney Margin 0.0 9010321
CC Mod Diff (ODO3920) 0.0 Normal Uterus 0.0 CC Margin (ODO3920) 0.0
Uterus Cancer 064011 1.0 CC Gr.2 ascend colon 0.0 Normal Thyroid
0.0 (ODO3921) CC Margin (ODO3921) 0.0 Thyroid Cancer 0.0 064010 CC
from Partial 7.7 Thyroid Cancer 0.0 Hepatectomy (ODO4309) A302152
Mets Liver Margin (ODO4309) 99.3 Thyroid Margin 0.0 A302153 Colon
mets to lung 0.1 Normal Breast 0.0 (OD04451-01) Lung Margin
(OD04451- 0.4 Breast Cancer 0.0 02) (OD04566) Normal Prostate
6546-1 0.0 Breast Cancer 0.0 (OD04590-01) Prostate Cancer 0.0
Breast Cancer Mets 0.0 (OD04410) (OD04590-03) Prostate Margin 0.0
Breast Cancer 0.0 (OD04410) Metastasis (OD04655-05) Prostate Cancer
0.0 Breast Cancer 064006 0.2 (OD04720-01) Prostate Margin 0.0
Breast Cancer 1024 0.0 (OD04720-02) Normal Lung 061010 4.0 Breast
Cancer 0.0 9100266 Lung Met to Muscle 0.0 Breast Margin 0.0
(ODO4286) 9100265 Muscle Margin 0.0 Breast Cancer 0.0 (ODO4286)
A209073 Lung Malignant Cancer 2.6 Breast Margin 0.0 (OD03126)
A2090734 Lung Margin (OD03126) 2.3 Normal Liver 57.8 Lung Cancer
(OD04404) 0.2 Liver Cancer 064003 0.0 Lung Margin (OD04404) 1.6
Liver Cancer 1025 0.4 Lung Cancer (OD04565) 0.1 Liver Cancer 1026
2.1 Lung Margin (OD04565) 0.8 Liver Cancer 6004-T 0.7 Lung Cancer
(OD04237- 0.1 Liver Tissue 6004-N 1.4 01) Lung Margin (OD04237- 1.2
Liver Cancer 6005-T 1.5 02) Ocular Mel Met to Liver 0.2 Liver
Tissue 6005-N 7.0 (ODO4310) Liver Margin (ODO4310) 100.0 Normal
Bladder 0.0 Melanoma Mets to Lung 0.0 Bladder Cancer 1023 0.0
(OD04321) Lung Margin (OD04321) 1.5 Bladder Cancer 0.4 A302173
Normal Kidney 0.0 Bladder Cancer 0.0 (OD04718-01) Kidney Ca,
Nuclear grade 0.0 Bladder Normal 0.1 2 (OD04338) Adjacent (OD04718-
03) Kidney Margin 0.0 Normal Ovary 0.0 (OD04338) Kidney Ca Nuclear
grade 0.0 Ovarian Cancer 0.0 1/2 (OD04339) 064008 Kidney Margin 0.0
Ovarian Cancer 0.0 (OD04339) (OD04768-07) Kidney Ca, Clear cell 0.0
Ovary Margin 0.0 type (OD04340) (OD04768-08) Kidney Margin 0.0
Normal Stomach 0.0 (OD04340) Kidney Ca, Nuclear grade 0.0 Gastric
Cancer 0.0 3 (OD04348) 9060358 Kidney Margin 0.0 Stomach Margin 0.0
(OD04348) 9060359 Kidney Cancer 0.0 Gastric Cancer 0.0 (OD04622-01)
9060395 Kidney Margin 0.0 Stomach Margin 0.0 (OD04622-03) 9060394
Kidney Cancer 0.0 Gastric Cancer 0.0 (OD04450-01) 9060397 Kidney
Margin 0.0 Stomach Margin 0.0 (OD04450-03) 9060396 Kidney Cancer
8120607 0.0 Gastric Cancer 0.0 064005
[0802] Panel 13D Summary: Ag2421 Expression of the NOV10 gene is
restricted to samples from liver, lung and trachea in this panel
(CTs=32-33), while none of the cancer cell lines appear to make
this protein. Thus, lack of expression of this gene might be
significant for cell proliferation and growth.
[0803] Furthermore, the difference in expression between adult
liver and fetal liver (CT=40) could be used to distinguish between
the two sources of liver tissue.
[0804] Panel 2D Summary: Ag2421 The NOV10 gene encodes a protein
homologous to pregnancy zone protein (PZP), a liver protein, and is
expressed primarily in liver tissue. This gene shows a higher level
of expression in normal liver than the matched tumor tissue,
metastatic melanoma and metastatic colon cancer. There is also
higher expression in normal lung compared to lung cancer samples.
This expression profile is in agreement with the results from Panel
1.3D. Thus, this expression could potentially be used as a
diagnostic marker for liver and lung cancer. Furthermore, the
protein product could potentially be used as a therapy for lung and
liver cancer.
REFERENCES
[0805] Mavroidis M, Sunyer J O, Lambris J D. Isolation, primary
structure, and evolution of the third component of chicken
complement and evidence for a new member of the alpha
2-macroglobulin family. J Immunol 1995 Mar. 1; 154 (5):2164-74
[0806] Although the third component of complement, C3, has been
isolated and its primary structure determined from most living
classes of vertebrate, limited information is available on its
structure and function for aves, which represent a significant
stage in complement evolution. In this study, we present the
complete cDNA sequence of chicken C3, the cDNA sequences of the
thioester region for two chicken alpha 2-macroglobulin (alpha
2M)-related proteins, a simplified method for purifying chicken C3,
and an analysis of the C3 convertase and factor I-mediated
cleavages in chicken C3. Using the reverse-transcriptase PCR, with
degenerate oligonucleotide primers derived from two conserved C3
sequences (GCGEQN/TM, TWLTAY/FV) and liver mRNA as template, we
isolated three distinct 220-bp PCR products, one with a high degree
of sequence similarity to C3 and two to alpha 2M and pregnancy zone
protein from other species. The complete cDNA sequence of chicken
C3 was obtained by screening a chicken liver lambda gt10 library
with the C3 PCR product and probes from the 5' end of the
partial-length C3 clones. The obtained sequence is in complete
agreement with the protein sequence of several tryptic peptides of
purified chicken C3 Chicken pro-C3 consists of an 18-residue
putative signal peptide, a 640-residue beta-chain (70 kDa), a
989-residue alpha-chain (111 kDa), and an RKRR linker region. It
contains an internal thioester and three potential N-glycosylation
sites, all in the alpha-chain. The convertase cleavage site,
predicted to be Arg-Ser, was confirmed by sequencing the
zymosan-bound C3 fragments generated upon complement activation.
NH2-terminal sequencing of the purified C3 chains showed that 1)
pro-C3 is indeed cleaved at the RKRR linker sequence to generate
the mature two-chain molecule, and 2) the beta-chain of chicken C3
is blocked. The deduced amino acid sequence shows 54, 54, 54, 53,
52, 57, and 55% amino acid identities to human, mouse, rat, guinea
pig, rabbit, cobra, and Xenopus C3, respectively, and an identity
of 44, 31, and 33% to trout, hagfish, and lamprey C3, respectively.
The identities to human C4, C5, and alpha 2M are 31, 29 and 23%,
respectively. A phylogenetic tree for C3, C4, C5, and alpha
2M-related proteins was constructed based on the sequence data and
is discussed.
PMID: 7532662
[0807] Panel 4D Summary: Ag2421 Results from one experiment with
the NOV10 gene are not included. The amp plot indicates that there
is a high probability of a probe failure. (Data not shown.)
NOV9
[0808] Expression of gene NOV9 was assessed using the primer-probe
set Ag2873, described in Table 44. TABLE-US-00127 TABLE 44 Probe
Name Ag2873 Primers Sequences Length Start Position SEQ ID NO:
Forward 5'-ccctgctcacaagactgactag-3' 22 1025 182 Probe
TET-5'-ctccacgcagtttcaggcatgaag-3'-TAMRA 24 1054 183 Reverse
5'-gacattaggagacaacctccaa-3' 22 1080 184
[0809] CNS_neurodegeneration_v1.0 Summary: Ag2873 Expression of the
NOV9 gene is low/undetectable in all samples on this panel.
(CTs>35). (Data not shown.)
[0810] Panel 13D Summary: Ag2873 Expression of the NOV9 gene is
low/undetectable in all samples on this panel. (CTs>35). (Data
not shown.)
[0811] Panel 2D Summary: Ag2873 Expression of the NOV9 gene is
low/undetectable in all samples on this panel. (CTs>35). (Data
not shown.)
[0812] Panel 4D Summary: Ag2873 Results from experiment with the
NOV9 gene are not included. The amp plot indicates that there were
experimental difficulties with this run.
NOV7a
[0813] Expression of gene NOV7a was assessed using the primer-probe
set Ag2878, described in Table 45. Results of the RTQ-PCR runs are
shown in Tables 46, 47, and 48. TABLE-US-00128 TABLE 45 Probe Name
Ag2878 Primers Sequences Length Start Position SEQ ID NO: Forward
5'-catctctaagaatgccctcaga-3' 22 490 185 Probe
TET-5'-cttcgctcgcttacacacctaagcct-3'-TAMRA 26 515 186 Reverse
5'-gagggtctccagatggttattg-3' 22 544 187
[0814] TABLE-US-00129 TABLE 46 CNS_neurodegeneration_v1.0 Rel.
Exp.(%) Ag2878, Rel. Exp.(%) Ag2878, Tissue Name Run 209058909
Tissue Name Run 209058909 AD 1 Hippo 14.7 Control (Path) 3 10.4
Temporal Ctx AD 2 Hippo 58.6 Control (Path) 4 36.9 Temporal Ctx AD
3 Hippo 4.9 AD 1 Occipital Ctx 8.0 AD 4 Hippo 38.2 AD 2 Occipital
Ctx 0.0 (Missing) AD 5 Hippo 44.1 AD 3 Occipital Ctx 6.0 AD 6 Hippo
100.0 AD 4 Occipital Ctx 27.0 Control 2 Hippo 18.8 AD 5 Occipital
Ctx 24.0 Control 4 Hippo 24.3 AD 6 Occipital Ctx 22.7 Control
(Path) 3 9.6 Control 1 Occipital 7.9 Hippo Ctx AD 1 Temporal Ctx
18.0 Control 2 Occipital 23.2 Ctx AD 2 Temporal Ctx 64.2 Control 3
Occipital 20.6 Ctx AD 3 Temporal Ctx 7.4 Control 4 Occipital 11.8
Ctx AD 4 Temporal Ctx 46.0 Control (Path) 1 57.8 Occipital Ctx AD 5
Inf Temporal 80.7 Control (Path) 2 6.6 Ctx Occipital Ctx AD 5 Sup
Temporal 46.3 Control (Path) 3 3.7 Ctx Occipital Ctx AD 6 Inf
Temporal 81.2 Control (Path) 4 7.6 Ctx Occipital Ctx AD 6 Sup
Temporal 97.3 Control 1 Parietal 18.9 Ctx Ctx Control 1 Temporal
18.4 Control 2 Parietal 59.0 Ctx Ctx Control 2 Temporal 38.7
Control 3 Parietal 20.2 Ctx Ctx Control 3 Temporal 28.3 Control
(Path) 1 65.1 Ctx Parietal Ctx Control 3 Temporal 26.8 Control
(Path) 2 23.2 Ctx Parietal Ctx Control (Path) 1 58.2 Control (Path)
3 13.4 Temporal Ctx Parietal Ctx Control (Path) 2 30.8 Control
(Path) 4 24.8 Temporal Ctx Parietal Ctx
[0815] TABLE-US-00130 TABLE 47 Panel 1.3D Rel. Exp.(%) Ag2878, Rel.
Exp.(%) Ag2878, Tissue Name Run 167646344 Tissue Name Run 167646344
Liver adenocarcinoma 0.0 Kidney (fetal) 10.1 Pancreas 0.0 Renal ca.
786-0 0.0 Pancreatic ca. CAPAN 2 0.0 Renal ca. A498 0.0 Adrenal
gland 0.0 Renal ca. RXF 393 0.0 Thyroid 0.0 Renal ca. ACHN 0.0
Salivary gland 0.0 Renal ca. UO-31 0.0 Pituitary gland 0.0 Renal
ca. TK-10 0.0 Brain (fetal) 60.7 Liver 2.1 Brain (whole) 34.2 Liver
(fetal) 2.4 Brain (amygdala) 43.5 Liver ca. 0.0 (hepatoblast) HepG2
Brain (cerebellum) 100.0 Lung 2.9 Brain (hippocampus) 17.9 Lung
(fetal) 3.2 Brain (substantia nigra) 81.2 Lung ca. (small cell) 0.0
LX-1 Brain (thalamus) 23.3 Lung ca. (small cell) 0.0 NCI-H69
Cerebral Cortex 51.4 Lung ca. (s.cell var.) 0.0 SHP-77 Spinal cord
69.7 Lung ca. (large 0.0 cell)NCI-H460 glio/astro U87-MG 0.0 Lung
ca. (non-sm. 0.0 cell) A549 glio/astro U-118-MG 0.0 Lung ca.
(non-s.cell) 0.0 NCI-H23 astrocytoma SW1783 0.0 Lung ca.
(non-s.cell) 0.0 HOP-62 neuro*; met SK-N-AS 0.0 Lung ca. (non-s.cl)
0.0 NCI-H522 astrocytoma SF-539 0.0 Lung ca. (squam.) 0.0 SW 900
astrocytoma SNB-75 0.0 Lung ca. (squam.) 0.0 NCI-H596 glioma SNB-19
0.0 Mammary gland 4.4 glioma U251 0.0 Breast ca.* (pl.ef) 0.0 MCF-7
glioma SF-295 0.0 Breast ca.* (pl.ef) 0.0 MDA-MB-231 Heart (fetal)
7.3 Breast ca.* (pl.ef) 0.0 T47D Heart 4.0 Breast ca. BT-549 0.0
Skeletal muscle (fetal) 8.6 Breast ca. MDA-N 0.0 Skeletal muscle
18.2 Ovary 0.0 Bone marrow 0.0 Ovarian ca. OVCAR-3 0.0 Thymus 0.0
Ovarian ca. OVCAR-4 0.0 Spleen 0.0 Ovarian ca. OVCAR-5 0.0 Lymph
node 0.0 Ovarian ca. OVCAR-8 0.0 Colorectal 5.8 Ovarian ca. IGROV-1
0.0 Stomach 0.0 Ovarian ca.* (ascites) 0.0 SK-OV-3 Small intestine
5.9 Uterus 17.0 Colon ca. SW480 0.0 Placenta 0.0 Colon ca.* 0.0
Prostate 3.0 SW620(SW480 met) Colon ca. HT29 0.0 Prostate ca.*
(bone 0.0 met)PC-3 Colon ca. HCT-116 0.0 Testis 0.0 Colon ca.
CaCo-2 0.0 Melanoma 0.0 Hs688(A).T Colon ca. 0.0 Melanoma* (met)
0.0 tissue(ODO3866) Hs688(B).T Colon ca. HCC-2998 0.0 Melanoma
UACC-62 0.0 Gastric ca.* (liver met) 0.0 Melanoma M14 0.0 NCI-N87
Bladder 0.0 Melanoma LOX 0.0 IMVI Trachea 0.0 Melanoma* (met) 0.0
SK-MEL-5 Kidney 0.0 Adipose 2.3
[0816] TABLE-US-00131 TABLE 48 Panel CNS_1 Rel. Exp.(%) Ag2878,
Rel. Exp.(%) Ag2878, Tissue Name Run 171688441 Tissue Name Run
171688441 BA4 Control 16.5 BA17 PSP 12.2 BA4 Control2 17.3 BA17
PSP2 2.2 BA4 4.5 Sub Nigra Control 23.0 Alzheimer's2 BA4
Parkinson's 49.7 Sub Nigra Control2 8.8 BA4 39.8 Sub Nigra 4.4
Parkinson's2 Alzheimer's2 BA4 50.7 Sub Nigra 42.0 Huntington's
Parkinson's2 BA4 11.3 Sub Nigra 41.2 Huntington's2 Huntington's BA4
PSP 15.9 Sub Nigra 13.6 Huntington's2 BA4 PSP2 13.9 Sub Nigra PSP2
3.7 BA4 Depression 18.0 Sub Nigra 5.5 Depression BA4 6.5 Sub Nigra
4.6 Depression2 Depression2 BA7 Control 15.0 Glob Palladus 12.1
Control BA7 Control2 4.8 Glob Palladus 4.5 Control2 BA7 7.7 Glob
Palladus 6.7 Alzheimer's2 Alzheimer's BA7 Parkinson's 13.4 Glob
Palladus 7.3 Alzheimer's2 BA7 32.1 Glob Palladus 100.0 Parkinson's2
Parkinson's BA7 43.2 Glob Palladus 7.9 Huntington's Parkinson's2
BA7 55.9 Glob Palladus PSP 1.6 Huntington's2 BA7 PSP 40.1 Glob
Palladus PSP2 3.4 BA7 PSP2 20.7 Glob Palladus 4.1 Depression BA7
Depression 18.0 Temp Pole Control 10.7 BA9 Control 18.7 Temp Pole
Control2 27.0 BA9 Control2 47.3 Temp Pole 5.0 Alzheimer's BA9
Alzheimer's 4.6 Temp Pole 10.7 Alzheimer's2 BA9 17.4 Temp Pole 27.9
Alzheimer's2 Parkinson's BA9 Parkinson's 35.6 Temp Pole 20.3
Parkinson's2 BA9 33.2 Temp Pole 59.0 Parkinson's2 Huntington's BA9
94.0 Temp Pole PSP 5.2 Huntington's BA9 20.6 Temp Pole PSP2 1.7
Huntington's2 BA9 PSP 16.0 Temp Pole 4.7 Depression2 BA9 PSP2 3.1
Cing Gyr Control 47.0 BA9 Depression 14.8 Cing Gyr Control2 16.8
BA9 9.5 Cing Gyr 11.8 Depression2 Alzheimer's BA17 Control 20.6
Cing Gyr 13.7 Alzheimer's2 BA17 Control2 7.3 Cing Gyr Parkinson's
31.4 BA17 4.3 Cing Gyr 27.2 Alzheimer's2 Parkinson's2 BA17 34.2
Cing Gyr 85.9 Parkinson's Huntington's BA17 12.9 Cing Gyr 15.4
Parkinson's2 Huntington's2 BA17 26.4 Cing Gyr PSP 14.4 Huntington's
BA17 7.0 Cing Gyr PSP2 3.5 Huntington's2 BA17 14.2 Cing Gyr
Depression 11.8 Depression BA17 11.2 Cing Gyr 7.3 Depression2
Depression2
[0817] CNS_neurodegeneration-v1.0 Summary: Ag2878 No differential
expression of the NOV7a gene is found between Alzheimer's disease
and control postmortem brains. This panel confirms the expression
of this gene at moderate level in the CNS in an independent group
of patients. Please see panel 1.3D for a discussion of utility of
this gene in the central nervous system.
[0818] Panel 1.3D Summary: Ag2878 The expression of the NOV7a gene
shows a CNS-preferential expression profile. Because it is not
detected in any cancers, this gene is an excellent diagnostic
device to differentiate normal CNS tissue from glioma. Furthermore,
it may be useful as a tumor suppressor gene in the treatment of
brain cancer.
[0819] Panel 4D Summary: Ag2878 Expression of the NOV7a gene is
low/undetectable in all samples on this panel. (CTs>35). (Data
not shown.)
[0820] Panel CNS.sub.--1 Summary: Ag2878 This panel confirms the
expression of the NOV7a gene at moderate level in the CNS in an
independent group of patients. Please see panel 1.3D for a
discussion of utility of this gene in the central nervous
system.
NOV6
[0821] Expression of gene NOV6 was assessed using the primer-probe
set Ag1799, described in Table 49. Results of the RTQ-PCR runs are
shown in Tables 50, 51 and 52. TABLE-US-00132 TABLE 49 Probe Name
Ag1799 Primers Sequences Length Start Position SEQ ID NO: Forward
5'-gaccaacggctttcttcaag-3' 20 680 188 Probe
TET-5'-accttccttcttgcgacttggatcct-3'-TAMRA 26 708 189 Reverse
5'-tcagttgttcaaagcacacaaa-3' 22 748 190
[0822] TABLE-US-00133 TABLE 50 Panel 1.3D Rel. Exp.(%) Ag1799, Rel.
Exp.(%) Ag1799, Tissue Name Run 156248690 Tissue Name Run 156248690
Liver adenocarcinoma 0.0 Kidney (fetal) 8.4 Pancreas 0.0 Renal ca.
786-0 0.0 Pancreatic ca. CAPAN 2 0.0 Renal ca. A498 1.3 Adrenal
gland 100.0 Renal ca. RXF 393 0.0 Thyroid 9.5 Renal ca. ACHN 0.0
Salivary gland 4.3 Renal ca. UO-31 0.0 Pituitary gland 0.6 Renal
ca. TK-10 0.0 Brain (fetal) 0.0 Liver 0.0 Brain (whole) 0.0 Liver
(fetal) 0.0 Brain (amygdala) 0.0 Liver ca. 6.3 (hepatoblast) HepG2
Brain (cerebellum) 0.0 Lung 0.6 Brain (hippocampus) 0.8 Lung
(fetal) 8.9 Brain (substantia nigra) 0.0 Lung ca. (small cell) 0.0
LX-1 Brain (thalamus) 5.4 Lung ca. (small cell) 11.8 NCI-H69
Cerebral Cortex 0.0 Lung ca. (s.cell var.) 0.0 SHP-77 Spinal cord
0.0 Lung ca. (large 0.0 cell)NCI-H460 glio/astro U87-MG 0.0 Lung
ca. (non-sm. 0.0 cell) A549 glio/astro U-118-MG 0.0 Lung ca.
(non-s.cell) 0.0 NCI-H23 astrocytoma SW1783 0.0 Lung ca.
(non-s.cell) 0.0 HOP-62 neuro*; met SK-N-AS 1.9 Lung ca. (non-s.cl)
0.0 NCI-H522 astrocytoma SF-539 0.0 Lung ca. (squam.) 0.0 SW 900
astrocytoma SNB-75 0.0 Lung ca. (squam.) 0.0 NCI-H596 glioma SNB-19
0.0 Mammary gland 0.0 glioma U251 0.0 Breast ca.* (pl.ef) 0.0 MCF-7
glioma SF-295 0.0 Breast ca.* (pl.ef) 0.0 MDA-MB-231 Heart (fetal)
0.0 Breast ca.* (pl.ef) 0.0 T47D Heart 0.8 Breast ca. BT-549 0.0
Skeletal muscle (fetal) 0.0 Breast ca. MDA-N 0.0 Skeletal muscle
0.0 Ovary 0.0 Bone marrow 0.0 Ovarian ca. OVCAR-3 0.0 Thymus 5.3
Ovarian ca. OVCAR-4 0.0 Spleen 0.0 Ovarian ca. OVCAR-5 0.0 Lymph
node 8.9 Ovarian ca. OVCAR-8 0.0 Colorectal 0.0 Ovarian ca. IGROV-1
0.0 Stomach 0.0 Ovarian ca.* (ascites) 2.0 SK-OV-3 Small intestine
0.0 Uterus 0.0 Colon ca. SW480 0.0 Placenta 0.0 Colon ca.* 0.0
Prostate 0.0 SW620(SW480 met) Colon ca. HT29 0.0 Prostate ca.*
(bone 0.0 met)PC-3 Colon ca. HCT-116 0.0 Testis 0.8 Colon ca.
CaCo-2 0.8 Melanoma 0.0 Hs688(A).T Colon ca. 0.0 Melanoma* (met)
0.0 tissue(ODO3866) Hs688(B).T Colon ca. HCC-2998 5.0 Melanoma
UACC-62 0.0 Gastric ca.* (liver met) 0.0 Melanoma M14 0.0 NCI-N87
Bladder 0.0 Melanoma LOX 0.0 IMVI Trachea 90.8 Melanoma* (met) 0.0
SK-MEL-5 Kidney 0.5 Adipose 0.0
[0823] TABLE-US-00134 TABLE 51 Panel 2D Rel. Exp.(%) Rel. Exp.(%)
Ag1799, Ag1799, Tissue Name Run 156251136 Tissue Name Run 156251136
Normal Colon 0.0 Kidney Margin 0.0 8120608 CC Well to Mod Diff 0.0
Kidney Cancer 0.0 (ODO3866) 8120613 CC Margin (ODO3866) 0.0 Kidney
Margin 0.0 8120614 CC Gr.2 rectosigmoid 0.0 Kidney Cancer 3.3
(ODO3868) 9010320 CC Margin (ODO3868) 0.0 Kidney Margin 0.0 9010321
CC Mod Diff (ODO3920) 0.0 Normal Uterus 0.0 CC Margin (ODO3920) 0.0
Uterus Cancer 064011 0.0 CC Gr.2 ascend colon 0.0 Normal Thyroid
100.0 (ODO3921) CC Margin (ODO3921) 0.0 Thyroid Cancer 0.0 064010
CC from Partial 0.0 Thyroid Cancer 25.2 Hepatectomy (ODO4309)
A302152 Mets Liver Margin (ODO4309) 0.0 Thyroid Margin 46.3 A302153
Colon mets to lung 0.0 Normal Breast 0.0 (OD04451-01) Lung Margin
(OD04451- 0.0 Breast Cancer 0.0 02) (OD04566) Normal Prostate
6546-1 15.6 Breast Cancer 0.0 (OD04590-01) Prostate Cancer 0.0
Breast Cancer Mets 0.0 (OD04410) (OD04590-03) Prostate Margin 0.0
Breast Cancer 0.0 (OD04410) Metastasis (OD04655-05) Prostate Cancer
0.0 Breast Cancer 064006 0.0 (OD04720-01) Prostate Margin 9.7
Breast Cancer 1024 0.0 (OD04720-02) Normal Lung 061010 15.6 Breast
Cancer 0.0 9100266 Lung Met to Muscle 0.0 Breast Margin 7.3
(ODO4286) 9100265 Muscle Margin 0.0 Breast Cancer 15.2 (ODO4286)
A209073 Lung Malignant Cancer 0.0 Breast Margin 0.0 (OD03126)
A2090734 Lung Margin (OD03126) 16.6 Normal Liver 0.0 Lung Cancer
(OD04404) 0.0 Liver Cancer 064003 0.0 Lung Margin (OD04404) 0.0
Liver Cancer 1025 0.0 Lung Cancer (OD04565) 0.0 Liver Cancer 1026
0.0 Lung Margin (OD04565) 0.0 Liver Cancer 6004-T 0.0 Lung Cancer
(OD04237- 8.5 Liver Tissue 6004-N 0.0 01) Lung Margin (OD04237- 0.0
Liver Cancer 6005-T 0.0 02) Ocular Mel Met to Liver 8.1 Liver
Tissue 6005-N 0.0 (ODO4310) Liver Margin (ODO4310) 0.0 Normal
Bladder 0.0 Melanoma Mets to Lung 15.8 Bladder Cancer 1023 0.0
(OD04321) Lung Margin (OD04321) 0.0 Bladder Cancer 0.0 A302173
Normal Kidney 0.0 Bladder Cancer 0.0 (OD04718-01) Kidney Ca,
Nuclear grade 0.0 Bladder Normal 0.0 2 (OD04338) Adjacent (OD04718-
03) Kidney Margin 0.0 Normal Ovary 6.8 (OD04338) Kidney Ca Nuclear
grade 0.0 Ovarian Cancer 0.0 1/2 (OD04339) 064008 Kidney Margin 0.0
Ovarian Cancer 0.0 (OD04339) (OD04768-07) Kidney Ca, Clear cell 0.0
Ovary Margin 0.0 type (OD04340) (OD04768-08) Kidney Margin 0.0
Normal Stomach 0.0 (OD04340) Kidney Ca, Nuclear grade 0.0 Gastric
Cancer 0.0 3 (OD04348) 9060358 Kidney Margin 0.0 Stomach Margin 0.0
(OD04348) 9060359 Kidney Cancer 0.0 Gastric Cancer 5.6 (OD04622-01)
9060395 Kidney Margin 0.0 Stomach Margin 0.0 (OD04622-03) 9060394
Kidney Cancer 0.0 Gastric Cancer 0.0 (OD04450-01) 9060397 Kidney
Margin 0.0 Stomach Margin 0.0 (OD04450-03) 9060396 Kidney Cancer
8120607 0.0 Gastric Cancer 0.0 064005
[0824] TABLE-US-00135 TABLE 52 Panel 4D Rel. Exp.(%) Rel. Exp.(%)
Ag1799, Run Ag1799, Run Tissue Name 156251137 Tissue Name 156251137
Secondary Th1 act 0.0 HUVEC IL-1beta 0.0 Secondary Th2 act 0.0
HUVEC IFN gamma 0.0 Secondary Tr1 act 0.0 HUVEC TNF alpha + IFN 0.0
gamma Secondary Th1 rest 0.0 HUVEC TNF alpha + IL4 0.0 Secondary
Th2 rest 0.0 HUVEC IL-11 0.0 Secondary Tr1 rest 0.0 Lung
Microvascular EC 0.0 none Primary Th1 act 0.0 Lung Microvascular EC
0.0 TNF alpha + IL-1beta Primary Th2 act 0.0 Microvascular Dermal
EC 0.0 none Primary Tr1 act 0.0 Microsvasular Dermal EC 0.0 TNF
alpha + IL-1beta Primary Th1 rest 0.0 Bronchial epithelium 0.0 TNF
alpha + IL1beta Primary Th2 rest 0.0 Small airway epithelium 0.0
none Primary Tr1 rest 0.0 Small airway epithelium 0.0 TNF alpha +
IL-1beta CD45RA CD4 0.0 Coronery artery SMC rest 0.0 lymphocyte act
CD45RO CD4 0.0 Coronery artery SMC 0.0 lymphocyte act TNF alpha +
IL-1beta CD8 lymphocyte act 0.0 Astrocytes rest 0.0 Secondary CD8
0.0 Astrocytes TNF alpha + IL- 0.0 lymphocyte rest 1beta Secondary
CD8 0.0 KU-812 (Basophil) rest 0.0 lymphocyte act CD4 lymphocyte
none 0.0 KU-812 (Basophil) 0.0 PMA/ionomycin 2ry Th1/Th2/Tr1_anti-
0.0 CCD1106 (Keratinocytes) 0.0 CD95 CH11 none LAK cells rest 0.0
CCD1106 (Keratinocytes) 0.0 TNF alpha + IL-1beta LAK cells IL-2 0.0
Liver cirrhosis 0.0 LAK cells IL-2 + IL-12 0.0 Lupus kidney 0.0 LAK
cells IL-2 + IFN 0.0 NCI-H292 none 0.0 gamma LAK cells IL-2 + IL-18
0.0 NCI-H292 IL-4 4.8 LAK cells 0.0 NCI-H292 IL-9 5.2 PMA/ionomycin
NK Cells IL-2 rest 0.0 NCI-H292 IL-13 0.0 Two Way MLR 3 day 0.0
NCI-H292 IFN gamma 0.0 Two Way MLR 5 day 0.0 HPAEC none 0.0 Two Way
MLR 7 day 0.0 HPAEC TNF alpha + IL- 0.0 1beta PBMC rest 0.0 Lung
fibroblast none 0.0 PBMC PWM 0.0 Lung fibroblast TNF alpha + IL-
0.0 1beta PBMC PHA-L 0.0 Lung fibroblast IL-4 0.0 Ramos (B cell)
none 0.0 Lung fibroblast IL-9 0.0 Ramos (B cell) 0.0 Lung
fibroblast IL-13 0.0 ionomycin B lymphocytes PWM 0.0 Lung
fibroblast IFN 0.0 gamma B lymphocytes CD40L 0.0 Dermal fibroblast
0.0 and IL-4 CCD1070 rest EOL-1 dbcAMP 0.0 Dermal fibroblast 0.0
CCD1070 TNF alpha EOL-1 dbcAMP 0.0 Dermal fibroblast 0.0
PMA/ionomycin CCD1070 IL-1beta Dendritic cells none 0.0 Dermal
fibroblast IFN 0.0 gamma Dendritic cells LPS 0.0 Dermal fibroblast
IL-4 0.0 Dendritic cells anti- 0.0 IBD Colitis 2 0.0 CD40 Monocytes
rest 0.0 IBD Crohn's 0.0 Monocytes LPS 0.0 Colon 0.0 Macrophages
rest 0.0 Lung 0.0 Macrophages LPS 0.0 Thymus 40.3 HUVEC none 0.0
Kidney 100.0 HUVEC starved 0.0
[0825] CNS_neurodegeneration_v1.0 Summary: Ag1799 Expression of the
NOV6 gene is low/undetectable in all samples on this panel.
(CTs>35). (Data not shown.) The amp plot indicates that there is
a high probability of a probe failure.
[0826] Panel 1.3D Summary: Ag1799 Expression of the NOV6 gene is
restricted to a few samples, with highest expression in the trachea
and adrenal gland (CTs=31). Thus, expression of this gene could be
used as a marker of these tissue types.
[0827] Panel 2D Summary: Ag1799 Expression of the NOV6 gene is
restricted to a samples derived from thyroid (CT=33.5). Thus,
expression of this gene could be used as a marker of thyroid
tissue.
[0828] Panel 4D Summary: Ag1799 Expression of the NOV6 gene is
restricted to a samples derived from thymus and kidney (CTs=33-34).
Thus, expression of this gene could be used as a marker of these
tissues.
[0829] Panel 5D Summary: Ag1799 Expression of the NOV6 gene is
low/undetectable in all samples on this panel. (CTs>35). (Data
not shown.) The amp plot indicates that there is a high probability
of a probe failure.
NOV5
[0830] Expression of gene NOV5 was assessed using the primer-probe
set Ag2911, described in Table 53. Results of the RTQ-PCR runs are
shown in Table 54. TABLE-US-00136 TABLE 53 Probe Name Ag2911
Primers Sequences Length Start Position SEQ ID NO: Forward
5'-cagggatggaatgcattatg-3' 20 349 191 Probe
TET-5'-caatgtcacctgtactcagatctgtga-3'-TAMRA 27 371 192 Reverse
5'-gctctccaaagcagtaaggaa-3' 21 422 193
[0831] TABLE-US-00137 TABLE 54 Panel 1.3D Rel. Exp.(%) Ag2911, Rel.
Exp.(%) Ag2911, Tissue Name Run 162292963 Tissue Name Run 162292963
Liver adenocarcinoma 0.0 Kidney (fetal) 0.0 Pancreas 0.0 Renal ca.
786-0 0.0 Pancreatic ca. CAPAN 2 0.0 Renal ca. A498 0.0 Adrenal
gland 0.0 Renal ca. RXF 393 0.0 Thyroid 0.0 Renal ca. ACHN 0.0
Salivary gland 0.0 Renal ca. UO-31 0.0 Pituitary gland 0.0 Renal
ca. TK-10 0.0 Brain (fetal) 0.0 Liver 0.0 Brain (whole) 21.5 Liver
(fetal) 0.0 Brain (amygdala) 15.8 Liver ca. 0.0 (hepatoblast) HepG2
Brain (cerebellum) 53.2 Lung 0.0 Brain (hippocampus) 10.5 Lung
(fetal) 0.0 Brain (substantia nigra) 3.2 Lung ca. (small cell) 0.0
LX-1 Brain (thalamus) 0.0 Lung ca. (small cell) 0.0 NCI-H69
Cerebral Cortex 54.0 Lung ca. (s.cell var.) 12.4 SHP-77 Spinal cord
0.0 Lung ca. (large 3.8 cell)NCI-H460 glio/astro U87-MG 0.0 Lung
ca. (non-sm. 0.0 cell) A549 glio/astro U-118-MG 0.0 Lung ca.
(non-s.cell) 0.0 NCI-H23 astrocytoma SW1783 2.7 Lung ca.
(non-s.cell) 0.0 HOP-62 neuro*; met SK-N-AS 0.0 Lung ca. (non-s.cl)
0.0 NCI-H522 astrocytoma SF-539 0.0 Lung ca. (squam.) 0.0 SW 900
astrocytoma SNB-75 0.0 Lung ca. (squam.) 0.0 NCI-H596 glioma SNB-19
0.0 Mammary gland 0.0 glioma U251 0.0 Breast ca.* (pl.ef) 0.0 MCF-7
glioma SF-295 0.0 Breast ca.* (pl.ef) 0.0 MDA-MB-231 Heart (fetal)
0.0 Breast ca.* (pl.ef) 4.2 T47D Heart 6.7 Breast ca. BT-549 0.0
Skeletal muscle (fetal) 100.0 Breast ca. MDA-N 0.0 Skeletal muscle
8.3 Ovary 0.0 Bone marrow 0.0 Ovarian ca. OVCAR-3 3.4 Thymus 15.0
Ovarian ca. OVCAR-4 0.0 Spleen 2.2 Ovarian ca. OVCAR-5 0.0 Lymph
node 0.0 Ovarian ca. OVCAR-8 0.0 Colorectal 3.3 Ovarian ca. IGROV-1
0.0 Stomach 0.0 Ovarian ca.* (ascites) 0.0 SK-OV-3 Small intestine
0.0 Uterus 2.5 Colon ca. SW480 0.0 Placenta 0.0 Colon ca.* 0.0
Prostate 0.0 SW620(SW480 met) Colon ca. HT29 0.0 Prostate ca.*
(bone 0.0 met)PC-3 Colon ca. HCT-116 0.0 Testis 6.7 Colon ca.
CaCo-2 0.0 Melanoma 12.9 Hs688(A).T Colon ca. 0.0 Melanoma* (met)
0.0 tissue(ODO3866) Hs688(B).T Colon ca. HCC-2998 0.0 Melanoma
UACC-62 0.0 Gastric ca.* (liver met) 0.0 Melanoma M14 0.0 NCI-N87
Bladder 1.8 Melanoma LOX 3.3 IMVI Trachea 3.4 Melanoma* (met) 0.0
SK-MEL-5 Kidney 0.0 Adipose 0.0
[0832] CNS_neurodegeneration_v1.0 Summary: Ag2911 Amp plot shows
that there were experimental difficulties with this run and gene
NOV5. (Data not shown.)
[0833] Panel 1.3D Summary: Ag2911 The NOV5 gene, a fatty acid
binding homolog, appears to be differentially expressed in adult
(CT value=34) vs fetal skeletal muscle (CT value=38). This gene
product may be useful for the differentiation of the adult from the
fetal source of this tissue. Fatty acid binding proteins sequester
fatty acid moieties thereby protecting against intracellular
lipotoxicity. Thus, an activator of this gene product may be a
treatment for the prevention of lipotoxicity in skeletal muscle.
Furthermore, increased intracellular triglyceride accumulation is
considered to be pathogenically important in skeletal muscle
insulin resistance and Type 2 diabetes. Thus, therapeutic
modulation of the expression or function of this gene may be
effective in the treatment of Type 2 diabetes.
REFERENCES
[0834] Unger R, Orci L. Diseases of liporegulation: new perspective
on obesity and related disorders. FASEB J. 2001 February; 15
(2):312-21. Review.
[0835] Obesity-related diseases now threaten to reach epidemic
proportions in the United States. Here we review in a rodent model
of genetic obesity, the fa/fa Zucker diabetic fatty (ZDF) rat, the
mechanisms involved in the most common complications of
diet-induced human obesity, i.e., noninsulin-dependent diabetes
mellitus, and myocardial dysfunction. In ZDF rats, hyperphagia
leads to hyperinsulinemia, which up-regulates transcription factors
that stimulate lipogenesis. This causes ectopic deposition of
triacylglycerol in nonadipocytes, providing fatty acid (FA)
substrate for damaging pathways of nonoxidative metabolism, such as
ceramide synthesis. In beta cells and myocardium, the resulting
functional impairment and apoptosis cause diabetes and
cardiomyopathy. Interventions that lower ectopic lipid accumulation
or block nonoxidative metabolism of FA and ceramide formation
completely prevent these complications. Given the evidence for a
similar etiology for the complications of human obesity, it would
be appropriate to develop strategies to avert the predicted
epidemic of lipotoxic disorders.
[0836] PMID: 11156947
[0837] Unger R, Orci L. Lipotoxic diseases of nonadipose tissues in
obesity. Int J Obes Relat Metab Disord. 2000 November; 24 Suppl
4:S28-32. Review.
[0838] It is proposed that an important function of leptin is to
confine the storage of triglycerides (TG) to the adipocytes, while
limiting TG storage in nonadipocytes. Excess TG deposition in
nonadipocytes leads to impairment of functions, increased ceramide
formation, which triggers nitric oxide-mediated lipotoxicity and
lipoapoptosis. The fact that TG content in nonadipocytes normally
remains within a very narrow range irrespective of excess caloric
intake, while TG content of adipocytes rises, is consistent with a
system of fatty acid (FA) homeostasis in nonadipose tissues. When
leptin is deficient or leptin receptors are dysfunctional, TG
content in nonadipose tissues such as pancreatic islets, heart and
skeletal muscle, can increase 10-50-fold, suggesting that leptin
controls the putative homeostatic system for intracellular TG. The
fact that function and viability of nonadipocytes is compromised
when their TG content rises above normal implies that normal
homeostasis of their intracellular FA is critical for prevention of
complications of obesity. FA overload of skeletal muscle,
myocardium and pancreatic islets cause, respectively, insulin
resistance, lipotoxic heart disease and adipogenic type 2 diabetes.
All can be completely prevented by treatment with antisteatotic
agents such as troglitazone. In diet-induced obesity, leptin
signaling is normal initially and lipotoxic changes are at first
prevented; later, however, post-receptor leptin resistance appears,
leading to dysfunction and lipoapoptosis in nonadipose tissues, the
familiar complications of obesity.
[0839] PMID: 11126236
[0840] Panel 2D Summary: Ag2911 Expression of gene NOV5 is
low/undetectable in all samples on this panel (CTs>35). (Data
not shown.)
[0841] Panel 4D Summary: Ag2911 Expression of gene NOV5 is
low/undetectable in all samples on this panel (CTs>35). (Data
not shown.)
NOV13
[0842] Expression of gene NOV13 was assessed using the primer-probe
set Ag1559, described in Table 55. Results of the RTQ-PCR runs are
shown in Table 56. TABLE-US-00138 TABLE 55 Probe Name Ag1559
Primers Sequences Length Start Position SEQ ID NO: Forward
5'-caggacctcggttatcaaca-3' 20 406 194 Probe
TET-5'-acctacgttgagcaaccgtgccg-3'-TAMRA 23 426 195 Reverse
5'-atcgtactcgctggcgtaa-3' 19 483 196
[0843] TABLE-US-00139 TABLE 56 Panel 5D Rel. Exp.(%) Rel. Exp.(%)
Ag1559, Run Ag1559, Run Tissue Name 169269222 Tissue Name 169269222
97457_Patient- 0.0 94709_Donor 2 AM - A_adipose 0.0 02go_adipose
97476_Patient- 0.0 94710_Donor 2 AM - B_adipose 0.0 07sk_skeletal
muscle 97477_Patient- 0.0 94711_Donor 2 AM - C_adipose 0.0
07ut_uterus 97478_Patient- 0.0 94712_Donor 2 AD - A_adipose 0.0
07pl_placenta 97481_Patient- 0.0 94713_Donor 2 AD - B_adipose 0.0
08sk_skeletal muscle 97482_Patient- 0.0 94714_Donor 2 AD -
C_adipose 0.0 08ut_uterus 97483_Patient- 0.0 94742_Donor 3 U - 0.0
08pl_placenta A_Mesenchymal Stem Cells 97486_Patient- 0.0
94743_Donor 3 U - 0.0 09sk_skeletal muscle B_Mesenchymal Stem Cells
97487_Patient- 100.0 94730_Donor 3 AM - A_adipose 0.0 09ut_uterus
97488_Patient- 0.0 94731_Donor 3 AM - B_adipose 0.0 09pl_placenta
97492_Patient- 0.0 94732_Donor 3 AM - C_adipose 0.0 10ut_uterus
97493_Patient- 0.0 94733_Donor 3 AD - A_adipose 0.0 10pl_placenta
97495_Patient- 0.0 94734_Donor 3 AD - B_adipose 0.0 11go_adipose
97496_Patient- 0.0 94735_Donor 3 AD - C_adipose 0.0 11sk_skeletal
muscle 97497_Patient- 0.0 77138_Liver_HepG2untreated 0.0
11ut_uterus 97498_Patient- 0.0 73556_Heart_Cardiac stromal 0.0
11pl_placenta cells (primary) 97500_Patient- 0.0 81735_Small
Intestine 0.0 12go_adipose 97501_Patient- 0.0 72409_Kidney_Proximal
0.0 12sk_skeletal muscle Convoluted Tubule 97502_Patient- 0.0
82685_Small intestine_Duodenum 0.0 12ut_uterus 97503_Patient- 0.0
90650_Adrenal_Adrenocortical 0.0 12pl_placenta adenoma 94721_Donor
2 U -- 0.0 72410_Kidney_HRCE 0.0 A_Mesenchymal Stem Cells
94722_Donor 2 U -- 0.0 72411_Kidney_HRE 0.0 B_Mesenchymal Stem
Cells 94723_Donor 2 U -- 0.0 73139_Uterus_Uterine smooth 0.0
C_Mesenchymal Stem muscle cells Cells
[0844] Panel 1.3D Summary: Ag1559 Expression of the NOV13 gene is
low/undetectable in all samples on this panel. (CTs>35). (Data
not shown.) The data suggest that there was a possible probe
failure.
[0845] Panel 2.2 Summary: Ag1559 Expression of the NOV13 gene is
low/undetectable in all samples on this panel. (CTs>35). (Data
not shown.) The data suggest that there was a possible probe
failure.
[0846] Panel 5D Summary: Ag1559 Expression of the NOV13 gene is
limited to placental tissue (CT=34.7). Thus, expression of this
gene could be used as a marker for this tissue. Furthermore, this
novel cytoplasmic protein may be important for the pathogenesis,
diagnosis, and/or treatment of reproductive diseases.
Example 3
SNP Analysis of NOVX Clones
[0847] SeqCalling.TM. Technology: cDNA was derived from various
human samples representing multiple tissue types, normal and
diseased states, physiological states, and developmental states
from different donors. Samples were obtained as whole tissue, cell
lines, primary cells or tissue cultured primary cells and cell
lines. Cells and cell lines may have been treated with biological
or chemical agents that regulate gene expression for example,
growth factors, chemokines, steroids. The cDNA thus derived was
then sequenced using CuraGen's proprietary SeqCalling technology.
Sequence traces were evaluated manually and edited for corrections
if appropriate. cDNA sequences from all samples were assembled with
themselves and with public ESTs using bioinformatics programs to
generate CuraGen's human SeqCalling database of SeqCalling
assemblies. Each assembly contains one or more overlapping cDNA
sequences derived from one or more human samples. Fragments and
ESTs were included as components for an assembly when the extent of
identity with another component of the assembly was at least 95%
over 50 bp. Each assembly can represent a gene and/or its variants
such as splice forms and/or single nucleotide polymorphisms (SNPs)
and their combinations.
[0848] Variant sequences are 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 silent, however, in the case
that a codon including a SNP encodes the same amino acid as a
result of the redundancy of the genetic code. SNPs occurring
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 for example,
alteration in temporal expression, physiological response
regulation, cell type expression regulation, intensity of
expression, stability of transcribed message.
[0849] Method of novel SNP Identification: SNPs are identified by
analyzing sequence assemblies using CuraGen's proprietary SNPTool
algorithm. SNPTool identifies variation in assemblies with the
following criteria: SNPs are not analyzed within 10 base pairs on
both ends of an alignment; Window size (number of bases in a view)
is 10; The allowed number of mismatches in a window is 2; Minimum
SNP base quality (PHRED score) is 23; Minimum number of changes to
score an SNP is 2/assembly position. SNPTool analyzes the assembly
and displays SNP positions, associated individual variant sequences
in the assembly, the depth of the assembly at that given position,
the putative assembly allele frequency, and the SNP sequence
variation. Sequence traces are then selected and brought into view
for manual validation. The consensus assembly sequence is imported
into CuraTools along with variant sequence changes to identify
potential amino acid changes resulting from the SNP sequence
variation. Comprehensive SNP data analysis is then exported into
the SNPCalling database.
[0850] Method of novel SNP Confirmation: SNPs are confirmed
employing a validated method know as Pyrosequencing
(Pyrosequencing, Westborough, Mass.). Detailed protocols for
Pyrosequencing can be found in: Alderbom et al. Determination of
Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA
Sequencing. (2000). Genome Research. 10, Issue 8, August.
1249-1265. In brief, Pyrosequencing is a real time primer extension
process of genotyping. This protocol takes double-stranded,
biotinylated PCR products from genomic DNA samples and binds them
to streptavidin beads. These beads are then denatured producing
single stranded bound DNA. SNPs are characterized utilizing a
technique based on an indirect bioluminometric assay of
pyrophosphate (PPi) that is released from each dNTP upon DNA chain
elongation. Following Klenow polymerase-mediated base
incorporation, PPi is released and used as a substrate, together
with adenosine 5'-phosphosulfate (APS), for ATP sulfurylase, which
results in the formation of ATP. Subsequently, the ATP accomplishes
the conversion of luciferin to its oxi-derivative by the action of
luciferase. The ensuing light output becomes proportional to the
number of added bases, up to about four bases. To allow
processivity of the method dNTP excess is degraded by apyrase,
which is also present in the starting reaction mixture, so that
only dNTPs are added to the template during the sequencing. The
process has been fully automated and adapted to a 96-well format,
which allows rapid screening of large SNP panels. The DNA and
protein sequences for the novel single nucleotide polymorphic
variants are reported. Variants are reported individually but any
combination of all or a select subset of variants are also
included. In addition, the positions of the variant bases and the
variant amino acid residues are underlined.
Results
[0851] Variants are reported individually but any combination of
all or a select subset of variants are also included as
contemplated NOVX embodiments of the invention.
[0852] NOV2a SNP Data:
[0853] NOV2a has two SNP variants, whose variant positions for its
nucleotide and amino acid sequences is numbered according to SEQ ID
NOs:9 and 10, respectively. The nucleotide sequence of the NOV2a
variant differs as shown in Table 57. TABLE-US-00140 TABLE 57 cSNP
and Coding Variants for NOV2a NT Position Wild Type Amino Acid
Amino Acid of cSNP NT Variant NT position Change 633 A G 199
E->G 941 G A 302 G->S 1156 T C 373 No change
[0854] NOV2b SNP Data:
[0855] NOV2b has four SNP variants, whose variant positions for its
nucleotide and amino acid sequences is numbered according to SEQ ID
NOs:11 and 12, respectively. The nucleotide sequence of the NOV2b
variant differs as shown in Table 58. TABLE-US-00141 TABLE 58 cSNP
and Coding Variants for NOV2b NT Position Wild Type Putative of
cSNP NT Variant NT Depth Allele Freq. 635 G A 37 0.216 786 C T 43
0.047 948 A G 37 0.162 1119 T C 26 0.231
[0856] NOV2c SNP Data:
[0857] NOV2c has four SNP variants, whose variant positions for its
nucleotide and amino acid sequences is numbered according to SEQ ID
NOs:13 and 14, respectively. The nucleotide sequence of the NOV2c
variant differs as shown in Table 59. TABLE-US-00142 TABLE 59 cSNP
and Coding Variants for NOV2c NT Position Wild Type Putative of
cSNP NT Variant NT Depth Allele Freq. 149 A G 16 0.375 174 T C 16
0.125 175 T C 16 0.125 320 C T 15 0.467 386 T C 15 0.133 435 G A 16
0.125
[0858] NOV6 SNP Data:
[0859] 6 has one SNP variant, whose variant position for its
nucleotide and amino acid numbered according to SEQ ID NOs:25 and
26, respectively. The nucleotide the NOV6 variant differs as shown
in Table 60. TABLE-US-00143 TABLE 60 cSNP and Coding Variants for
NOV6 NT Position Wild Type Amino Acid Amino Acid of cSNP NT Variant
NT position Change 190 G S 64 A->T 396 A G 132 No change
[0860] NOV7a SNP Data:
[0861] NOV7a has one SNP variant, whose variant position for its
nucleotide and amino acid sequences is numbered according to SEQ ID
NOs:27 and 28, respectively. The nucleotide sequence of the NOV7a
variant differs as shown in Table 61. TABLE-US-00144 TABLE 61 cSNP
and Coding Variants for NOV7a NT Position Wild Type Amino Acid
Amino Acid of cSNP NT Variant NT position Change 1638 C T 513
P->L
[0862] NOV8 SNP Data:
[0863] NOV8 has one SNP variant, whose variant position for its
nucleotide and amino acid sequences is numbered according to SEQ ID
NOs:31 and 32, respectively. The nucleotide sequence of the NOV8
variant differs as shown in Table 62. TABLE-US-00145 TABLE 62 cSNP
and Coding Variants for NOV8 NT Position Wild Type Amino Acid Amino
Acid of cSNP NT Variant NT position Change 102 T C 28 C->R 185 A
G 55 No change 210 G A 64 A->T 225 T C 69 F->L 395 T C 125 No
change
Example 4
In-Frame Cloning
NOV2e
[0864] For NOV2e the cDNA coding for the DOMAIN of NOV1a from
residues 51 to 400 was targeted for "in-frame" cloning by PCR. The
PCR template was based on the previously identified plasmid, when
available, or on human cDNA(s). TABLE-US-00146 TABLE 99
Oligonucleotide primers used to clone the target cDNA sequence:
Primers Sequences F1 5'-GGATCC TCCCAGTTGGAGGAGGTGTTTCACTCT-3' (SEQ
ID NO:199) R1 5'-CTCGAG AGGAGAAGAAAATCTGCCGAAGAAGAGGATGC-3' (SEQ ID
NO:200) SF1 5'-ATGAACTGAACATAACCAACAGGCT-3' (SEQ ID NO:201) SF2
5'-GGACTTGTTCCCAGATGGCTCTA-3' (SEQ ID NO:202) SF3
5'-TTTAGCTTCACTTTCCTGGAGGACT-3' (SEQ ID NO:203) SF4
5'-AAAGAAAGGTGAATCTGCACTTGCCC-3' (SEQ ID NO:204) SF5
5'-TTGTGGCAGTAACTGAGGAAGGC-3' (SEQ ID NO:205) SR1
5'-AGCCTGTTGGTTATGTTCAGTTCAT-3' (SEQ ID NO:206) SR2
5'-TTTTTCATTTGTTTTGCTTTCAACC-3' (SEQ ID NO:207) SR3
5'-AGGAATGGCTCTGTGTCATCATCTG-3' (SEQ ID NO:209) SR4
5'-CTTTCTTCCATATGCCCTGGACTA-3' (SEQ ID NO:210) SR5
5'-CAAAGGAACTGTGCAGGAACTTCT-3' (SEQ ID NO:211)
[0865] For downstream cloning purposes, the forward primer includes
an in-frame Hind III restriction site and the reverse primer
contains an in-frame Xho I restriction site.
[0866] Two parallel PCR reactions were set up using a total of
0.5-1.0 ng human pooled cDNAs as template for each reaction. The
pool is composed of 5 micrograms of each of the following human
tissue cDNAs: adrenal gland, whole brain, amygdala, cerebellum,
thalamus, bone marrow, fetal brain, fetal kidney, fetal liver,
fetal lung, heart, kidney, liver, lymphoma, Burkitt's Raji cell
line, mammary gland, pancreas, pituitary gland, placenta, prostate,
salivary gland, skeletal muscle, small Intestine, spleen, stomach,
thyroid, trachea, uterus.
[0867] When the tissue of expression is known and available, the
second PCR was performed using the above primers and 0.5 ng-1.0 ng
of one of the following human tissue cDNAs: [0868] skeleton muscle,
testis, mammary gland, adrenal gland, ovary, colon, normal
cerebellum, normal adipose, normal skin, bone marrow, brain
amygdala, brain hippocampus, brain substantia nigra, brain
thalamus, thyroid, fetal lung, fetal liver, fetal brain, kidney,
heart, spleen, uterus, pituitary gland, lymph node, salivary gland,
small intestine, prostate, placenta, spinal cord, peripheral blood,
trachea, stomach, pancreas, hypothalamus.
[0869] The reaction mixtures contained 2 microliters of each of the
primers (original concentration: 5 pmol/ul), 1 microliter of 10 mM
dNTP (Clontech Laboratories, Palo Alto Calif.) and 1 microliter of
50.times. Advantage-HF 2 polymerase (Clontech Laboratories) in 50
microliter-reaction volume. The following reaction conditions were
used: TABLE-US-00147 PCR condition 1: a) 96.degree. C. 3 minutes b)
96.degree. C. 30 seconds denaturation c) 60.degree. C. 30 seconds,
primer annealing d) 72.degree. C. 6 minutes extension Repeat steps
b-d 15 times e) 96.degree. C. 15 seconds denaturation f) 60.degree.
C. 30 seconds, primer annealing g) 72.degree. C. 6 minutes
extension Repeat steps e-g 29 times e) 72.degree. C. 10 minutes
final extension PCR condition 2: a) 96.degree. C. 3 minutes b)
96.degree. C. 15 seconds denaturation c) 76.degree. C. 30 seconds,
primer annealing, reducing the temperature by 1.degree. C. per
cycle d) 72.degree. C. 4 minutes extension Repeat steps b-d 34
times e) 72.degree. C. 10 minutes final extension
[0870] An amplified product was detected by agarose gel
electrophoresis. The fragment was gel-purified and ligated into the
pCR2.1 vector (Invitrogen, Carlsbad, Calif.) following the
manufacturer's recommendation. Twelve clones per PCR reaction were
picked and sequenced. The inserts were sequenced using
vector-specific M13 Forward and M13 Reverse primers and the
gene-specific primers in Tables 88 and 89. TABLE-US-00148 TABLE 88
Gene-specific Primers NOV Primers Sequences NOV11c SF1
GCCCTCCCGGTCCAGGTC (SEQ ID NO:200) SF2 GGCGACGGCACCAGCATGT (SEQ ID
NO:201) SR1 GCCTGGCCTGCCGGGTTCT (SEQ ID NO:202) SR2
CATGAGCACGTGGTAAGCG (SEQ ID NO:203)
[0871] TABLE-US-00149 TABLE 89 Gene-specific Primers NOV Primers
Sequences NOV1b SF1 GTGCTGGCATTGGAGTGTT (SEQ ID NO:204) TAGTG SF2
ATCAAGCACGTTGACACAG (SEQ ID NO:205) AATGAG SF3 GCATTCACTAACCTAACAC
(SEQ ID NO:206) CATTTACA SF4 GTTCAGCAGAGATGTCGTC (SEQ ID NO:207)
TGACCTTC SF5 GGGATCCTCCAGATCCTGT (SEQ ID NO:208) ATTTTT SF6
TGAAGAACACATCAACAAC (SEQ ID NO:209) AGACATAA SR1
ACTGTTTTCAGCAGCTACC (SEQ ID NO:210) TTAATTTC SR2
CTTGATGAATGTGTGGTAC (SEQ ID NO:211) GCGAT SR3 GTGAATGCAAACTTGAGGT
(SEQ ID NO:212) CTTTTGT SR4 CCTCATATAATCCTACCAT (SEQ ID NO:213)
TGGCTGTACT SR5 GAGGATCCCAGTGTAAAAA (SEQ ID NO:214) TACTTCTG SR6
TAGCACTTCATAAGCAATA (SEQ ID NO:215) ATGATCCC SR7
TGAGTGTACTAGCAGACAC (SEQ ID NO:216) CTCAATGAT
Other Embodiments
[0872] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims, which follow. In particular,
it is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims. The choice of nucleic acid starting material, clone of
interest, or library type is believed to be a matter of routine for
a person of ordinary skill in the art with knowledge of the
embodiments described herein. Other aspects, advantages, and
modifications considered to be within the scope of the following
claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060009634A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060009634A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References