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CA 02447935 2003-11-25
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NOVEL ANTIBODIES THAT BIND TO ANTIGENIC
POLYPEPTIDES, NUCLEIC ACIDS ENCODING THE ANTIGENS,
AND METHODS OF USE
FIELD OF THE INVENTION
The present invention relates to novel antibodies that bind immunospecifically
to
antigenic polypeptides, wherein the polypeptides have characteristic
properties related to
biochemical or physiological responses in a cell, a tissue, an organ or an
organism. The
novel polypeptides are gene products of novel genes, or are specified
biologically active
fragments or derivatives thereof. Methods of use of the antibodies encompass
procedures
for diagnostic and prognostic assay of the polypeptides, as well as methods of
treating
diverse pathological conditions.
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BACKGROUND OF THE INVENTION
Eukaryotic cells are characterized by biochemical and physiological processes
which under normal conditions are exquisitely balanced to achieve the
preservation and
propagation of the cells. When such cells are components of multicellular
organisms such
as vertebrates, or more particularly organisms such as mammals, the regulation
of the
biochemical and physiological processes involves intricate signaling pathways.
Frequently,
such signaling pathways involve extracellulax signaling proteins, cellular
receptors that
bind the signaling proteins, and signal transducing components located within
the cells.
Signaling proteins may be classified as endocrine effectors, paracrine
effectors or
autocrine effectors. Endocrine effectors are signaling molecules secreted by a
given organ
into the circulatory system, which are then transported to a distant target
organ or tissue.
The target cells include the receptors for the endocrine effector, and when
the endocrine
effector binds, a signaling cascade is induced. Paracrine effectors involve
secreting cells
and receptor cells in close proximity to each other, for example two different
classes of
cells in the same tissue or organ. One class of cells secretes the paracrine
effector, which
then reaches the second class of cells, for example by diffusion through the
extracellular
fluid. The second class of cells contains the receptors for the paracrine
effector; binding of
the effector results in induction of the signaling cascade that elicits the
corresponding
biochemical or physiological effect. Autocrine effectors are highly analogous
to paracrine
effectors, except that the same cell type that secretes the autocrine effector
also contains the
receptor. Thus the autocrine effector binds to receptors on the same cell, or
on identical
neighboring cells. The binding process then elicits the characteristic
biochemical or
physiological effect.
Signaling processes may elicit a variety of effects on cells and tissues
including by
way of nonlimiting example induction of cell or tissue proliferation,
suppression of growth
or proliferation, induction of differentiation or maturation of a cell or
tissue, and
suppression of differentiation or maturation of a cell or tissue.
Many pathological conditions involve dysregulation of expression of important
effector proteins. In certain classes of pathologies the dysregulation is
manifested as
elevated or excessive synthesis and secretion of protein effectors. In a
clinical setting a
subject may be suspected of suffering from a condition brought on by elevated
or excessive
levels of a protein effector of interest.
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Antibodies are multichain proteins that bind specifncally to a gmen antigen,
and
bind poorly, or not at all, to substances deemed not to be cognate antigens.
Antibodies are
comprised of two short chains termed light chains and two long chains termed
heavy
chains. These chains are constituted of immunoglobulin domains, of which
generally there
are two classes: one variable domain per chain, one constant domain in light
chains, and
three or more constant domains in heavy chains. The antigen-specific portion
of the
immunoglobulin molecules resides in the variable domains; the variable domains
of one
light chain and one heavy chain associate with each other to generate the
antigen-binding
moiety. Antibodies that bind immunospecifically to a cognate or target antigen
bind with
high affinities. Accordingly, they are useful in assaying specifically for the
presence of the
antigen in a sample. In addition, they have the potential of inactivating the
activity of the
antigen.
Therefore there is a need to assay for the level of a protein effector of
interest in a
biological sample from such a subject, and to compare this level with that
characteristic of
a nonpathological condition. In particular, there is a need for such an assay
based on the
use of an antibody that binds immunospecifically to the antigen. There fm-ther
is a need to
inhibit the activity of the protein effector in cases where a pathological
condition arises
from elevated or excessive levels of the effector based on the use of an
antibody that binds
immunospecifically to the effector. Thus, there is a need for the antibody as
a product of
manufacture. There further is a need for a method of treatment of a
pathological condition
brought on by an elevated or excessive level of the protein effector of
interest based on
administering the antibody to the subject.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences
encoding novel polypeptides. The novel nucleic acids and polypeptides are
referred to
herein as NOVX, or NOV l, NOV2, NOV3, etc., 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.
In one aspect, the invention provides an isolated polypeptide comprising a
mature
form of a NOVX amino acid. The polypeptide can be, for example, a NOVX amino
acid
sequence or a variant of a NOVX amino acid sequence, wherein any amino acid
specified
in the chosen sequence is changed to a different amino acid, provided that no
more than
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15% of the amino acid residues in the sequence are so cha~igea. We-invention
also
includes fragments of any of NOVX polypeptides. In another aspect, the
invention also
includes an isolated nucleic acid that encodes a NOVX polypeptide, or a
fragment,
homolog, analog or derivative thereof.
Also included in the invention is a NOVX polypeptide that is a naturally
occurring
variant of a NOVX sequence. In one embodiment, the variant includes an amino
acid
sequence that is the translation of a nucleic acid sequence differing by a
single nucleotide
from a NOVX nucleic acid sequence. In another embodiment, the NOVX polypeptide
is a
variant polypeptide described therein, wherein any amino acid specified in the
chosen
sequence is changed to provide a conservative substitution.
In another aspect, invention provides a method for determining the presence or
amount of the NOVX polypeptide in a sample by providing a sample; introducing
the
sample to an antibody that binds immunospecifically to the polypeptide; and
determining
the presence or amount of antibody bound to the NOVX polypeptide, thereby
determining
the presence or amount of the NOVX polypeptide in the sample.
In yet another aspect, the invention includes a method for deternuning the
presence
of or predisposition to a disease associated with altered levels of a NOVX
polypeptide in a
mammalian subject by measuring the level of expression of the polypeptide in a
sample
from the first mammalian subject; and comparing the amount of the polypeptide
in the
sample of the first step to the amount of the polypeptide present in a control
sample from a
second mammalian subject known not to have, or not to be predisposed to, the
disease. An
alteration in the expression level ofthe polypeptide in the first subject as
compared to the
control sample indicates the presence of or predisposition to the disease.
In another aspect, the invention includes pharmaceutical compositions that
include
therapeutically- or prophylactically-effective amounts of a therapeutic and a
pharmaceutically-acceptable carrier. The therapeutic can be, e.g., a NOVX
nucleic acid, a
NOVX polypeptide, or an antibody specific for a NOVX polypeptide. In a further
aspect,
the invention includes, in one or more containers, a therapeutically- or
prophylactically-
effective amount of this pharmaceutical composition.
In still another aspect, the invention provides the use of a therapeutic in
the
manufacture of a medicament for treating a syndrome associated with a human
disease that
is associated with a NOVX polypeptide.
In a further aspect, the invention provides a method for modulating the
activity of a
NOVX polypeptide by contacting a cell sample expressing the NOVX polypeptide
with
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antibody that binds the NOVX polypeptide in an amount suiiicierit-to moautate
the activity
of the polypeptide.
The invention also includes an isolated nucleic acid that encodes a NOVX
polypeptide, or a fragment, homolog, analog or derivative thereof. In a
preferred
embodiment, the nucleic acid molecule comprises the nucleotide sequence of a
naturally
occurring allelic nucleic acid variant. In another embodiment, the nucleic
acid encodes a
variant polypeptide, wherein the variant polypeptide has the polypeptide
sequence of a
naturally occurring polypeptide variant. In another embodiment, the nucleic
acid molecule
differs by a single nucleotide from a NOVX nucleic acid sequence. In one
embodiment,
the NOVX nucleic acid molecule hybridizes under stringent conditions to the
nucleotide
sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is
an integer
between l and 73, or a complement of the nucleotide sequence. In one
embodiment, the
invention provides a nucleic acid molecule wherein the nucleic acid includes
the nucleotide
sequence of a naturally occurring allelic nucleic acid variant.
Also included in the invention is a vector containing one or more of the
nucleic
acids described herein, and a cell containing the vectors or nucleic acids
described herein.
The invention is also directed to host cells transformed with a vector
comprising any of the
nucleic acid molecules described above.
In yet another aspect, the invention provides for a method for determining the
presence or amount of a nucleic acid molecule in a sample by contacting a
sample with a
probe that binds a NOVX nucleic acid and determining the amount of the probe
that is
bound to the NOVX nucleic acid. For example the NOVX nucleic may be a marker
for cell
or tissue type such as a cell or tissue type that is cancerous.
In yet a further aspect, the invention provides a method for determining the
presence of or predisposition to a disease associated with altered levels of a
nucleic acid
molecule in a first mammalian subject, wherein an alteration in the level of
the nucleic acid
in the first subject as compared to the control sample indicates the presence
of or
predisposition to the disease.
The invention further provides an antibody that binds immunospecifically to a
NOVX polypeptide. The NOVX antibody may be monoclonal, humanized, or a fully
human antibody. Preferably, the antibody has a dissociation constant for the
binding of the
NOVX polypeptide to the antibody less than 1 x 10-9 M. More preferably, the
NOVX
antibody neutralizes the activity of the NOVX polypeptide.
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In a further aspect, the invention provides for the use of a t~lierapeutic in
the
manufacture of a medicament for treating a syndrome associated with a human
disease,
associated with a NOVX polypeptide. Preferably the therapeutic is a NOVX
antibody.
In yet a further aspect, the invention provides a method of treating or
preventing a
NOVX-associated disorder, a method of treating a pathological state in a
mammal, and a
method of treating or preventing a pathology associated with a polypeptide by
administering a NOVX antibody to a subject in an amount sufficient to treat or
prevent the
disorder.
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 are
not intended to
be limiting.
Other features and advantages of the invention will be apparent from the
following
detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded
thereby. Included in the invention are the novel nucleic acid sequences, their
encoded
polypeptides, antibodies, and other related compunds. 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 1 provides a summary of the NOVX nucleic
acids and
their encoded polypeptides.
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TABLE 1. NOVX Polynucleotide and Polypeptide Sequences and
Corresponding SEQ ID Numbers
SEQ
Internal IdentificationID SEQ ID Homology
NO NO
(nucleic(polypeptide)
acid
la CG100653-O1 1 2 Cadherin Associated
Protein-like
2a CG100689-O1 3 4 Leucine Rich Repeat-like
3a CG100760-O1 5 6 Leucine Rich Repeat-like
4a 7 8 Leukocyte Surface Antigen
CG 100851-02 CD53-like
5a CG101068-O1 9 10 Claudin-9-like
6a 11 12 Integral Membrane Protein
CG 2 01231-O IsOform
1 2-like
6b 13 14 Integral Membrane Protein
CG 101231-02 Isoform
2-like
7a CG101362-Oi 15 16 Prion Protein-like
8a 17 18 Von Willebrand Domain
CG101458-O1 Containin Protein-like
9a CG101475-O1 19 20 Plasma Membrane Protein-like
9b CG 101475-02 21 22 Plasma Membrane Protein-like
IOa CG101772-01 23 24 RAGE-like
11 a CG 102532-O1 25 26 Emerin-like
12a CG102575-O1 27 28 ATPase-like
12b CG102575-02 29 30 ATPase-like
13a 31 32 Mat8 (Mammary Tumor
CG 102615-01 8 kDa)
Protein-like
13b 33 34 MatB (Mammary Tumor
CG 102615-04 8 kDa)
Protein-like
14a CG102646-01 35 36 High Affinity Proline
Permease-like
15a CG102878-Ol 37 38 Transmembrane-like
15b CG102878-02 39 40 Transmembrane-like
16a CG 103459-O 1 41 42 Pe tide/Histidine Transporter-like
17a CG104210-O1 43 44 Type III Membrane Protein-like
17b CG104210-02 45 46 Type III Membrane Protein-like
17c 272249075 47 48 Type III Membrane Protein-like
18a CG104251-Ol 49 50 Type III Membrane Protein-like
19a 51 52 Phospholipid-Transporting
CG 104934-O1 ATPase
IH-like
20a 53 54 Meningioma-Expressed
CG105463-OI Antigen
6/11 (MEA6) (MEAI1)-like
20b 55 56 Meningioma-Expressed
CG 105463-02 Antigen
6/ 1 I (MEA6) (MEA
11 )-like
21a CG105491-O1 57 58 Serine Protease-like
22a CG105954-O1 59 60 Neurofascin Precursor-like
23a CG105963-O1 61 62 Cadherin-like
24a CG 105973-O1 63 64 Integrin A1 ha 8-like
24b CG 105973-02 65 66 Inte rin Alpha 8-like
25a CG106915-O1 67 68 No o Rece for Isoform-1-like
26a CG 106924-O I 69 70 Nogo Receptor Isoform-2-Iike
26b 210062144 71 72 Nogo Receptor Isoform-2-like
27a CG106942-O1 73 74 NRAMP-like Membrane
Protein
28a 75 76 Syntaxin Domain Containing
CG 107513-01 Protein-like
29a CG107533-02 77 78 Tumor Necrosis Factor-like
30a CG107562-O1 ~ 79 80 Leucine-Rich Repeat
Type III
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Transmembrane-like
30b 81 82 Leucine-Rich Repeat
Type III
CG 107562-02 Transmembrane-1 ike
30c 83 84 Leucine-Rich Repeat
Type 11I
210086373 Transmembrane-like
30d 85 86 Leucine-Rich Repeat
Type III
210086403 Transmembrane-like
30e 87 88 Leucine-Rich Repeat
Type II1
210086422 Transmembrane-like
31 a CG 108184-O1 89 90 Transmembrane Protein
Tm7-like
3I b CG 108184-02 91 92 Transmembrane Protein
Tm7-like
31c CG108184-03 93 94 Transmembrane Protein
Tm7-like
32a 95 96 Sialic Acid Binding
CG 108238-O1 Immunoglobulin-like
33a CG108695-O1 97 98 OB binding rotein (SIGLEC)-like
34a CG109505-O1 99 100 Aldehyde Dehydro enase-like
35a 101 102 Latent Transforming
Growth Factor
CG109742-O1 Beta Bindin Protein
3-like
35b 103 104 Latent Transforming
Growth Factor
207639410 Beta Bindin Protein
3-like
35c 105 106 Latent Transforming
Growth Factor
207639427 Beta Binding Protein
3-like
35d 107 108 Latent Transforming
Growth Factor
207639438 Beta Bindin~ Protein
3-like
35e 109 110 Latent Transforming
Growth Factor
207639448 Beta Bindin Protein
3-like
36a CG109844-O1 11 112 C4B-Binding Protein-like
I
37a CG110014-02 113 114 Colon Carcinoma kinase
4-like
37b CG 110014-03 115 1 I6 Colon Carcinoma kinase
4-like
37c CGl 10014-04 117 118 Colon Carcinoma kinase
4-like
38a CG110187-O1 119 120 AI ha Cl-like Protocadherin
38b CG110I87-03 121 122 Alpha C1-like Protocadherin
39a 123 124 Disintegrin-like l
Metalloprotease
(Reprolysin Type) with
CG110205-O1 Tlu-ombospondin Type
I Motif like
39b 125 126 Disintegrin-like /
Metalloprotease
(Reprolysin Type) with
CG110205-02 Thrombos ondin Type
I Motif like
39c 127 128 Disintegrin-Iike l
Metalloprotease
(Reprolysin Type) with
207756942 Thrombospondin Type
I Motif lilee
39d 129 130 Disintegrin-like /
Metalloprotease
(Reprolysin Type) with
207756946 Thrombospondin Type
I Motif like
39e 131 132 Disintegrin-like /
Metalloprotease
(Reprolysin Type) with
207756950 Thrombospondin Type
I Motif like
39f 133 134 Disintegrin-like l
Metalloprotease
(Reprolysin Type) with
207756966 Thrombospondin Type
I Motif like
40a CGII0242-O1 135 136 Ebnerin-like
40b 207728344 137 138 Ebnerin-like
40c 207728348 139 140 Ebnerin-like
40d 207728354 141 142 Ebnerin-like
40e 207728365 143 144 Ebnerin-like
41a 145 146 Endosomal Glycoprotein
CG99598-O 1 Precursor-like
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Table 1 indicates the homology of NOVX polypeptides ~o known protein families.
Thus, the nucleic acids and polypeptides, antibodies and related compounds
according to
the invention corresponding to a NOVX as identified in column 1 of Table 1
will be useful
in therapeutic and diagnostic applications implicated in, for example,
pathologies and
disorders associated with the known protein families identified in column 5 of
Table 1.
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.
Consistent with other known members of the family of proteins, identified in
column 5 of Table l, the NOVX polypeptides of the present invention show
homology to,
and contain domains that are characteristic of, other members of such protein
families. Details of the sequence relatedness and domain analysis for each
NOVX are
presented in Example A.
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 diseases associated with the protein
families listed
in Table 1.
The NOVX nucleic acids and polypeptides are also useful for detecting specific
cell
types. Details of the expression analysis for each NOVX are presented in
Example C.
Accordingly, the NOVX nucleic acids, polypeptides, antibodies and related
compounds
according to the invention will have diagnostic and therapeutic applications
in the detection
of a variety of diseases with differential expression in normal vs. diseased
tissues, e.g.
detection of a variety of cancers.
Additional utilities for NOVX nucleic acids and polypeptides according to the
invention are disclosed herein.
NOVX clones
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
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of domains and sequence relatedness to previously descrilie-d prot~tris.
Acrctitionally;
NOVX nucleic acids and polypeptides can also be used to identify proteins that
are
members of the family to which the NOVX polypeptides belong.
The NOVX genes and their corresponding encoded proteins are useful for
preventing, treating or ameliorating medical conditions, e.g., by protein or
gene therapy.
Pathological conditions can be diagnosed by determining the amount of the new
protein in
a sample or by determining the presence of mutations in the new genes.
Specific uses are
described for each of the NOVX genes, based on the tissues in which they are
most highly
expressed. Uses include developing products for the diagnosis or treatment of
a variety of
diseases and disorders.
The NOVX nucleic acids and proteins of the invention are useful in potential
diagnostic and therapeutic applications and as research tools. 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 (vi) a
biological defense weapon.
In one specific embodiment, the invention includes an isolated polypeptide
comprising an amino acid sequence selected from the group consisting of: (a) a
mature
form of the amino acid sequence selected from the group consisting of SEQ ID
NO: 2n,
wherein n is an integer between l and 73; (b) a variant of a mature form of
the amino acid
sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an
integer
between 1 and 73, wherein any amino acid in the mature form is changed to a
different
amino acid, provided that no more than 15% of the amino acid residues in the
sequence of
the mature form are so changed; (c) an amino acid sequence selected from the
group
consisting of SEQ ID NO: Zn, wherein n is an integer between 1 and 73; (d) a
variant of the
amino acid sequence selected from the group consisting of SEQ ID N0:2n,
wherein n is an
integer between 1 and 73 wherein any amino acid specified in the chosen
sequence is
changed to a different amino acid, provided that no more than 15% of the amino
acid
residues in the sequence are so changed; and (e) a fragment of any of (a)
through (d).
In another specific embodiment, the invention includes an isolated nucleic
acid
molecule comprising a nucleic acid sequence encoding a polypeptide comprising
an amino
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acid sequence selected from the group consisting of (a)"a mature torrii o~-the
amino acid
sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 73; (b) a
variant of
a mature form of the amino acid sequence selected from the group consisting of
SEQ ID
NO: 2n, wherein n is an integer between 1 and 73 wherein any amino acid in the
mature
form of the chosen sequence is changed to a different amino acid, provided
that no more
than 15% of the amino acid residues in the sequence of the mature form are so
changed; (c)
the amino acid sequence selected from the group consisting of SEQ ID NO: 2n,
wherein n
is an integer between 1 and 73; (d) a variant of the amino acid sequence
selected from the
group consisting of SEQ ID NO: 2n, wherein n is an integer between I and 73,
in which
any amino acid specified in the chosen sequence is changed to a different
amino acid,
provided that no more than 15% of the amino acid residues in the sequence are
so changed;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide
comprising the
amino acid sequence selected from the group consisting of SEQ ID NO: 2n,
wherein n is an
integer between 1 and 73 or any variant of said polypeptide wherein any amino
acid of the
chosen sequence is changed to a different amino acid, provided that no more
than 10% of
the amino acid residues in the sequence are so changed; and (f) the complement
of any of
said nucleic acid molecules.
In yet another specific embodiment, the invention includes an isolated nucleic
acid
molecule, wherein said nucleic acid molecule comprises a nucleotide sequence
selected
from the group consisting of: (a) the nucleotide sequence selected from the
group
consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 73; (b) a
nucleotide sequence wherein one or more nucleotides in the nucleotide sequence
selected
from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between
l and 73
is changed from that selected from the group consisting of the chosen sequence
to a
different nucleotide provided that no more than 15% of the nucleotides are so
changed; (c)
a nucleic acid fragment of the sequence selected from the group consisting of
SEQ ID NO:
2n-1, wherein n is an integer between 1 and 73; and (d) a nucleic acid
fragment wherein
one or more nucleotides in the nucleotide sequence selected from the group
consisting of
SEQ ID NO: 2n-1, wherein n is an integer between l and 73 is changed from that
selected
from the group consisting of the chosen sequence to a different nucleotide
provided that no
more than 15% of the nucleotides are so changed.
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NOVX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that
encode
NOVX polypeptides or biologically active portions thereof. Also included in
the invention
are nucleic acid fragments suff cient for use as hybridization probes to
identify NOVX-
encoding nucleic acids (e.g., NOVX mRNA's) 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
I O acid molecule may be single-stranded or double-stranded, but preferably is
comprised
double-stranded DNA. ' -
A 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,
myristylation or
phosphorylation. In general, a mature polypeptide or protein may result from
the operation
of only one of these processes, or a combination of any of them.
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The term "probes", as utilized herein, refers to nucleic acid sequences of
vamable
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.
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. Fpr
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 cellltissue from which
the nucleic
acid is derived (e.g., brain, heart, Liver, spleen, etc.). Moreover, an
"isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of other cellular
material or
culture medium when produced by recombinant techniques, or of chemical
precursors or
other chemicals when chemically synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having
the
nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1-73,
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 N0:2h-l, wherein n is an
integer between
1-73, 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°a Ed., Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT
PROTOCOLS IN
3O MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers
according to standard PCR amplification techniques. The nucleic acid so
amplified can be
cloned into an appropriate vector and characterized by DNA sequence analysis.
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Furthermore, oligonucleotides corresponding to NOVX~nuc~eotnte sequences can
be
prepared by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a
S 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, SO nt, or
100 nt in length, preferably about 15 nt to 30 ntin length. In one embodiment
of the
invention, an oligonucleotide comprising a nucleic acid molecule less than 100
nt in length
would further comprise at least 6 contiguous nucleotides of SEQ ID N0:2n-1,
wherein n is
an integer between 1-73, or a complement thereof. Oligonucleotides may be
chemically
synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
1 S comprises a nucleic acid molecule that is a complement of the nucleotide
sequence SEQ ID
N0:2~c-l, wherein n is an integer between 1-73, or a portion of this
nucleotide sequence
(e.g., a fragment that can be used as a probe or primer or a fragment encoding
a
biologically-active portion of a NOVX polypeptide). A nucleic acid molecule
that is
complementary to the nucleotide sequence of SEQ ID N0:2n-I, wherein ~ is an
integer
between 1-73, is one that is sufficiently complementary to the nucleotide
sequence of SEQ
ID N0:2n-l, wherein n is an integer between 1-73, that it can hydrogen bond
with little or
no mismatches to the nucleotide sequence of SEQ ID N0:2n-l, wherein h is an
integer
between 1-73, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
2S base pairing between nucleotides units of a nucleic acid molecule, and the
term "binding"
means the physical or chemical interaction between two polypeptides or
compounds or
associated polypeptides or compounds or combinations thereof. Binding includes
ionic,
non-ionic, van der Waals, hydrophobic interactions, and the like. A physical
interaction
can be either direct or indirect. Indirect interactions may be through or due
to the effects of
another polypeptide or compound. Direct binding refers to interactions that do
not take
place through, or due to, the effect of another polypeptide or compound, but
instead are
without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous)
nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to
allow for specific
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hybridization in the case of nucleic acids or for specific recognition o~ 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.
A full-length NOVX clone is identified as containing an ATG translation start
colon and an in-frame stop colon. Any disclosed NOVX nucleotide sequence
lacking an
ATG start colon therefore encodes a truncated C-terminal fragment of the
respective
NOVX polypeptide, and requires that the corresponding full-length cDNA extend
in the 5'
direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence
lacking an
in-frame stop colon similarly encodes a truncated N-terminal fragment of the
respective
NOVX polypeptide, and requires that the corresponding full-length cDNA extend
in the 3'
direction of the disclosed sequence.
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°10) 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 8c Sons, New York, NY,
1993,
and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode
those
CA 02447935 2003-11-25
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sequences coding for isoforms of NOVX polypeptides. -Isofoiins 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 a
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
N0:2~-l, wherein h is an integer between 1-73, as well as a polypeptide
possessing NOVX
biological activity. Various biological activities of the NOVX proteins are
described
below.
A NOVX polypeptide is encoded by the open reading frame ("ORF") of a 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.
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 of SEQ
ID NO:2n-
l, wherein rz is an integer between 1-73; or an anti-sense strand nucleotide
sequence of
SEQ ID N0:2h-1, wherein ~ is an integer between 1-73; or of a naturally
occurring mutant
of SEQ ID NO:2n-l, wherein ~ is an integer between 1-73.
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Probes based on the human NOVX nucleotide sequence's can be usea to etetect
transcripts or genomic sequences encoding the same or homologous proteins. In
various
embodiments, the probe further comprises a label group attached thereto, e.g.
the label
group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor.
Such probes can be used as a part of a diagnostic test kit for identifying
cells or tissues
which mis-express a NOVX protein, such as by measuring a level of a NOVX-
encoding
nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA
levels or
determining whether a genomic NOVX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of a 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 of SEQ ID N0:2n-
1,
wherein n is an integer between .l -73, that encodes a polypeptide having a
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 i~ vitro)
and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
2Q nucleotide sequences of SEQ ID N0:2fz-l, wherein n is an integer between 1-
73, due to
degeneracy of the genetic code and thus encode the same NOVX proteins as that
encoded
by the nucleotide sequences of SEQ ID N0:2n-1, wherein ~ is an integer between
1-73. In
another embodiment, an isolated nucleic acid molecule of the invention has a
nucleotide
sequence encoding a protein having an amino acid sequence of SEQ ID N0:2h,
wherein n
is an integer between 1-73.
In addition to the human NOVX nucleotide sequences of SEQ ID N0:2h-1, wherein
~ is an integer between 1-73, 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 a NOVX
protein, preferably a vertebrate NOVX protein. Such natural allelic variations
can typically
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result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and
all such
nucleotide variations and resulting amino acid polymorphisms in the NOVX
polypeptides,
which are the result of natural allelic variation and that do not alter the
functional activity
of the NOVX polypeptides, are intended to be within the scope of the
invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species,
and
thus that have a nucleotide sequence that differs from any one of the human
SEQ ID
N0:2h-1, wherein h is an integer between 1-73, are intended to be within the
scope of the
invention. Nucleic acid molecules corresponding to natural allelic variants
and
homologues of the NOVX cDNAs of the invention can be isolated based on their
homology
to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a
portion
thereof, as a hybridization probe according to standard hybridization
techniques under
stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 6 nucleotides in length and hybridizes under stringent
conditions to the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2~c-l,
wherein ~
is an integer between I-73. In another embodiment, the nucleic acid is at
least 10, 25, 50,
100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet
another
embodiment, an isolated nucleic acid molecule of the invention hybridizes to
the coding
region. As used herein, the term "hybridizes under stringent conditions" is
intended to
describe conditions for hybridization and washing under which nucleotide
sequences at
least 60% homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding 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.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to
no other sequences. Stringent conditions are sequence-dependent and will be
different in
different circumstances. Longer sequences hybridize specifically at higher
temperatures
than shorter sequences. Generally, stringent conditions are selected to be
about 5 °C lower
than the thermal melting point (Tm) for the specific sequence at a defined
ionic strength
and pH. The Tm is the temperature (under defined ionic strength, pH and
nucleic acid
concentration) at which 50% of the probes complementary to the target sequence
hybridize
to the target sequence at equilibrium. Since the target sequences are
generally present at
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excess, at Tm, 50% of the probes are occupied at equilitinurii. ' T'-
ypical'Iy;"-Strut gent
conditions will be those in which the salt concentration is less than about
1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and
the
temperature is at least about 30 °C for short probes, primers or
oligonucleotides (e.g., 10 nt
to 50 nt) and at least about 60 °C for longer probes, primers and
oligonucleotides.
Stringent conditions may also be achieved with the addition of destabilizing
agents, such as
formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons,
N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences
at least about
65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically
remain
hybridized to each other. A non-limiting example of stringent hybridization
conditions are
hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCI (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% FicoII, 0.02% BSA, and 500 mg/ml denatured salmon sperm
DNA at 65 °C, followed by one or more washes in 0.2X SSC, 0.01 % BSA at
50 °C. An
isolated nucleic acid molecule of the invention that hybridizes under
stringent conditions to
any one of the sequences of SEQ ID N0:2n-1, wherein n is an integer between 1-
73,
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).
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of SEQ ID N0:2n-l, wherein fZ
is an
integer between 1-73, or fragments, analogs or derivatives thereof, under
conditions of
moderate stringency is provided. A non-limiting example of moderate stringency
hybridization conditions axe hybridization in 6X SSC, SX Reinhardt's solution,
0.5% SDS
and 100 mg/ml denatured salmon sperm DNA at 55 °C, followed by one or
more washes in
1X SSC, 0.1% SDS at 37 °C. Other conditions of moderate stringency that
may be used
are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993,
CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, I 990; GENE TRANSFER
AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule comprising the nucleotide sequences of SEQ ID NO:Zn-l, wherein n is
an integer
between 1-73, or fragments, analogs or derivatives thereof, under conditions
of low
stringency, is provided. A non-limiting example of low stringency
hybridization conditions
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WO 02/099062 PCT/US02/17559
are hybridization in 35% formamide, 5X SSC, 50 mM Tris=HCI' (pT=I 7:5), 5- inM
EDTA,
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/m1 denatured salmon sperm DNA, IO%
(wt/volt) dextran sulfate at 40 °C, followed by one or more washes in
2X SSC, 25 mM
Tris-HCl (pH 7.4), 5 mM EDTA, and 0. I % SDS at 50 °C. Other conditions
of low
stringency that may be used are well known in the art (e.g., as employed for
cross-species
hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER
AND
EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981.
Pt~oc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may
exist in the population, the skilled artisan will further appreciate that
changes can be
introduced by mutation into the nucleotide sequences of SEQ ID N0:2~z-l,
wherein h is an
integer between 1-73, 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 of SEQ ID N0:2h, wherein h is
an integer
between I-73. 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 particularly non-amenable to alteration. Amino acids for which
conservative
substitutions can be made are well-known within the art.
2S 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 any one of SEQ ID NO:2h-l,
wherein
n is an integer between 1-73, yet retain biological activity. In one
embodiment, the isolated
nucleic acid molecule comprises a nucleotide sequence encoding a protein,
wherein the
protein comprises an amino acid sequence at least about 45% homologous to the
amino
acid sequences of SEQ ID N0:2~, wherein n is an integer between 1-73.
Preferably, the
protein encoded by the nucleic acid molecule is at least about 60% homologous
to SEQ ID
N0:2n, wherein v~ is an integer between 1-73; more preferably at least about
70%
homologous to SEQ ID N0:2n, wherein n is an integer between 1-73; still more
preferably
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at least about 80% homologous to SEQ ID N0:2TZ, wherein ~ is an
int'eger~~etween 1-73
even more preferably at least about 90% homologous to SEQ ID N0:2n, wherein to
is an
integer between 1-73; and most preferably at least about 95% homologous to SEQ
ID
N0:2n, wherein r~ is an integer between 1-73.
An isolated nucleic acid molecule encoding a NOVX protein homologous to the
protein of SEQ ID N0:2~c, wherein n is an integer between 1-73, can be created
by
introducing one or more nucleotide substitutions, additions or deletions into
the nucleotide
sequence of SEQ ID N0:2n-1, wherein n is an integer between 1-73, such that
one or more
amino acid substitutions, additions or deletions are introduced into the
encoded protein.
Mutations can be introduced into any of SEQ ID N0:2fz-1, wherein fZ is an
integer
between 1-73, 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),
nonpolax 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, txyptophan, 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 a NOVX coding
sequence, such
as by saturation mutagenesis, and the resultant mutants can be screened for
NOVX
biological activity to identify mutants that retain activity. Following
mutagenesis of any
one of SEQ ID N0:2rc-1, wherein n is an integer between 1-73, the encoded
protein can be
expressed by any recombinant technology known in the art and the activity of
the protein
can be determined.
The relatedness of amino acid families may also be determined based on side
chain
interactions. Substituted amino acids may be fully conserved "strong" residues
or fully
conserved "weak" residues. The "strong" group of conserved amino acid residues
may be
any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY,
FYW, wherein the single letter amino acid codes are grouped by those amino
acids that
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may be substituted fox each other. Likewise, the "weak'j' group of conserved
xesidues may
be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK,
NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the
single
letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to
form protein:protein interactions with other NOVX proteins, other cell-surface
proteins, or
biologically-active portions thereof, (ii) complex formation between a mutant
NOVX
protein and a NOVX ligand; or (iii) the ability of a mutant NOVX protein to
bind to an
intracellular target protein or biologically-active portion thereof; (e.g.
avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the
ability
to regulate a specific biological function (e.g., regulation of insulin
release).
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to the nucleic acid molecule
comprising the
nucleotide sequence of SEQ ID N0:2~-l, wherein n is an integer between 1-73,
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
a NOVX protein of SEQ ID N0:2n, wherein ~ is an integer between 1-73, or
antisense
nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID N0:2r~-
l,
wherein ~e is an integer between 1-73, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence encoding a 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).
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WO 02/099062 PCT/US02/17559
Given the coding strand sequences encoding the NOVX protein disclosed herein,
antisense nucleic acids of the invention cambe 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
S 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 S, 10, 1S, 20, 2S, 30, 3S, 40, 4S
or SO
nucleotides in length. An antisense nucleic acid of the invention can be
constructed using
chemical synthesis or enzymatic Iigation 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.,
1 S phosphorothioate derivatives and acxidine substituted nucleotides can be
used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: S-fluorouracil, S-bromouracil, S-chlorouracil, S-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, S-(carboxyhydroxylmethyl) uracil,
S-carboxymethylaminomethyl-2-thiouridine, S-carboxymethylaminomethyluracil,
dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
I-methylguanine, I-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, S-methylcytosine, N6-adenine, 7-
methylguanine,
S-methylaminomethyluracil, S-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, S'-methoxycarboxymethyluracil, S-methoxyuracil,
2S 2-methylthio-N6-isopentenyladenine, uracil-S-oxyacetic acid (v),
wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, S-methyl-2-thiouracil, 2-thiouracil, 4-
thiouracil,
S-methyluracil, uracil-S-oxyacetic acid methylester, uracil-5-oxyacetic acid
(v),
S-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).
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WO 02/099062 PCT/US02/17559
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated i~z situ such that they hybridize with or bind to
cellular mRNA and/or
genomic DNA encoding a 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
I 5 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
axe preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is
an a-anomeric nucleic acid molecule. An cc-anomeric nucleic acid molecule
forms specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
(3-units,
the strands run parallel to each other. See, e.g., Gaultier, et al., 1987.
Nucl. Acids Res. 15:
6625-6641. The antisense nucleic acid molecule can also comprise a
2'-o-methylxibonucleotide (See, e.g., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or
a chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987. FEBS Lett. 215:
327-330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modif ed
bases, and nucleic acids whose sugar phosphate backbones are modified or
derivatized.
These modifications are carried out at least in part to enhance the chemical
stability of the
modified nucleic acid, such that they may be used, for example, as antisense
binding
nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
24
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WO 02/099062 PCT/US02/17559
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 a NOVX-encoding nucleic acid can be designed based upon
the
nucleotide sequence of a NOVX cDNA disclosed herein (i.e., any one of SEQ ID
N0:2n-1,
wherein rz is an integer between 1-73). For example, a derivative of a
Tetrahynae~a L-19
IVS RNA can be constructed in which the nucleotide sequence of the active site
is
complementary to the nucleotide sequence to be cleaved in a NOVX-encoding
mRNA.
See, e.g., U.S. Patent 4,987,071 to Cech, et al, and U.S. Patent 5,116,742 to
Cech, et al.
NOVX mRNA can also be used to select a catalytic RNA having a specific
ribonuclease
activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993)
Science
261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the NOVX nucleic acid
(e.g., the
NOVX promoter and/or enhancers) to form triple helical structures that prevent
transcription of the NOVX gene in target cells. See, e.g., Helene, 1991.
Ahtica~zcer D~°ug
Des. 6: 569-84; Helene, et al. 1992. Ar~~. N. Y. Acad. Sci. 660: 27-36; Maher,
1992.
Bioassays 14: 807-15. '
In various embodiments, the NOVX nucleic acids can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization,
or solubility of the molecule. For example, the deoxyribose phosphate backbone
of the
nucleic acids can be modified to generate peptide nucleic acids. See, e.g.,
Hyrup, et al.,
1996. Bioorg Med Cl2eyvc 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
nucleotide bases 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 oligomer can be performed using standard solid phase peptide
synthesis
protocols as described in Hyrup, et al., 1996. supra; Perry-O'I~eefe, et al.,
1996. Proc. Natl.
Acad. Sci. LISA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For
example, PNAs can be used as antisense or antigene agents for sequence-
specific
modulation of gene expression by, e.g., inducing transcription or translation
arrest or
inhibiting replication. PNAs of NOVX can also be used, for example, in the
analysis of
CA 02447935 2003-11-25
WO 02/099062 PCT/US02/17559
single base pair mutations in a gene (e.g., PNA directed"PC'R ctampmg; as
araticiat
restriction enzymes when used in combination with other enzymes, e.g., S~
nucleases (See,
Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and
hybridization
(See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of NOVX can be
generated
that may combine the advantageous properties of PNA and DNA. Such chimeras
allow
DNA recognition enzymes (e.g., RNase H and DNA polymerises) 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 nucleotide bases, 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. Nuel 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-S'-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. Clzem. Lett. 5: 1119-
11124.
In other embodiments, the oligonucleotide may include other appended groups
such
as peptides (e.g., for targeting host cell receptors ire vivo), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acid. Sci. U.S.A. 86:
6553-6556; Lemaitre, et al., I 987. Proc. Natl. Acid. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89110134). In
addition, oligonucleotides can be modified with hybridization triggered
cleavage agents
(see, e.g., Krol, et al., 1988. BioTechuiques 6:958-976) or intercalating
agents (see, e.g.,
Zon, 1988. PharMZ. 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.
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WO 02/099062 PCT/US02/17559
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino acid sequence of NOVX polypeptides whose sequences are provided in any
one of
SEQ ID N0:2n, whexein n is an integex between 1-73. The invention also
includes a
mutant or variant protein any of whose residues may be changed from the
corresponding
residues shown in any one of SEQ ID N0:2n, wherein n is an integer between 1-
73, while
still encoding a protein that maintains its NOVX activities and physiological
functions, or a
functional fxagment thereof.
In general, a NOVX variant that preserves NOVX-like function includes any
variant
in which residues at a particular position in the sequence have been
substituted by other
amino acids, and further include the possibility of inserting an additional
residue or
residues between two residues of the parent protein as well as the possibility
of deleting
one or more residues from the parent sequence. Any amino acid substitution,
insertion, or
deletion is encompassed by the invention. In favorable circumstances, the
substitution is a
conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and
biologically-
active portions thereof, ox 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, a NOVX protein or
polypeptide can be
synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active
portion
thereof is substantially free of 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 fxom cellular components of the cells from which it
is isolated or
recombinantly-produced. In one embodiment, the language "substantially free of
cellular
material" includes pxeparations 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 Iess than about 20% of non-NOVX proteins, still more preferably
Less than about
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WO 02/099062 PCT/US02/17559
10% of non-NOVX proteins, and most preferably less than about 5% of non-NUVX
proteins. When the NOVX protein or biologically-active portion thereof is
recombinantly-
produced, it is also preferably substantially free of culture medium, i.e.,
culture medium
represents less than about 20%, more preferably less than about 10%, and most
preferably
less than about 5% of the volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from
chemical
precursors or other chemicals that are involved in the synthesis of the
protein. In one
embodiment, the language "substantially free of chemical precursors or other
chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry
weight) of
chemical precursors or non-NOVX chemicals, more preferably less than about 20%
chemical precursors or non-NOVX chemicals, still more preferably less than
about 10%
chemical precursors or non-NOVX chemicals, and most preferably less than about
5%
chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequences of the
NOVX proteins (e.g., the amino acid sequence of SEQ ID N0:2n, wherein n is an
integer
between 1-73) that include fewer amino acids than the full-length NOVX
proteins, and
exhibit at least one activity of a 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 a NOVX protein can be a polypeptide which is, for example,
10, 25, 50,
100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the
protein
are deleted, can be prepared by recombinant techniques and evaluated for one
or more of
the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID
N0:2~, wherein h is an integer between 1-73. In other embodiments, the NOVX
protein is
substantially homologous to SEQ ID N0:2n, wherein ~ is an integer between 1-
73, and
retains the functional activity of the protein of SEQ ID NO:2h, wherein n is
an integer
between 1-73, 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 of SEQ ID NO:2n, wherein n is an integer
between
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WO 02/099062 PCT/US02/17559
1-73, and retains the functional activity of the NOVX proteins of S~~-'~D
~I~b:2n, wherein
h is an integer between 1-73.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be
introduced in the sequence of a first amino acid or nucleic acid sequence for
optimal
alignment with a second amino or nucleic acid sequence). The amino acid
residues or
nucleotides at corresponding amino acid positions or nucleotide positions are
then
compared. When a position in the first sequence is occupied by the same amino
acid
residue or nucleotide as the corresponding position in the second sequence,
then the
molecules are homologous at that position (i. e., as used herein amino acid or
nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known in the art, such as GAP software provided in the GCG program package.
See,
Needleman and Wunsch, 1970. JMoI 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
of SEQ ID NO:2n-l, wherein ra is an integer between 1-73.
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 I00 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
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WO 02/099062 PCT/US02/17559
identity and often 90 to 95 percent sequence identity, mode it~u~lly a~-feast
f9 percent
sequence identity as compared to a reference sequence over a comparison
region.
Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein,
a
NOVX "chimeric protein" or "fusion protein" comprises a NOVX polypeptide
operatively-
linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a
polypeptide
having an amino acid sequence corresponding to a NOVX protein of SEQ ID N0:2n,
wherein n is an integer between 1-73, 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
a NOVX
fusion protein the NOVX polypeptide can correspond to all or a portion of a
NOVX
protein. In one embodiment, a NOVX fusion protein comprises at least one
biologically-
active portion of a NOVX protein. In another embodiment, a NOVX fusion protein
comprises at least two biologically-active portions of a NOVX protein. In yet
another
embodiment, a NOVX fusion protein comprises at Ieast three biologically-active
portions
of a NOVX protein. Within the fusion protein, the term "operatively-linked" is
intended to
indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-
frame
with one another. The non-NOVX polypeptide can be fused to the N-terminus or
C-terminus of the NOVX polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which
the
NOVX sequences axe fused to the C-terminus of the GST (glutathione S-
transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
NOVX
polypeptides.
In another embodiment, the fusion protein is a NOVX protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host
cells), expression and/or secretion of NOVX can be increased through use of a
heterologous signal sequence.
In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion
protein in which the NOVX sequences axe 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 a NOVX ligand and a NOVX protein on
the
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WO 02/099062 PCT/US02/17559
surface of a cell, to thereby suppress NOVX-mediated sign'a'l trarisduction in
vivo. The
NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability
of a
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 a NOVX
ligand.
A 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). A 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.
NOYX Agonists and Antagonists
The invention also pertains to variants of the NOVX proteins that function as
either
NOVX agonists (i. e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein
can be generated by mutagenesis (e.g., discrete point mutation or truncation
of the NOVX
protein). An agonist of the NOVX protein can retain substantially the same, or
a subset of,
the biological activities of the naturally occurring form of the NOVX protein.
An
antagonist of the NOVX protein can inhibit one or more of the activities of
the naturally
occurring form of the NOVX protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which includes
the NOVX
protein. Thus, specific biological effects can be elicited by treatment with a
variant of
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WO 02/099062 PCT/US02/17559
limited function. In one embodiment, treatment of a subject'vit'h a'"vai~ianf
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.
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. An~u. Rev.
Biochenz. 53: 323;
Itakura, et al., 1984. Science I98: 1056; Ike, et al., 1983. Nucl. Acids Res.
11: 477.
Polypeptide Libraries
In addition, libraries of fragments of the NOVX protein coding sequences can
be
used to generate a variegated population of NOVX fragments for screening and
subsequent
selection of variants of a NOVX protein. In one embodiment, a library of
coding sequence
fragments can be generated by treating a double stranded PCR fragment of a
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~
nuclease,
and ligating the resulting fragment library into an expression vector. By this
method,
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WO 02/099062 PCT/US02/17559
expression libraries can be derived which encodes N-terminal and internal
fragments of
various sizes of the NOVX proteins.
Various techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of 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.
NOVX Antibodies
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, Fab, Fab° and Ftab~)a fragments, and an Fab 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 IgGi, IgG2, and others.
Furthermore, in
humans, the light chain may be a kappa chain or a lambda chain. Reference
herein to
antibodies includes a reference to all such classes, subclasses and types of
human antibody
species.
An isolated 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
33
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antigenic peptide fragment comprises at least 6 amino acid"residues"ot-~the
ammo acid
sequence of the full length protein, such as an amino acid sequence of SEQ ID
N0:2~,
wherein rz is an integer between I-73, 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 IO 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 xegions of the protein that are located on its
surface; commonly
these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of NOVX 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,
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, P~°oc. Nat. Acad. Sci.
USA 78: 3824-
3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-I42, 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.
The term "epitope" includes any protein determinant capable of specif c
binding to
an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of
chemically
active surface groupings of molecules such as amino acids or sugar side chains
and usually
have specific three dimensional structural characteristics, as well as
specific charge
characteristics. A NOVX polyppeptide or a fragment thereof comprises at least
one
antigenic epitope. An anti-NOVX antibody of the present invention is said to
specifically
bind to antigen NOVX when the equilibrium binding constant (KD) is S1 ~.M,
preferably 5
100 nM, more preferably <_ 10 nM, and most preferably _< 100 pM to about 1 pM,
as
measured by assays such as radioligand binding assays or similar assays known
to those
skilled in the art.
'34
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A protein of the invention, or a derivative, fragment, analog, riomolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
Various procedures known within the art may be used fox 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, NY, incorporated herein by reference).
Some of
these antibodies are discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals
(e.g.,
rabbit, goat, mouse or other mammal) may be immunized by one or more
injections with
the native protein, a synthetic variant thereof, or a derivative of the
foregoing. An
appropriate immunogenic preparation can contain, for example, the naturally
occurring
immunogenic protein, a chemically synthesized polypeptide representing the
irnmunogenic
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
paxvum,
or similar immunostimulatory agents. Additional examples of adjuvants which
can be
employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the irrimunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
CA 02447935 2003-11-25
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chromatography. Purification of immunoglobulins is discu'~"sed,"ror example;
by'b.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA,
VoI. 14, No. 8
(April I7, 2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs)
of the monoclonal antibody axe identical iri 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.
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.
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 axe 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.
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Preferred immortalized cell lines are those that ~use~ef~cieritly, 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 marine
rnyeloma lines, which can be obtained, for instance, from the Salk Institute
Cell
Distribution Center, San Diego, California and the American Type Culture
Collection,
Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines
also
have been described for the production of human monoclonal antibodies (Kozbor,
J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques
and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be
assayed
for the presence of monoclonal antibodies directed against the antigen.
Preferably, the
binding specificity of monoclonal antibodies produced by the hybridoma cells
is
determined by immunoprecipitation or by an 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.
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.
The monoclonal antibodies secreted by the subclones can be isolated or
purified
from the culture medium or ascites fluid by conventional immunoglobulin
purification
procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography,
gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such
as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies
of the invention can be readily isolated and sequenced using conventional
procedures (e.g.,
by using oligonucleotide probes that are capable of binding specifically to
genes encoding
the heavy and light chains of marine antibodies). The hybridoma cells of the
invention
serve as a preferred source of such DNA. Once isolated, the DNA can be placed
into
37
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WO 02/099062 PCT/US02/17559
expression vectors, which are then transfected into host 'cel'~s such as siiW
ari (:O'~lcells,
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 modif ed, for example, by
substituting the
coding sequence for human heavy and light chain constant domains in place of
the
homologous marine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368,
812-13
(1994)) or by covalently joining to the immunoglobulin coding sequence aI1 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
I 0 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
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, ilnmunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
F(ab')a 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. Patent No. 5,225,539.) In some instances, Fv framework residues
of the
human immunoglobulin are replaced by corresponding non-human residues.
Humanized
antibodies can also comprise residues which are found neither in the recipient
antibody nor
in the imported CDR or framework sequences. In general, the humanized antibody
will
comprise substantially all of at Least one, and typically two, variable
domains, in which all
or substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the framework regions are those
of a human
innmunoglobulin consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
38
CA 02447935 2003-11-25
WO 02/099062 PCT/US02/17559
human immunoglobulin (Jones et al., 1986; Riechmann et ~'t, '198$; and
I~resla, Curr. Z7p.
Struct. Biol., 2:593-596 (1992)).
Human Antibodies
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 I~ozbor, 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).
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 alI respects, including gene rearrangement, assembly, and antibody
repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technology 10, 779-
783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature
368, 812-I3
(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)).
Human antibodies may additionally be produced using transgenic nonhuman
animals which are modified so as to produce fully human antibodies rather than
the
animal's endogenous antibodies in response to challenge by an antigen. (See
PCT
publication W094/02602). The endogenous genes encoding the heavy and light
39
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WO 02/099062 PCT/US02/17559
immunoglobulin chains in the nonhuman host have been incapacitated, and active
loci
encoding human heavy and Iight chain immunogIobulins 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
xenomouseTM 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
1 S obtain analogs of antibodies such as, for example, single chain Fv
molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S.
Patent No. 5,939,598. It can be obtained by a method including deleting the J
segment
genes from at least one endogenous heavy chain locus in an embryonic stem cell
to prevent
rearrangement of the locus and to prevent formation of a transcript of a
rearranged
immunoglobulin heavy 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.
A method for producing an antibody of interest, such as a human antibody, is
disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression
vector that
contains a nucleotide sequence encoding a heavy chain into one mammalian host
cell in
culture, introducing an expression vector containing a nucleotide sequence
encoding a light
chain into another mammalian host cell, and fusing the two cells to form a
hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and the light
chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99/53049.
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WO 02/099062 PCT/US02/17559
Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an antigenic protein of the invention (see
e.g., U.S.
Patent No. 4,946,778). In addition, methods can be adapted for the
construction of Fab
expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to
allow rapid and
effective identification of monoclonal Fab fragments with the desired
specificity for a
protein or derivatives, fragments, analogs or homologs thereof. Antibody
fragments that
contain the idiotypes to a protein antigen may be produced by techniques known
in the art
including, but not limited to: (i) an F(ab~~~ fragment produced by pepsin
digestion of an
antibody molecule; (ii) an Fab fragment generated by reducing the disulfide
bridges of an
F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the
antibody molecule
with papain and a reducing agent and (iv) F,, fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies
that have binding specificities for at least two different antigens. In the
present case, one of
the binding specificities is for an antigenic protein of the invention. The
second binding
target is any other antigen, and advantageously is a cell-surface protein or
receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas)
produce a potential mixture of ten different antibody molecules, of which only
one has the
correct bispecific structure. The purification of the correct molecule is
usually
accomplished by affinity chromatography steps. Similar procedures are
disclosed in WO
93108829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-
3659
(1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to imrnunoglobulin 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
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WO 02/099062 PCT/US02/17559
least one of the fusions. L~NAs encoding the immunogiobuim heavy-chain iusions
and, u-
desired, the immunoglobulin light chain, are inserted into separate expression
vectors, and
are co-transfected into a suitable host organism. For further details of
generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210
(1986).
According to another approach described in WO 96/2701 l, the interface between
a
pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
which are recovered from recombinant cell culture. The preferred interface
comprises at
least a part of the CH3 region of an antibody constant domain. In this method,
one or more
small amino acid side chains from the interface of the first antibody molecule
are replaced
with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical
or similar size to the large side chains) are created on the interface of the
second antibody
molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or
threonine). This provides a mechanism for increasing the yield of the
heterodimer over
other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments.(e.g. F(ab')2 bispecific antibodies). Techniques for generating
bispecific
antibodies from antibody fragments have been described in the literature. For
example,
bispecif c 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')~, 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 bispecif c antibody. The bispecific antibodies
produced
can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')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.
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WO 02/099062 PCT/US02/17559
Various techniques for making and isolating bispecifac'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 (Vu)
connected to a light-chain variable domain (VL) by a linker which is too short
to allow
pairing between the two domains on the same chain. Accordingly, the VH and VL
domains
of one fragment are forced to pair with the complementary VL and VH domains of
another
fragment, thereby forming two antigen-binding sites. Another strategy for
making
bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has
also been
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60
(1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of
which originates in the protein antigen of the invention. Alternatively, an
anti-antigenic
arm of an immunoglobulin molecule can be combined with an arm which binds to a
triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.
CD2, CD3,
CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII
(CD32) and
Fc7RIII (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
Heteroconjugate antibodies axe 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
43
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WO 02/099062 PCT/US02/17559
cells (U.S. Patent No. 4,676,980), and for treatment of HIV''irifectiori ('W~C-
9170036~0;-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.
Patent No. 4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing
interchain disulfide bond formation in this region. The homodimeric antibody
thus
generated can have improved internalization capability and/or increased
complement-
mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See
Caron et
al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-
2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity can also be prepared
using
heterobifunctional cross-linkers as described in Wolff et al. Cancer Research,
53: 2560-
2565 (1993). Alternatively, an antibody can be engineered that has dual Fc
regions and can
thereby have enhanced complement Iysis and ADCC capabilities. See Stevenson et
al.,
Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g.,
an
enzymatically active toxin of bacterial, fungal, plant, or animal origin, or
fragments
thereof), or a radioactive isotope (i. e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above. Enzyrnatically 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
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CA 02447935 2003-11-25
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tricothecenes. A variety of radionuclides are available for flie
prodizct'i~ori ~of~
radioconjugated antibodies. Examples include 212Bi, 1311, ~3iln, 9oY, and
~86Re.
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 I,5-difluoro-2,4-dinitrobenzene). Fox example, a ricin
immunotoxin
can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
Carbon-I4-
labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-
DTPA)
is an exemplary chelating agent for conjugation of radionucleotide to the
antibody. See
W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate
is 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
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. Patent No.
5,013,556.
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 Iiposomes as
described in Martin
et al ., J. Biol. Chern., 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 (I989).
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Diagnostic Applications of Antibodies Directed Against the Proteins of the
Invention
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).
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 Iysate 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
pxotein 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, [3-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 IuminoI;
examples of
bioluminescent materials include luciferase, luciferin, and aequorin, and
examples of
suitable radioactive material include lash i3ih ass or 3H.
Antibody Therapeutics
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Antibodies of the invention, including polyclonaI;
rrioizdclorial;"liuiii'aiiized"arld' 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 Iigand to which it naturally binds. In this case, the antibody
binds to the target
and masks a binding site of the naturally occurring Iigand, wherein the ligand
serves as an
effector molecule. Thus the receptor mediates a signal transduction pathway
for which
ligand is responsible.
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.
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
xate 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
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
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WO 02/099062 PCT/US02/17559
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.
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. Natl. Acad. Sci. LISA, 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 axe suitably present in combination in amounts that are effective
for the purpose
intended.
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.
The formulations to be used for in vivo administration must be sterile. This
is
readily accomplished by filtration through sterile filtration membranes.
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 y ethyl-L-glutamate,
non-
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WO 02/099062 PCT/US02/17559
degradable ethylene-vinyl acetate, degradable lactic acid-
~l~'c'~l~c°~aci~d ~~~~i~l7I~iriers«~s~c~~Fa~~°
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 hydrogels release proteins for shorter time periods.
ELISA Assay
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., Fab or F~ab~2) can be used. The term "labeled", with regard to the
probe or antibody, is
intended to encompass direct labeling of the probe or antibody by coupling
(i.e., physically
linking) a detectable substance to the probe or antibody, as well as indirect
labeling of the
probe or antibody by reactivity with another reagent that is directly labeled.
Examples of
indirect labeling include detection of a primary antibody using a
fluorescently-labeled
secondary antibody and end-labeling of a DNA probe with biotin such that it
can be
detected with fluorescently-labeled streptavidin. The term "biological sample"
is intended
to include tissues, cells and biological fluids isolated from a subject, as
well as tissues, cells
and fluids present within a subject. 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 ivy vivo. For example, in vitro techniques for detection of an analyte mRNA
include
Northern hybridizations and iu situ hybridizations. Ih vitro techniques for
detection of an
analyte protein include enzyme linked immunosorbent assays (ELISAs), Western
blots,
immunoprecipitations, and immunofluorescence. I~ 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, NJ,
1995;
"Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San
Diego,
CA, 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.
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For example, the antibody can be labeled with a radioactme marker whose
presence and
location in a subject can be detected by standard imaging techniques.
NOVX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding a 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.
The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means
that the recombinant expression vectors include one or more regulatory
sequences, selected
on the basis of the host cells to be used for expression, that is operatively-
linked to the
nucleic acid sequence to be expressed. Within a recombinant expression vector,
"operably-
linked" is intended to mean that the nucleotide sequence of interest is linked
to the
regulatory sequences) in a manner that allows for expression of the nucleotide
sequence
(e.g., in an irc vitro transcription/translation system or in a host cell when
the vector is
introduced into the host cell).
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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 ofNOVX proteins,
fusion
proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression
of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX
proteins can be
expressed in bacterial cells such as Eschef~ichia coli, insect cells (using
baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host cells are
discussed further
in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic
Press, San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be
~0 transcribed and translated in vitro, for example using T7 promoter
regulatory sequences
and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia
coli
with vectors containing constitutive or inducible promoters directing the
expression of
either fusion or non-fusion proteins. Fusion vectors add a number of amino
acids to a
protein encoded therein, usually to the amino terminus of the recombinant
protein. Such
fusion vectors typically serve three purposes: (i) to increase expression of
recombinant
protein; (ii) to increase the solubility of the recombinant protein; and (iii)
to aid in the
purification of the recombinant protein by acting as a ligand in affinity
purification. Often,
in fusion expression vectors, a proteolytic cleavage site is introduced at the
junction of the
fusion moiety and the recombinant protein to enable separation of the
recombinant protein
from the fusion moiety subsequent to purification of the fusion protein. 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
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CA 02447935 2003-11-25
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pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione 5-transterase (CJ5~1
), maltose E
'binding protein, or protein A, respectively, to the target recombinant
protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET 1 1d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990)
60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the
recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN
1O 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: 211 I-2118). Such
alteration of nucleic
acid sequences of the invention can be carried out by standard DNA synthesis
techniques.
In another embodiment, the NOVX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Sacchanornyces cef°ivisae
include pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,
Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells
(e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell.
Biol. 3: 2156-2165)
and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC
(Kaufinan,
et al., 1987. EMBO J. 6: 187-I 95). 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.
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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 (Calaine and Eaton, 1988. Adv.
Imrrzunol. 43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore,
1989. EMBO
J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;
Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the
neurofilament
promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad Sci. USA 86: 5473-5477),
pancreas-specific promoters (Edlund, et al., 1985. Sciefzce 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 marine hox promoters (I~essel and Grass, 1990. Science
249:
374-379) and the a,-fetoprotein promoter (Camper and Tilghman, 1989. Genes
Dev. 3:
537-546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation.
That is, the DNA molecule is operatively-linked to a regulatory sequence in a
manner that
allows for expression (by transcription of the DNA molecule) of an RNA
molecule that is
antisense to NOVX mRNA. Regulatory sequences. operatively linked to a nucleic
acid
cloned in the antisense orientation can be chosen that direct the continuous
expression of
the antisense RNA molecule in a variety of cell types, for instance viral
promoters and/or
enhancers, or regulatory sequences can be chosen that direct constitutive,
tissue specific or
cell type specific expression of antisense RNA. The antisense expression
vector can be in
the form of a recombinant plasmid, phagemid or attenuated virus in which
antisense nucleic
acids are produced under the control of a high efficiency regulatory region,
the activity of
which can be determined by the cell type into which the vector is introduced.
For a
discussion of the regulation of gene expression using antisense genes see,
e.g., Weintraub,
et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-
Trends in
Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
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"recombinant host cell" are used interchangeably herein: lf~is understood that
such terms
refer not only to the particular subject cell but also to the progeny or
potential progeny of
such a cell. Because certain modifications may occur in succeeding generations
due to
either mutation or environmental influences, such progeny may not, in fact, be
identical to
the parent cell, but are still included within the scope of the term as used
herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX
protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells
(such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host
cells are
known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the 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.
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 (~.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Various selectable
markers include those that confer resistance to drugs, such as 6418,
hygromycin and
methotrexate. Nucleic acid encoding a selectable marker can be introduced into
a host cell
on the same vector as that encoding 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).
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
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WO 02/099062 PCT/US02/17559
medium such that NOVX protein is produced. In another eimhodirrient, the
method further
comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte
or an embryonic stem cell into which NOVX protein-coding sequences have been
introduced. Such host cells can then be used to create non-human transgenie
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 wluch one or more of the cells
of the animal
includes a transgene. Other examples of transgenic animals include non-human
primates,
sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous
DNA that is
integrated into the genome of a cell from which a transgenic animal develops
and that
remains in the genome of the mature animal, thereby directing the expression
of an
encoded gene product in one or more cell types or tissues of the transgenic
animal. As used
herein, a "homologous recombinant animal" is a non-human animal, preferably a
mammal,
more preferably a mouse, in which an endogenous NOVX gene has been altered by
homologous recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic cell of the
animal, prior to
development of the animal.
A transgenic animal of the invention can be created by introducing
NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte
(e.g., by
microinjection, retroviral infection) and allowing the oocyte to develop in a
pseudopregnant
female foster animal. The human NOVX cDNA sequences, i. e., any one of SEQ ID
N0:2n-l, wherein n is an integer between 1-73, can be introduced as a
transgene into the
genome of a non-human animal. Alternatively, a non-human homologue of the
human
NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization
to the
human NOVX cDNA (described further supra) and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the transgene to
increase the
efficiency of expression of the transgene. A tissue-specific regulatory
sequences) can be
CA 02447935 2003-11-25
WO 02/099062 PCT/US02/17559
operably-linked to the NOVX transgene to direct expressioi~~bf NO'~X"'protein
to'particuTar~~
cells. Methods for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become conventional in
the art and
are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and
4,873,191; and
Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. Similar methods are used for production of
other
transgenic animals. A transgenic founder animal can be identified based upon
the presence
of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues
or
cells of the animals. A transgenic founder animal can then be used to breed
additional
animals carrying the transgene. Moreover, transgenic animals carrying a
transgene-
encoding NOVX protein can further be bred to other transgenic animals carrying
other
transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at
least a portion of a 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 any one of SEQ ID N0:2~-1, wherein n is
an
integer between I-73), 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 N0:2~-1,
wherein h is an integer between 1-73, can be used to construct a homologous
recombination vector suitable for altering an endogenous NOVX gene in the
mouse
genome. In one embodiment, the vector is designed such that, upon homologous
recombination, the endogenous NOVX gene is functionally disrupted (i. e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination, the endogenous NOVX gene is mutated or otherwise altered but
still
encodes functional protein (e.g., the upstream regulatory region can be
altered to thereby
alter the expression of the endogenous NOVX protein). In the homologous
recombination
vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-
termini by
additional nucleic acid of the NOVX gene to allow for homologous recombination
to occur
between the exogenous NOVX gene carried by the vector and an endogenous NOVX
gene
in an embryonic stem cell. The additional flanking NOVX nucleic acid is of
suffcient
length for successful homologous recombination with the endogenous gene.
Typically,
several kilobases of flanking DNA (both at the S'- and 3'-termini) are
included in the
vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of
homologous
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recombination vectors. The vector is ten introduced into'
aii°'~rlibryo'3ii'c"stein cell°"line ~e.g.,
by electroporation) and cells in which the introduced NOVX gene has
homologously-
recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al.,
1992. Cell
69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to
form aggregation chimeras. See, e. g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152. A chimeric embryo can then be implanted into a suitable
pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the
homologously-
recombined DNA in their germ cells can be used to breed animals in which all
cells of the
animal contain the homologously-recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination vectors and
homologous
recombinant animals are described further in Bradley, 1991. Curr. Opin.
Biotechnol. 2:
823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO
92/0968;
and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain selected systems that allow for regulated expression of the transgene.
One example
of such a system is the cre/loxP recombinase system of bacteriophage P1. For a
description
of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc.
Natl. Acad. Sci.
LISA 89: 6232-6236. Another example of a recombinase system is the FLP
recombinase
system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science
251:1351-1355.
If a cre/loxP recombinase system is used to regulate expression of the
transgene, animals
containing transgenes encoding both the Cre recombinase and a selected protein
are
required. Such animals can be provided through the construction of "double"
transgenic
animals, e.g., by mating two transgenic animals, one containing a transgene
encoding a
selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in 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
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animal. The offspring borne of this female foster animal"will lie ~a crone of
the aroma! from
which the cell (e. g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies
(also referred to herein as "active compounds") of the invention, and
derivatives, fragments,
analogs and homologs thereof, can be incorporated into pharmaceutical
compositions
suitable for administration. Such compositions typically comprise the nucleic
acid
molecule, protein, or antibody and a pharmaceutically acceptable carrier. As
used herein,
"pharmaceutically acceptable 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,
forger's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous
vehicles
such as fixed oils may also be used. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active compound, use
thereof in the
compositions is contemplated. Supplementary active compounds can also be
incorporated
into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with
its intended route of administration. Examples of routes of administration
include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation), transdermal
(i.e., topical), transmucosal, and rectal administration. Solutions or
suspensions used for
parenteral, intradermal, or subcutaneous application can include the following
components:
a sterile diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents; antibacterial agents
such as beaizyl
alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate;
chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such
as acetates,
citrates or phosphates, and agents for the adjustment of tonicity such as
sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampoules, disposable
syringes or
multiple dose vials made of glass or plastic.
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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 ELT" (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
(fox example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and
suitable
mixtures thereof. The proper fluidity can be maintained, for example, by the
use of a
coating such as lecithin, by the maintenance of the required particle size in
the case of
dispersion and by the use of surfactants. Prevention of the action of
microorganisms can be
achieved by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases,
it will be
preferable to include isotonic agents, for example, sugars, polyalcohols such
as manitol,
sorbitol, sodium chloride in the composition. Prolonged absorption of the
injectable
compositions can be brought about by including in the composition an agent
which delays
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound
(e.g., aNOVX protein or anti-NOVX antibody) in the required annount in an
appropriate
solvent with one or a combination of ingredients enumerated above, as
required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active
compound into a sterile vehicle that contains a basic dispersion medium and
the required
other ingredients from those enumerated above. In the case of sterile powders
for the
preparation of sterile injectable solutions, methods of preparation are vacuum
drying and
freeze-drying that yields a powder of the active ingredient plus any
additional desired
ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent ox 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
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WO 02/099062 PCT/US02/17559
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.
For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant,
e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic
acid' derivatives. Transmucosal administration can be accomplished through the
use of
nasal sprays or suppositories. For transdermal administration, the active
compounds are
formulated into ointments, salves, gels, or creams as generally known in the
art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention
enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will
protect the compound against rapid elimination from the body, such as a
controlled release
formulation, 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. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as used
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WO 02/099062 PCT/US02/17559
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.
The nucleic acid molecules of the invention can be inserted into vectors and
used as
gene therapy vectors. Gene therapy vectors can be delivered to a subject by,
for example,
intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by
stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci.
USA 91:
3054-3057). The pharmaceutical preparation of the gene therapy vector can
include the
gene therapy vector in an acceptable diluent, or can comprise a slow release
matrix in
which the gene delivery vehicle is imbedded. Alternatively, where the complete
gene
delivery vector can be produced intact from recombinant cells, e.g.,
retroviral vectors, the
pharmaceutical preparation can include one or more cells that produce the gene
delivery
system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
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 a
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
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and modulate NOVX activity. In yet a further aspect, the invention can be used
in methods
to influence appetite, absorption of nutrients and the disposition of
metabolic substrates in
both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening
assays
described herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a "screening
assay") for
identifying modulators, i.e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or
have a
stimulatory or inhibitory effect on, e.g., NOVX protein expression orNOVX
protein
activity. The invention also includes compounds identified in the screening
assays
described herein.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which bind to or modulate the activity of the membrane-bound form of
a
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.
A~tica~zcer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about 5 kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules. Libraries of
chemical and/or
biological mixtures, such as fungal, bacterial, or algal extracts, are known
in the art and can
be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in
the
art, for example in: DeWitt, et al., 1993. Pi°oc. Natl. Acad. Sci.
U.S.A. 90: 6909; Erb, et al.,
1994. Proc. Natl. Acad. Sci. U.S.A. 9I: 11422; Zuckermann, et al., 1994. J.
Med. Chem. 37:
2678; Cho, et al., 1993. Seience 261: 1303; Carrell, et al., 1994. Angew.
Clzenz. Int. Ed.
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Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061;
and Gallop, et
al., 1994. J. Med. Chern. 37: 1233.
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. Patent No. 5,223,409),
spores (Ladner,
U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. P~oc. 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.SA. 87:
6378-6382;
Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a
membrane-bound form of NOVX protein, or a biologically-active portion thereof,
on the
cell surface is contacted with a test compound and the ability of the test
compound to bind
to a 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 1251, 3sS, iaC, or
3H, either
directly or indirectly, and the radioisotope detected by direct counting of
radioemission or
by scintillation counting. Alternatively, test compounds can be enzymatically-
labeled with,
for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic
label detected by determination of conversion of an appropriate substrate to
product. In
one embodiment, the assay comprises contacting a cell which expresses a
membrane-bound
form of NOVX protein, or a biologically-active portion thereof, on the cell
surface with a
known compound which binds NOVX to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the test compound
to interact
with a NOVX protein, wherein determining the ability of the test compound to
interact with
a NOVX protein comprises determining the ability of the test compound to
preferentially
bind to NOVX protein or a biologically-active portion thereof as compared to
the known
compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of NOVX protein, or a biologically-active
portion
thereof, on the cell surface with a test compound and determining the ability
of the test
compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX
protein or
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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 a NOVX target molecule. As used herein, a "target molecule" is a
molecule
with which a NOVX protein binds or interacts in nature, for example, a
molecule on the
suxface of a cell which expresses a 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. A NOVX target molecule
can be a
non-NOVX molecule or a NOVX protein or polypeptide of the invention. In one
embodiment, a NOVX target molecule is a component of a signal transduction
pathway
that facilitates transduction of an extracellular signal (e.g. a signal
generated by binding of
a compound to a membrane-bound NOVX molecule) through the cell membrane and
into
the cell. The target, for example, can be a second intercellular protein that
has catalytic
activity or a protein that facilitates the association of downstream signaling
molecules with
NOVX.
Determining the ability of the NOVX protein to bind to or interact with a 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 a NOVX target molecule can be accomplished
by
determining the activity of the target molecule. For example, the activity of
the target
molecule can be determined by detecting induction of a cellular second
messenger of the
target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting
catalytic/enzymatic
activity of the target an appropriate substrate, detecting the induction of a
reporter gene
(comprising a NOVX-responsive regulatory element operatively linked to a
nucleic acid
encoding a detectable marker, e.g., luciferase), or detecting a cellular
response, for
example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay
comprising
contacting a 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
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with a NOVX protein, wherein determining the ability of the test compound to
interact with
a 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.
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 a 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 a
NOVX target molecule. For example, the catalytic/enzymatic activity of the
target
molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the 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 a NOVX protein,
wherein
determining the ability of the test compound to interact with a NOVX protein
comprises
determining the ability of the NOVX protein to preferentially bind to or
modulate the
activity of a NOVX target molecule.
The cell-free assays of the invention axe 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° X-100, Triton~ X-114,
Thesit°,
Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-
propane
sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it
may
be desirable to immobilize either NOVX protein or its target molecule to
facilitate
separation of complexed from uncomplexed forms of one or both of the proteins,
as well as
CA 02447935 2003-11-25
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to accommodate automation of the assay. Binding of a test' coiizpound'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
microtiter
plates, that are then combined with the test compound or the test compound and
either the
non-adsorbed target protein or NOVX protein, and the mixture is incubated
under
conditions conducive to complex formation (e.g., at physiological conditions
for salt and
pH). Following incubation, the beads or microtiter plate wells are washed to
remove any
unbound components, the matrix immobilized in the case of beads, complex
determined
either directly or indirectly, for example, as described, supra.
Alternatively, the complexes
can be dissociated from the matrix, and the level of NOVX protein binding or
activity
determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example, either the NOVX protein or its
target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g.,
biotinylation
kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated
96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX
protein or
target molecules, but which do not interfere with binding of the NOVX protein
to its 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.
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
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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.
In yet another aspect of the invention, the NOVX proteins can be used as "bait
proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent
No. 5,283,3I7;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268:
12046-12054; Bartel, et al., 1993. Bioteehnigues 14: 920-924; Iwabuchi, et
al., 1993.
O~cogeue 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that
bind to or
interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX
activity. Such NOVX-binding proteins are also involved in the propagation of
signals by
the NOVX proteins as, for example, upstream or downstream elements of the NOVX
pathway.
The two-hybrid system is based on the modular nature of most transcription
factors,
which consist of separable DNA-binding and activation domains. Briefly, the
assay utilizes
two different DNA constructs. In one construct, the gene that codes for NOVX
is fused to a
gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-4). In
the other construct, a DNA sequence, from a library of DNA sequences, that
encodes an
unidentified protein ("prey" or "sample") is fused to a gene that codes for
the activation
domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to
interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and
activation
domains of the transcription factor are brought into close proximity. This
proximity allows
transcription of a reporter gene (e.g., LacZ) that is operably linked to a
transcriptional
regulatory site responsive to the transcription factor. Expression of the
reporter gene can
be detected and cell colonies containing the functional transcription factor
can be isolated
and used to obtain the cloned gene that encodes the protein which interacts
with NOVX.
The invention further pertains to novel agents identified by the
aforementioned
screening assays and uses thereof for treatments as described herein.
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Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding complete gene sequences) can be used in numerous ways as
polynucleotide
reagents. By way of example, and not of limitation, these sequences can be
used to: (i)
map their respective genes on a chromosome; and, thus, locate gene regions
associated with
genetic disease; (ii) identify an individual from a minute biological sample
(tissue typing);
and (iii) aid in forensic identification of a biological sample. Some of these
applications
are described in the subsections, below.
Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated,
this
sequence can be used to map the location of the gene on a chromosome. This
process is
called chromosome mapping. Accordingly, portions or fragments of the NOVX
sequences
of SEQ ID NO:2n-l, wherein n is an integer between 1-73, or fragments or
derivatives
thereof, can be used to map the location of the NOVX genes, respectively, on a
chromosome. The mapping of the NOVX sequences to chromosomes is an important
first
step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the NOVX sequences. Computer analysis of
the
NOVX, sequences can be used to rapidly select primers that do not span more
than one
exon in the genomic DNA, thus complicating the amplification process. These
primers can
then be used for PCR screening of somatic cell hybrids containing individual
human
chromosomes. Only those hybrids containing the human gene corresponding to the
NOVX
sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different
mammals
(e.g., human and mouse cells). As hybrids of human and mouse cells grow and
divide, they
gradually lose human chromosomes in random order, but retain the mouse
chromosomes.
By using media in which mouse cells cannot grow, because they lack a
particular enzyme,
but in which human cells can, the one human chromosome that contains the gene
encoding
the needed enzyme will be retained. By using various media, panels of hybrid
cell lines
can be established. Each cell line in a panel contains either a single human
chromosome or
a small number of human chromosomes, and a full set of mouse chromosomes,
allowing
easy mapping of individual genes to specific human chromosomes. See, e.g.,
D'Eustachio,
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et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only
fragments of
human chromosomes can also be produced by using human chromosomes with
translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
sequence to a particular chromosome. Three or more sequences can be assigned
per day
using a single thermal cycler. Using the NOVX sequences to design
oligonucleotide
. primers, sub-localization can be achieved with panels of fragments from
specific
chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise chromosomal
location in one
step. Chromosome spreads can be made using cells whose division has been
blocked in
metaphase by a chemical like colcemid that disrupts the mitotic spindle. The
chromosomes
can be treated briefly with trypsin, and then stained with Giemsa. A pattern
of light and
dark bands develops on each chromosome, so that the chromosomes can be
identified
individually. The FISH technique can be used with a DNA sequence as short as
500 or 600
bases. However, clones larger than 1,000 bases have a higher likelihood of
binding to a
unique chromosomal location with sufficient signal intensity for simple
detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get
good results at
a reasonable amount of time. For a review of this technique, see, Verma, et
al., HUMAN
CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding regions of the genes actually are preferred for mapping purposes.
Coding
sequences are more likely to be conserved within gene families, thus
increasing the chance
of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such
data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-
line
through Johns Hopkins University Welch Medical Library). The relationship
between
genes and disease, mapped to the same chromosomal region, can then be
identified through
linkage analysis (co-inheritance of physically adjacent genes), described in,
e.g., Egeland,
et al., 1987. Nature, 325: 783-787.
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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
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 poIymorphisrns," described in U.S. Patent
No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an
alternative
technique that determines the actual base-by-base DNA sequence of selected
portions of an
individual's genome. Thus, the NOVX sequences described herein can be used to
prepare
two PCR primers from the 5'- and 3'-termini of the sequences. These primers
can then be
used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner,
can provide unique individual identifications, as each individual will have a
unique set of
such DNA sequences due to allelic differences. The sequences of the invention
can'be used
to obtain such identification sequences from individuals and from tissue. The
NOVX
sequences of the invention uniquely represent portions of the human genome.
Allelic
variation occurs to some degree in the coding regions of these sequences, and
to a greater
degree in the noncoding regions. It is estimated that allelic variation
between individual
humans occurs with a frequency of about once per each 500 bases. Much of the
allelic
variation is due to single nucleotide polyrnorphisms (SNPs), which include
restriction
fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes.
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Because greater numbers of polymorphisms occur in the noncocling 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 coding
sequences,
such as those of SEQ ID N0:2tZ-l, wherein n is an integer between 1-73, are
used, a more
appropriate number of primers for positive individual identification would be
500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which
diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring clinical trials
are used for
prognostic (predictive) purposes to thereby treat an individual
praphylactically.
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 fox determining whether an individual is at
risk of
developing a disorder associated with NOVX protein, nucleic acid expression or
activity.
For example, mutations in a NOVX gene can be assayed in a biological sample.
Such
assays can be used for prognostic or predictive purpose to thereby
prophylactically treat an
individual prior to the onset of a disorder characterized by or associated
with NOVX
protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein,
nucleic acid expression or activity in an individual to thereby select
appropriate therapeutic
or prophylactic agents for that individual (referred to herein as
"pharmacogenomics").
Pharmacogenomics allows far 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.)
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Yet another aspect of the invention pertains to monitoring the influence of
agents
(e.g., drugs, compounds) on the expression or activity of NOVX in clinical
trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assays
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 N0:2n-l, wherein ~ is an
integer between
1-73, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50,
100, 250 or 500
nucleotides in length and sufficient to specifically hybridize under stringent
conditions to
NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic
assays of
the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be
polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,
Fab or
F(ab')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 ivy vitro as
well as
i~ vivo. For example, i~ vitro techniques for detection of NOVX mRNA include
Northern
hybridizations and in situ hybridizations. Ih vitro techniques for detection
of NOVX
protein include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence. l~ vitro techniques for
detection of
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NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques
for detection of NOVX protein include introducing into a subject a labeled
anti NOVX
antibody. For example, the antibody can be labeled with a radioactive marker
whose
presence and location in a subject can be detected by standard imaging
techniques.
In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test
subject or genomic DNA molecules from the test subject. A preferred biological
sample is
a peripheral blood leukocyte sample isolated by conventional means from a
subject.
In another embodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
capable of detecting 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.
The invention also encompasses kits for detecting the presence of NOVX in a
biological sample. For example, the kit can comprise: a labeled compound or
agent
capable of detecting NOVX protein or mRNA in a biological sample; means for
determining the amount of NOVX in the sample; and means for comparing the
amount of
NOVX in the sample with a standard. The compound or agent can be packaged in a
suitable container. The kit can further comprise instructions for using the
kit to detect
NOVX protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant
NOVX expression or activity. For example, the assays described herein, such as
the
preceding diagnostic assays or the following assays, can be utilized to
identify a subject
having or at risk of developing a disorder associated with NOVX protein,
nucleic acid
expression or activity. Alternatively, the prognostic assays can be utilized
to identify a
subject having or at risk for developing a disease or disorder. Thus, the
invention provides
a method for identifying a disease or disorder associated with aberrant NOVX
expression
or activity in which a test sample is obtained from a subject and NOVX protein
or nucleic
acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX
protein or
nucleic acid is diagnostic for a subject having or at risk of developing a
disease or disorder
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associated with aberrant NOVX expression or activity. As used herein, a "test
sample"
refers to a biological sample obtained from a subject of interest. For
example, a test sample
can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug
candidate) to
treat a disease or disorder associated with aberrant NOVX expression or
activity. For
example, such methods can be used to determine whether a subject can be
effectively
treated with an agent for a disorder. Thus, the invention provides methods for
determining
whether a subject can be effectively treated with an agent for a disorder
associated with
aberrant NOVX expression or activity in which a test sample is obtained and
NOVX
protein or nucleic acid is detected (e.g., wherein the presence of NOVX
protein or nucleic
acid is diagnostic for a subject that can be administered the agent to treat a
disorder
associated with aberrant NOVX expression or activity).
The methods of the invention can also be used to detect genetic lesions in a
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 a 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 a NOVX gene; (ii) an
addition of one
or more nucleotides to a NOVX gene; (iii) a substitution of one or more
nucleotides of a
NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration
in the
level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification
of a
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 a NOVX gene,
(viii) a
non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and
(x)
inappropriate post-translational modification of a 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 a 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.
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In certain embodiments, detection of the lesion involves the use of a
probe/primer in
a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
4,683,202),
such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain
reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et
al., 1994.
Pr~oc. 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 a
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.
Alternative amplification methods include: self sustained sequence replication
(see,
Guatelli, et al., 1990. Pr~oc. Natl. Acaa'. Sci. USA 87: 1874-1878),
transcriptional
amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sei. USA 86:
1173-1177);
Qj3 Replicase (see, Lizardi, et al, 1988. BioTechf2ology 6: 1197), or any
other nucleic acid
amplification method, followed by the detection of the amplified molecules
using
techniques well known to those of skill in the art. These detection schemes
are especially
useful for the detection of nucleic acid molecules if such molecules are
present in very low
numbers.
In an alternative embodiment, mutations in a NOVX gene from a sample cell can
be
identified by alterations in restriction enzyme cleavage patterns. For
example, sample and
control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and
compared. Differences in fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes
(see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence
of specific
mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by
hybridizing
a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays
containing
CA 02447935 2003-11-25
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hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al.,
1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example,
genetic
mutations in NOVX can be identified in two dimensional arrays containing light-
generated
DNA probes as described in Cronin, et al., supra. Briefly, a first
hybridization array of
probes can be used to scan through Long stretches of DNA in a sample and
control to
identify base changes between the sequences by making linear arrays of
sequential
overlapping probes. This step allows the identification of point mutations.
This is
followed by a second hybridization array that allows the characterization of
specific
mutations by using smaller, specialized probe arrays complementary to all
variants or
mutations detected. Each mutation array is composed of parallel probe sets,
one
complementary to the wild-type gene and the other complementary to the mutant
gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the
art'can be used to directly sequence the NOVX gene and detect mutations by
comparing the
sequence of the sample NOVX with the corresponding wild-type (control)
sequence.
Examples of sequencing reactions include those based on techniques developed
by Maxim
and 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. Biotech~iques 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. Biotechhol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In
general,
the art technique of "mismatch cleavage" starts by providing heteroduplexes of
formed by
hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded
duplexes axe 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~ 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
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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., I
992. Methods
Enzynzol. 217: 286-295. In an embodiment, the contxol DNA or RNA can be
labeled for
detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch xepair" enzymes) in defined systems for detecting and mapping point
mutations
in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of
E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa
cells
I O cleaves T at G/T mismatches. See, e.g., Hsu, et al., 1994.
Ca~cirzoge~zesis I5: 1657-I662.
According to an exemplary embodiment, a probe based on a NOVX sequence, e.g.,
a
wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a
test
cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the
cleavage
products, if any, can be detected from electrophoresis protocols or the like.
See, e.g., U.S.
Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify mutations in NOVX genes. For example, single strand conformation
polymorphism (SSCP) may be used 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 hetexoduplex molecules on
the basis of
changes in electrophoretic mobility. See, e.g., Keen, et al., 1991. Trefzds
Gefzet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gxadient of denaturant is assayed using
denaturing
gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature
313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure
that it does
not completely denature, for example by adding a GC clamp of approximately 40
by of
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high-melting GC-rich DNA by PCR. In a further embodirrierit; 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. Cl~e~rz. 265:
12753.
Examples of other techniques fox 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.
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.
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 a NOVX
gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes,
in
which NOVX is expressed may be utilized in the prognostic assays described
herein.
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However, any biological sample containing nucleated cells may be used,
including, for
example, buccaI mucosaI cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity
(e.g., NOVX gene expression), as identified by a screening assay described
herein can be
administered to individuals to treat (prophylactically or therapeutically)
disorders (The
disorders include metabolic disorders, diabetes, obesity, infectious disease,
anorexia,
cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's
Disease,
Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the
various
dyslipidemias, metabolic disturbances associated with obesity, the metabolic
syndrome X
and wasting disorders associated with chronic diseases and various cancers.)
In
conjunction with such treatment, the pharmacogenomics (i. e., the study of the
relationship
between an individual's genotype and that individual's response to a foreign
compound or
drug) of the individual may be considered. Differences in metabolism of
therapeutics can
lead to severe toxicity or therapeutic failure by altering the relation
between dose and blood
concentration of the pharmacologically active drug. Thus, the pharmacogenomics
of the
individual permits the selection of effective agents (e.g., drugs) for
prophylactic or
therapeutic treatments based on a consideration of the individual's genotype.
Such
pharmacogenornics 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 agents) for therapeutic or prophylactic treatment of the
individual.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected persons.
See e.g., Eichelbaum, 1996. Clin. Exp. Phc~rmacol. Physiol., 23: 983-985;
Linden 1997.
Clin. Chem.; 43: 254-266. In general, two types of pharmacogenetic conditions
can be
differentiated. Genetic conditions transmitted as a single factor altering the
way drugs act
on the body (altered drug action) or genetic conditions transmitted as single
factors altering
the way the body acts on drugs (altered drug metabolism). These
pharmacogenetic
conditions can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited
enzymopathy in which the main clinical complication is hemolysis after
ingestion of
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oxidant drugs (anti-malarials, sulfonamides, analgesics, nits~furans) and
consumption of
fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
determinant of both the intensity and duration of drug action. The discovery
of genetic
polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and
cytochrome pregnancy zone protein precursor enzymes CYP2D6 and CYP2C 19) 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
amplifcation.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or
mutation
content of NOVX genes in an individual can be determined to thereby select
appropriate
agents) for therapeutic or prophylactic treatment of the individual. In
addition,
pharmacogenetic studies can be used to apply genotyping of polymorphic alleles
encoding
drug-metabolizing enzymes to the identification of an individual's drug
responsiveness
phenotype. This knowledge, when applied to dosing or drug selection, can avoid
adverse
reactions or therapeutic failure and thus enhance therapeutic_or prophylactic
efficiency
when treating a subject with a NOVX modulator, such as a modulator identified
by one of
the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity ofNOVX (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
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herein to increase NOVX gene expression, protein levels, or upregulate NOVX
activity,
can be monitored in clinical trails of subjects exhibiting decreased NOVX gene
expression,
protein levels, or downregulated NOVX activity. Alternatively, the
effectiveness of an
agent determined by a screening assay to decrease NOVX gene expression,
protein levels,
or downregulate NOVX activity, can be monitored in clinical trails of subjects
exhibiting
increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such
clinical trials, the expression or activity of NOVX and, preferably, other
genes that have
been implicated in, for example, a cellular proliferation or immune disorder
can be used as
a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule)
that modulates NOVX activity (e.g., identified in a screening assay as
described herein) can
be identified. Thus, to study the effect of agents on cellular proliferation
disorders, for
example, in a clinical trial, cells can be isolated and RNA prepared and
analyzed for the
levels of expression of NOVX and other genes implicated in the disorder. The
levels of
gene expression (i.e., a gene expression pattern) can be quantified by
Northern blot analysis
or RT-PCR, as described herein, or alternatively by measuring the amount of
protein
produced, by one of the methods as described herein, or by measuring the
levels of activity
of NOVX or other genes. In this manner, the gene expression pattern can serve
as a
marker, indicative of the physiological response of the cells to the agent.
Accordingly, this
response state may be determined before, and at various points during,
treatment of the
individual with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness of treatment of a subject with an agent (e.g., an agonist,
antagonist, protein,
peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified by
the screening assays described herein) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of the agent;
(ii) detecting
the level of expression of a 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
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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.
Methods of Treatment
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; adenocarcinorna, lymphoma, uterus cancer, fertility,
hemophilia,
hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies,
graft versus
host disease, AIDS, bronchial astluna, Crohn's disease; multiple sclerosis,
treatment of
Albright Hereditary Ostoeodystrophy, and other diseases, disorders and
conditions of the
like.
These methods of treatment will be discussed more fully, below.
Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity 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 axe "dysfunctional" (i. e., due to a
heterologous insertion
within the coding sequences of coding sequences to an aforementioned peptide)
that axe
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
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or antibodies specific to a peptide of the invention) that alter the
interaction between an
aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a
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.
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).
Prophylactic Methods
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 abenrancy, for example, a 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
Another aspect of the invention pertains to methods of modulating NOVX
expression or activity for therapeutic purposes. The modulatory method of the
invention
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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 a NOVX protein, a peptide, a 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 a 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 a NOVX protein or nucleic acid
molecule
as therapy to compensate for reduced or aberrant NOVX expression or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is
abnormally downregulated andlor in which increased NOVX activity has 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 subj ect has a
gestational
disease (e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or irc vivo assays
are
performed to determine the effect of a specific Therapeutic and whether its
administration
is indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with
representative cells of the types) involved in the patient's disorder, to
determine if a given
Therapeutic exerts the desired effect upon the cell type(s). Compounds for use
in therapy
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,
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for in vivo testing, any of the animal model system known in the art may be
used prior to
administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
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.
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.
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.
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EXAMPLES
Example A: Polynucleotide and Polypeptide Sequences, and Homology Data
Example 1.
The NOV 1 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table I A.
Table 1A. NOVl Sequence Analysis
SEQ ID NO: 1 3430 by
NOVla, ,GGGCTGCAGGAATTCCCCCACAGAGGGAGCATGACTTCGGCAACTTCACCTATCATTC
CG100653-Ol TGAAATGGGACCCCAAAAGTTTGGAAATCCGGACGCTAACAGTGGAAAGGCTGTTGGA
DNA Se 118nC~ GCCACTTGTTACACAGGTGACTACACTTGTCAACACAAGCAACAAAGGCCCATCTGGT
AAAAAGAAAGGGAGGTCAAAGAAAGCCCATGTACTAGCTGCCTCTGTAGAGCAAGCCA
CTCAGAATTTCCTGGAAAAGGGTGAACAGATCGCTAAGGAGAGTCAAGATCTCAAAGA
AGAGTTGGTGGCTGCTGTAGAGGATGTGCGCAAACAAGGTGAGACGATGCGGATCGCC
CGGCAAGGGCTTTGCTCTCCGCGGTGACACGCTTACTCATCCTGGCGGACATGGCAGA
TGTCATGAGACTTTTATCCCATCTGAAAATTGTGGAAGAGGCCCTGGAAGCTGTCAAA
AATGCTACAAATGAGCAAGACCTTGCAAACCGTTTTAAAGAGTTTGGGAAAAAGATGG
GGATGAGATGGCAGCCGCCCGAGGGGCTCTGAAGAAGAATGCCACAATGCTGTACACG
GCCTCTCAAGCATTTCTCCGCCACCCAGATGTCGCCGCTACGAGAGCCAACCGAGATT
ATGTGTTCAAACAAGTCCAGGAGGCCATCGCCGGCATCTCCAATGCTGCTCAAGCTAC
CTCGCCCACTGACGAAGCCAAGGGCCACACGGGCATCGGCGAGCTGGCTGCGGCTCTT
AATGAGTTTGACAATAAGATTATCCTGGACCCCATGACGTTCAGCGAGGCCAGGTTCC
GGCCGTCCCTGGAGGAGAGGCTGGAGAGCATCATCAGCGGCGCAGCGCTGATGGCCGA
CTCCTCCTGCACGCGAGACGACCGGCGCGAGAGGATCGTGGCGGAGTGCAACGCCGTG
CGGCAGGCGCTCCAGGACCTGCTCAGCGAGTACATGAATAATACTGGAAGGAAAGAAA
AAGGAGATCCTCTCAACATTGCGATTGATAAGATGACTAAGAAAACAAGAGATCTAAG
GAGACAGCTTCGGAAAGCAGTGATGGATCACATATCTGACTCTTTCCTGGAAACCAAT
GTTCCTTTGCTAGTTCTCATTGAGGCTGCAAAGAGCGGAAATGAAAAGGAAGTGAAAG
AATATGCCCAAGTTTTCCGTGAGCATGCCAACAAACTGGTAGAGGTTGCCAATTTGGC
CTGTTCCATCTCCAACAATGAAGAAGGGGTGAAATTAGTTCGGATGGCAGCCACCCAG
ATTGACAGCCTGTGTCCCCAGGTCATCAATGCCGCTCTGACACTGGCTGCCCGGCCAC
. AGAGCAAAGTTGCTCAGGATAACATGGACGTCTTCAAAGACCAGTGGGAGAAGCAGGT
CCGAGTGTTGACAGAGGCCGTGGATGACATCACCTCAGTGGATGACTTCCTCTCTGTC
TCAGAAAATCACATCTTGGAGGATGTGAACAAGTGTGTGATAGCCCTCCAAGAGGGCG
ATGTGGACACTCTGGACCGGACTGCAGGGGCCATCAGGGGCCGGGCAGCTCGAGTCAT
ACACATCATCAATGCTGAGATGGAGAACTATGAAGCTGGGGTTTATACTGAGAAGGTG
~TTGGAAGCTACAAAATTGCTTTCTGAAACAGTGATGCCACGCTTCGCTGAACAAGTAG
CATCGATGCCTCTCGCCTGGTGTATGATGGCGTTCGGGACATCAGAAAGGCTGTGCTG
ATGATCAGGACCCCAGAAGAACTAGAGGATGATTCTGACTTTGAGCAGGAAGATTATG
ATGTGCGTAGAGGGACAAGTGTTCAGACTGAGGATGACCAGCTCATTGCAGGGCAGAG
CGCACGGGCCATCATGGCGCAACTACCGCAGGAGGAGAAGGCAAAAATAGCTGAGCAG
GTGGAGATATTCCATCAAGAGAAAAGCAAGCTGGATGCAGAAGTGGCCAAATGGGACG
ATCATTGTACTGGCCAAGCAGATGTGTATGATCATGATGGAAAT
GACAGACTTCACAAGAGGCAAAGGCCCATTGAAAAATACATCTGATGTCATTAATGCT
GCCAAGAAAATTGCCGAAGCAGGTTCTCGAATGGACAAATTAGCTCGTGCTGTGGCTG
ATCAGCTGGACAGTGCCACATCGCTTATCCAGGCAGCTAAAAACCTGATGAATGCTGT
TGTCCTCACGGTGAAAGCATCCTATGTGGCCTCAACCAAATACCAGAAGGTCTATGGG
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CCCTTGTGAAGAGAGAAA.AGCCTGAAGAATTCCAGACACGAGTTCGACGAGGTTCTCA
GAAGAAACACATTTCGCCTGTACAGGCTTTAAGTGAATTCAAAGCAATGGATTCCTTC
TAGGACGATAGGTTTTAACAAGAAAGCTTTTTCTTTCTTTTCTTTCTTTCTTTTTCTT
TTTAATTCCATTTTTGTATGCATACCTGCCAGCTCGTATGCCTCTGGCATGGGGAAAT
TAAGGGAACAGTGTCTGTTTGCATGTAAGATGAGATGAGATCAATACTACTGATCCAT
CTGTAGCCTGGGAAGGAGACAGGACATTCCTGTACTAAGGTGGCACAGAGCTGTCCTT
TGCAACATTCTCATAAA.ATTGGGCACAGAGTTCGCATTGGCGCAATATTTATGGGAGT
GGGAGGGATGGGGAAAATAAACTTAACTCTACAAA.AGCAAACTCTAATGCATGCAAGA
ATCATTAGGTTGGCAGGTATATGCATAAGTGAAAAATCTGGAAGTGTAATGGTAGAAC
ATAAAACTTGTATTGCTTCTGTTTCAGTGCAAAAATGTACTAGCCAATACGCTTAAGT
GTGTGGCCCATGAATTGAACAATTTAACCTTGAAGTCTATATCCGTGATATTATGTCG
ATTTTTAACTGAGGGGAAATTAACTAGTCCAGCCTAAAATGCTTCTTTTAATCTGCAT
TCTGTTTCCTCTTCTAGTTGTGCCATTACTAGTGATCATGTTTTTTTCCCCCCTTTAA
TGAAA.ACAATAAACATCTATTTGAGACAATTAAAATCCTTCTGGGGGCACTGGAAGCA
CAATACGGTGACCAATCTTGCTTTCATTTTTTTTTCTTTTTAATTTGAACCATGATTT
TGCTAGAAATAGAAGGCCCAGTGGTGGAATATTAGAGGGAAGGAAACTGACAACGTGT
GAAAGTTA
ORF Start: ATG at 31 ORF Stop: TAG at 2611
SEQ ID NO: 2 860 as MW at 9SS2S.9kD
NOVIa, MTSATSPIILKWDPKSLEIRTLTVERLLEPLVTQVTTLVNTSNKGPSGKKKGRSKKAH
CGIOO6S3-OI V~SVEQATQNFLEKGEQIAKESQDLKEELVAAVEDVRKQGETMRIASSEFADDPCS
Protein SequenceSVKRGTMVRAARALLSAVTRLLILADMADVMRLLSHLKIVEEALEAVKNATNEQDLAN
RFKEFGKKMVKLNYVAARRQQELKDPHCRDEMAAARGALKKNATMLYTASQAFLRHPD
VAATRANRDYVFKQVQEAIAGISNAAQATSPTDEAKGHTGIGELAAALNEFDNKIILD
PMTFSEARFRPSLEERLESIISGAALMADSSCTRDDRRERIVAECNAVRQALQDLLSE
YMNNTGRKEKGDPLNIAIDKMTKKTRDLRRQLRKAVMDHISDSFLETNVPLLVLIEAA
KSGNEKEVKEYAQVFREHANKLVEVANLACSISNNEEGVKLVRMAATQIDSLCPQVIN
AALTLAARPQSKVAQDNMDVFKDQWEKQVRVLTEAVDDITSVDDFLSVSENHILEDVN
KCVIALQEGDVDTLDRTAGAIRGRAARVIHIINAEMENYEAGVYTEKVLEATKLLSET
VMPRFAEQVEVAIEALSANVPQPFEENEFIDASRLVYDGVRDIRKAVLMIRTPEELED
DSDFEQEDYDVRRGTSVQTEDDQLIAGQSARAIMAQLPQEEKAKIAEQVEIFHQEKSK
LDAEVAKWDDSGNDIIVLAKQMCMIMMEMTDFTRGKGPLKNTSDVINAAKKIAEAGSR
MDKLARAVADQLDSATSLIQAAKNLMNAVVLTVKASYVASTKYQKVYGTAAVNSPVVS
WKMKAPEKKPLVKREKPEEFQTRVRRGSQKKHISPVQALSEFKAMDSF
Further analysis of the NOV 1 a protein yielded the following properties shown
in Table 1 B.
Table 1B. Protein Sequence Properties NOVla
PSort 0.3600 probability located in mitochondrial matrix space; 0.3000
probability
analysis: located in microbody (peroxisome); 0.1000 probability located in
lysosome
(lumen); 0.0000 probability located in endoplasmic reticulum (membrane)
SignalP No Known Signal Sequence Predicted
analysis:
A search of the NOV 1 a protein against the Geneseq database, a proprietary
database that contains sequences published in patents and patent publication,
yielded
several homologous proteins shown in Table 1 C.
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Table 1C. Geneseq Results for NOVla
NOVla Identities/
Geneseq Protein/Organism/Length [PatentResidues!Similarities Expect
for
Identifier ~ #, Date] Match the Matched Value
ResiduesRegion
AAR58778 ~ Neural alpha-catenin protein1..860 8511906 (93%)0.0
-
Homo Sapiens, 906 aa. 1..906 855/906 (93%)
[JP06211898-A, 02-AUG-1994]
T
AAY07060 a Renal cancer associated 8..859 694/899 (77%)0.0
antigen
precursor sequence - Homo Sapiens, 9..905 773/899 (85%)
'~ 906 aa. [W09904265-A2, 28-JAN-
1999]
AAU32945 Novel human secreted protein8..769 611/766 (79%)0.0
#3436 - Homo sapiens, 932 aa. 10..773 683/766 (88%)
[W0200179449-A2, 25-OCT-2001]
ABG10622 i Novel human diagnostic 8..769 610/766 (79%)0.0
protein
#10613 - Homo Sapiens, 932 aa. 10..773 682/766 (88%)
j [WO200175067-A2, 11-OCT-2001]
ABGI 0622 Novel human diagnostic 8..769 610/766 (79%)0.0
protein
#10613 - Homo Sapiens, 932 aa. 10..773 682/766 (88%)
[WO200175067-A2, 11-OCT-2001]
In a BLAST search of public sequence databases, the NOV 1 a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 1D.
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Table 1D. Public BLASTP Results for NOVla
~OVIa
Identities/
Protein f Residues/Similarities' Expect
for
AccessionProtein/Organism/Length Match the Matched Value
Number Residues Portion
P30997 ~ Alpha-2 catenin (Alpha 1..860 8511906 (93%)~ 0.0
N-catenin)
(Neural alpha-catenin) 1..906 855/906 (93%)
- Gallus
'~ gallus (Chicken), 906 .
aa.
I49499 alpha N-catenin I - mouse,~ 1..860 850/905 (93%)0.0
905 aa.
_ .
l ..
505 854/905 (93
/)
A45011 ~ alpha-catenin 2 - human,1..769 768/770 (99%)0.0
945 aa.
1..770 7681770 (99%)
P26232 ' Alpha-2 catenin (Alpha-catenin~ 1..769 768/770 (99%)~ 0.0
related protein) (Alpha 1..770 768/770 (99%)
N-catenin) -
Homo Sapiens (Human), 953
aa.
Q61301 ; Alpha-2 catenin (Alpha-catenin1..769 759/770 (98%)~ 0.0
related protein) (Alpha 1..770 762/770 (98%)
N-catenin) -
Mus musculus (Mouse), 953
aa.
PFam analysis predicts that the NOV 1 a protein contains the domains shown in
the Table
1 E.
Table 1E. Domain Analysis of NOVla
Identities/
Pfam Domain NOVIa Match Re ion : Similarities Ex ect Value
P
for the Matched Region
Vinculin 18..765 424/948 (45%) ~ 0
736/948 (78°l°)
Vinculin 766..821 ~ 32/57 (56%) 5.4e-30
~~~.w,.,.,.__~.~~,....~._~..~ ~, . 56/57 (98%) ~~_~
Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 2A.
Table 2A. NOV2 Seguence Analysis
SEQ ID NO: 3 2883 by
NOV2a, CGTGAATGGTGTAGTGAGTTCTAATGAAACTTTATTTACAAGAGGAGACTGACCAGGT~
CG100689-O1 ~Z'TGGCCTGGGGGCCACAGTGTGTAGACCCCTGGAAAGATACATCCTGAGAAGAAAAAA
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DNA SeCjLteriCe AGAATATATGCAGGAATGCTTAACTTTGTGGGTTTTCTCTCCTCTTGCCCTCACTGAC
TCAGGATACACAAAGACCTATCAAGCTCACGCAAAGCAGAAATTCAGCCGCTTATGGT
CCAGCAAGTCTGTCACTGAGATTCACCTATACTTTGAGGAGGAAGTCAAGCAAGAAGA
ATGTGACCATTTGGACCGCCTTTTTGCTCCCAAGGAAGCTGGGAAACAGCCACGTACA
GTGATCATTCAAGGACCACAAGGAATTGGAAAAACGACACTCCTGATGAAGCTGATGA
TGGCCTGGTCGGACAACAAGATCTTTCGGGATAGGTTCCTGTACACGTTCTATTTCTG
CTGCAGAGAACTGAGGGAGTTGCCGCCAACGAGTTTGGCTGACTTGATTTCCAGAGAG
TGGCCTGACCCCGCTGCTCCTATAACAGAGATCGTGTCTCAACCGGAGAGACTCTTGT
TCGTCATCGACAGCTTCGAAGAGCTGCAGGGCGGCTTGAACGAACCCGATTCGGATCT
GTGTGGTGACTTGATGGAGAAACGGCCGGTGCAGGTGCTTCTGAGCAGTTTGCTGAGG
AAGAAGATGCTCCCGGAGGCCTCCCTGCTCATCGCTATCAAACCCGTGTGCCCGAAGG
AGCTCCGGGATCAGGTGACGATCTCAGAAATCTACCAGCCCCGGGGATTCAACGAGAG
TGATAGGTTAGTGTATTTCTGCTGTTTCTTCAAAGACCCGAAAAGAGCCATGGAAGCC
TTCAATCTTGTAAGAGAAAGTGAACAGCTGTTTTCCATATGCCAAATCCCGCTCCTCT
GCTGGATCCTGTGTACCAGTCTGAAGCAAGAGATGCAGAAAGGAAAAGACCTGGCCCT
GACCTGCCAGAGCACTACCTCTGTGTACTCCTCTTTCGTCTTTAACCTGTTCACACCT
GAGGGTGCCGAGGGCCCGACTCCGCAAACCCAGCACCAGCTGAAGGCCCTGTGCTCCC
TGGCTGCAGAGGGTATGTGGACAGACACATTTGAGTTTTGTGAAGACGACCTCCGGAG
AAATGGGGTTGTTGACGCTGACATCCCTGCGCTGCTGGGCACCAAGATACTTCTGAAG
TACGGGGAGCGTGAGAGCTCCTACGTGTTCCTCCACGTGTGTATCCAGGAGTTCTGTG
CCGCCTTGTTCTATTTGCTCAAGAGCCACCTTGATCATCCTCACCCAGCTGTGAGATG
TGTACAGGAATTGCTAGTTGCCAATTTTGAAAAAGCAAGGAGAGCACATTGGATTTTT
TTGGGGTGTTTTCTAACTGGCCTTTTAAATAAAAAGGAACAAGAAAAACTGGATGCGT
TTTTTGGCTTCCAACTGTCCCAAGAGATAAAGCAGCAAATTCACCAGTGCCTGAAGAG
CTTAGGGGAGCGTGGCAATCCTCAGGGACAGGTGGATTCCTTGGCGATATTTTACTGT
CTCTTTGAAATGCAGGATCCTGCCTTTGTGAAGCAGGCAGTGAACCTCCTCCAAGAAG
CTAACTTTCATATTATTGACAACGTGGACTTGGTGGTTTCTGCCTACTGCTTAAAATA
CTGCTCCAGCTTGAGGAAACTCTGTTTTTCCGTTCAAAATGTCTTTAAGAAAGAGGAT
GAACACAGCTCTACGTCGGATTACAGCCTCATCTGTTGGCATCACATCTGCTCTGTGC
TCACCACCAGCGGGCACCTCAGAGAGCTCCAGGTGCAGGACAGCACCCTCAGCGAGTC
GACCTTTGTGACCTGGTGTAACCAGCTGAGGCATCCCAGCTGTCGCCTTCAGAAGCTT
GGAATAAATAACGTTTCCTTTTCTGGCCAGAGTGTTCTGCTCTTTGAGGTGCTCTTTT
CAGGTCCCTCTGTGATGCCTTGAACTACCCAGCAGGCAACGTCAAAGAGCTAGCGCTG
GTAAATTGTCACCTCTCACCCATTGATTGTGAAGTCCTTGCTGGCCTTCTAACCAACA
ACAAGAAGCTGACGTATCTGAATGTATCCTGCAACCAGTTAGACACAGGCGTGCCCCT
TTTGTGTGAAGCCCTGTGCAGCCCAGACACGGTCCTGGTATACCTGATGTTGGCTTTC
TGCCACCTCAGCGAGCAGTGCTGCGAATACATCTCTGAAATGCTTCTGCGTAACAAGA
GCGTGCGCTATCTAGACCTCAGTGCCAATGTCCTGAAGGACGAAGGACTGAAAACTCT
CTGCGAGGCCTTGAAACATCCGGACTGCTGCCTGGATTCACTGTGTTTGGTAAAATGT
TTTATCACTGCTGCTGGCTGTGAAGACCTCGCCTCTGCTCTCATCAGCAATCAAAACC
TGAAGATTCTGCAAATTGGGTGCAATGAAATCGGAGATGTGGGTGTGCAGCTGTTGTG
TCGGGCTCTGACGCATACGGATTGCCGCTTAGAGATTCTTGGGTTGGAAGAATGTGGG
TTAACGAGCACCTGCTGTAAGGATCTCGCGTCTGTTCTCACCTGCAGTAAGACCCTGC
AGCAGCTCAACCTGACCTTGAACACCTTGGACCACACAGGGGTGGTTGTACTCTGTGA
GGCCCTGAGACACCCAGAGTGTGCCCTGCAGGTGCTCGGGCTGAGAAAAACT'GATTTT
GATGAGGAAACCCAGGCACTTCTGACGGCTGAGGAAGAGAGAAATCCTAACCTGACCA
TCACAGACGACTGTGACACAATCACAAGGGTAGAGATCTGA
ORF Start ATG at 124 ORF Stop: TGA at 2881
~ 919 as _ . . .. _.."~,:
SE ID NO. 4 MW at 103966.7kD
NOV2a, MQECLTLWVFSPLALTDSGYTKTYQAHAKQKFSRLWSSKSVTEIHLYFEEEVKQEECD
CG100689-01 HLDRLFAPKEAGKQPRTVIIQGPQGIGKTTLLMKLMMAWSDNKIFRDRFLYTFYFCCR
Protein SeCjlleriCe ELRELPPTSLADLISREWPDPAAPITEIVSQPERLLFVIDSFEELQGGLNEPDSDLCG
DLMEKRPVQVLLSSLLRKKMLPEASLLIAIKPVCPKELRDQVTISEIYQPRGFNESDR
LVYFCCFFKDPKRAMEAFNLVRESEQLFSICQIPLLCWILCTSLKQEMQKGKDLALTC
QSTTSVYSSFVFNLFTPEGAEGPTPQTQHQLKALCSLAAEGMWTDTFEFCEDDLRRNG
VVDAI7IPALLGTKILLKYGERESSYVFLHVCIQEFCAALFYLLKSHLDHPHPAVRCVQ
ELLVANFEKARRAHWIFLGCFLTGLLNKKEQEKLDAFFGFQLSQEIKQQIHQCLKSLG
ERGNPQGQVDSLAIFYCLFEMQDPAFVKQAVNLLQEANFHIIDNVDLVVSAYCLKXCS
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SLRKLCFSVQNVFKKEDEHSSTSDYSLICWHHICSVLTTSGHLRELQVQDSTLSESTF
VTWCNQLRHPSCRLQKLGINNVSFSGQSVLLFEVLFYQPDLKYLSFTLTKLSRDDIRS
LCDALNYPAGNVKELALVNCHLSPIDCEVLAGLLTNNKKLTYLNVSCNQLDTGVPLLC
EALCSPDTVLVYLMLAFCHLSEQCCEYISEMLLRNKSVRYLDLSANVLKDEGLKTLCE
ALKHPDCCLDSLCLVKCFITAAGCEDLASALISNQNLKILQIGCNEIGDVGVQLLCRA
LTHTDCRLEILGLEECGLTSTCCKDLASVLTCSKTLQQLNLTLNTLDHTGVVVLCEAL
RHPECALQVLGLRKTDFDEETQALLTAEEERNPNLTITDDCDTITRVEI
Further analysis of the NOV2a protein yielded the following properties shown
in Table 2B.
Table 2B. Protein Sequence Properties NOV2a
PSort 0.6000 probability located in nucleus; 0.3000 probability located in
microbody
analysis: (peroxisome); 0.2000 probability located in endoplasmic reticulum
(membrane); 0.1000 probability located in mitochondria! inner membrane
SignalP Cleavage site between residues 17 and 18
analysis.
A search of the NOV2a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 2C.
Table 2C. Geneseq Results for NOV2a
NOVZa Identities/
Geneseq Protein/OrganismlLength Residues!SimilaritiesExpect
[Patent ~ for
Identifier#, Date] Match the lYlatchedValue
Residues Region
AAM50328 Human nucleotide binding 33..882 849/850 (99%)0.0
site
protein NBS-5 - Homo Sapiens,1..850 850/850 (99%)
858
aa. [WO200183753-A2, 08-NOV-
2001]
_ __ ~ _____
AAU07878 Polypeptide sequence for 165..907 375/743 (50%): 0.0
mammalian Spg65 - Mammalia,5..744 528/743 (70%)
~
748 aa. [WO200166752-A2,
I3-
_SEP.-2001]. ~ ~
.... .....
AAE07514 Human PYRIN-1 protein - 20..907~~320/926 (34%)e-146
Homo '
i sapiens, 1034 aa. [W0200161005-134..1028491/926 (52%)
p'2' ~3-AUG-2001 ] ...
. . [
___
AAG65895 Amino acid sequence of ~75..907 301/849 (35%)e-137
GSI~ gene ~~
Id 97078 - Homo Sapiens, 208..1043460/849 (53%)
1062 aa. ~
[WO200172961-A2, 04-OCT-
2001]
AAE07513 Human nucleotide binding 75..907 299/849 (35%)e-134
site 1
(NBS-1) protein - Homo 180..1014459/849 (53%)
Sapiens,
1033 aa. [W0200I61005-A2,
23-
AUG-2001] _. _.
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In a BLAST search of public sequence databases, the NOV2a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 2D.
Table 2D. Public BLASTP Results
for NOV2a
NOV2a Identities/
Protein , SimilaritiesExpect
Residues/
AccessionProtein/OrganismlLength for the
Number es Matched ~ Value
R Portion
idues
__
Q96MN2 ' CDNA FLJ32126 FIS, CLONE 1..919 918/919 (99%)0.0
~
PEBLM2000112, WEAKLY 1..919 918/919 (99%)
SIMILAR TO HOMO SAPIENS
NUCLEOTIDE-BINDING SITE
PROTEIN 1 MRNA - Homo Sapiens
(Human), 919 aa.
.~~. ..._._..~.._....-~......-...-,.~..",~.-..,..-..,...,_
Q96MN2 NACHT-, LRR- and PYD-containing18..919 900/902 (99%)_
~ 0.0
protein 4 (PAAD and NACHT- 93..994 901/902 (99%)
containing protein 2) (PYRIN-
containing APAF 1-like protein
4)
(Ribonuclease inhibitor 2)
- Homo
Sapiens (Human), 994 aa.
AAL88672 RIBONUCLEASE INHIBITOR 2 18..919 894/902 (99%)0.0
- ~
Homo Sapiens (Human), 916 15..916 897/902 (99%)
aa.
CAD19386 SEQUENCE 7 FROM PATENT 33..882 849/850 (99%)0.0
~
W00183753 - Homo Sapiens 1..850 850/850 (99%)
(Human), ~
858 as (fragment).
Q99MW0 RIBONUCLEASE/ANGIOGENIN 165..907374/743 (50%)0.0
=
INHIBITOR 2 - Mus musculus 5..744 528/743 (70%)
(Mouse), 748 aa.
PFam analysis predicts that the NOV2a protein contains the domains shown in
the Table
2E.
Table 2E. Domain Analysis of NOV2a
Identities/
Pfam Domain NOVZa Match Region Similarities Expect Value
for the Matched Region
SRP54 71..93 11/23 (48%) ~~~ 0.1 g.. .
. . . ~. _. .17/23...(74%)
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Exanipte 3.
The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 3A.
Table 3A. NOV3 Sequence Analysis
SEQ ID NO: 5 2142 by
NOV3a, ~TATTATTCAGCAAACAATCTCAATGTGTTCCTGATGGGAGAGAGAGCATCTGGAAAAA
CG100760-Ol CTATTGTTAT.AAATCTGGCTGTGTTGAGGTGGATCAAGGGTGAGATGTGGCAGAACAT
DNA Se ueriCe GATCTCGTACGTCGTTCACCTCACTGCTCACGAAATAAACCAGATGACCAACAGCAGC
TTGGCTGAGCTAATCGCCAAGGACTGGCCTGACGGCCAGGCTCCCATTGCAGACATCC
TGTCTGATCCCAAGAAACTCCTTTTCATCCTCGAGGACTTGGACAACATAAGATTCGA
GTTAAATGTCAATGAAAGTGCTTTGTGTAGTAACAGCACCCAGAAAGTTCCCATTCCA
GTTCTCCTGGTCAGTTTGCTGAAGAGAAAAATGGCTCCAGGCTGCTGGTTCCTCATCT
CCTCAAGGCCCACACGTGGGAATAATGTAAAAACGTTCTTGAAAGAGGTAGATTGCTG
CACGACCTTGCAGCTGTCGAATGGGAAGAGGGAGATATATTTTAACTCTTTCTTTAAA
~GACCGCCAGAGGGCGTCGGCAGCCCTCCAGCTTGTACATGAGGATGAAATACTCGTGG
GGACAAGGGGCGTGACTTCCAGCTCTGCTGCCAAACACCCACTGATCTACATGCCCAC
TTTCTTGCTGATGCGTTGACATCAGAGGCTGGACTTACTGCCAATCAGTATCACCTAG
TTTCAGTGGTGAAGACCTCAGATGTGTTGGGTTTACTGAGGCTGATGTCTCTGTGTTG
CAGGCCGCGAATATTCTTTTGCCGAGCAACACTCATAAAGACCGTTACAAGTTCATAC
ACTTGAACGTCCAGGAGTTTTGTACAGCCATTGCATTTCTGATGGCAGTACCCAACTA
TCTGATCCCCTCAGGCAGCAGAGAGTATAAAGAGAAGAGAGAACAATACTCTGACTTT
AGACATCCTTTGGATACCAGCTACCGATGGTAGACAGCTTCAAGTGGTACTCGGTGGG
ATACATGAAACATTTGGACCGTGACCCGGAAAAGTTGACGCACCATATGCCTTTGTTT
TACTGTCTCTATGAGAATCGGGAAGAAGAATTTGTGAAGACGATTGTGGATGCTCTCA
TGGAGGTTACAGTTTACCTTCAATCAGACAAGGATATGATGGTCTCATTATACTGTCT
GGATTACTGCTGTCACCTGAGGACACTTAAGTTGAGTGTTCAGCGCATCTTTCAAAAC
CCAGCATCCAACACGTAACTCGATTGTGCCTGGGATTTAATCGGCTCCAAGATGATGG
CATAAAGCTATTGTGTGCGGCCCTGACTCACCCCAAGTGTGCCTTAGAGAGACTGGAG
CTCTGGTTTTGCCAGCTGGCAGCACCCGCTTGCAAGCACTTGTCAGATGCTCTCCTGC
AGAACAGGAGCCTGACACACCTGAATCTGAGCAAGAACAGCCTGAGAGACGAGGGAGT
CAAGTTCCTGTGTGAGGCCTTGGGTCGCCCAGATGGTAACCTGCAGAGCCTGAGTTTG
TCAGGTTGTTCTTTCACAAGAGAGGGCTGTGGAGAGCTGGCTAATGCCCTCAGCCATA
ATCATAATGTGAAAATCTTGGATTTGGGAGAAAATGATCTTCAGGATGATGGAGTGAA
GCTACTGTGTGAGGCTCTGAAACCACATCGTGCATTGCACACACTTGGGTTGGCGAAA
GCCTGGTCAATCTGAACCTTCTAGGCAATGAATTGGATACTGATGGTGTCAAGATGCT
ATGTAAGGCTTTGAAAAAGTCGACATGCAGGCTGCAGAAACTCGGGTAAACCTCACTG
ORF Start: ATG at 34 ORF Stop: TAA at 2077
SEQ ID NO: 6 X681 as MW at 76724.1kD
NOV3a, MGERASGKTIVINLAVLRWIKGEMWQNMISYVVHLTAHEINQMTNSSLAELIAKDWPD
CGlOO76O-O1 GQAPIADILSDPKKLLFILEDLDNIRFELNVNESALCSNSTQKVPIPVLLVSLLKRKM
Protein SeqilenCe'nPGCWFI'ISSRPTRGNNVKTFLKEVDCCTTLQLSNGKREIYFNSFFKDRQRASAALQL
VHEDEILVGLCRVAILCWITCTVLKRQMDKGRDFQLCCQTPTDLHAHFLADALTSEAG
LTANQYHLGLLKRLCLLAAGGLFLSTLNFSGEDLRCVGFTEADVSVLQAANILLPSNT
HKDRYKFIHLNVQEFCTAIAFLMAVPNYLIPSGSREYKEKREQYSDFNQVFTFIFGLL
NANRRKILETSFGYQLPMVDSFKWYSVGYMKHLDRDPEKLTHHMPLFYCLYENREEEF
VKTIVDALMEVTVYLQSDKDMMVSLYCLDYCCHLRTLKLSVQRIFQNKLEKCNLSAAS
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CQDLALFLTSIQHVTRLCLGFNRLQDDGIKLLCAALTHPKCALERLELWFCQLAAPAC
KHLSDALLQNRSLTHLNLSKNSLRDEGVKFLCEALGRPDGNLQSLSLSGCSFTREGCG
ELANALSHNHNVKILDLGENDLQDDGVKLLCEALKPHRALHTLGLAKCNLTTACCQHL
FSVLSSSKSLVNLNLLGNELDTDGVKMLCKALKKSTCRLQKLG
Further analysis of the NOV3a protein yielded the following properties shown
in Table 3B.
y4 ~~~~~~ Table 3B. Protein Sequence~Properties NOV3a
PSort ; 0.8200 probability located in endoplasmic reticulum (membrane); 0.1900
analysis: . probability located in plasma membrane; 0.1000 probability located
in
endoplasmic reticulum (lumen); 0.1000 probability located in outside
SignalP Cleavage site between residues 23 and 24
analysis:
A search of the NOV3a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 3C.
Table 3C. Geneseq Results for NOV3a
NOV3a Identities)
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent for
Identifier#, Date) Match the Matched Value
~ Residueslegion . ..
. .
AAM50330 Human nucleotide binding 1..681 539/745 (72%)0.0
site
protein NBS-3 - Homo Sapiens,116..859587/745 (78%)
875
aa. [W0200183753-A2, 08
NOV-
2001 ]
AAM50326 Human nucleotide binding 1..510 469/517 (90%)0.0
site
protein NBS-3 - Homo sapiens,116..631479/517 (91%)
631
aa. [W0200183753-A2, 08-NOV-
2001
AAM50328 Human nucleotide binding 2..681 247/750 (32%)e-1
site OS
protein NBS-5 - Homo sapiens,48..792 381/750 (49%)
858
aa. [WO200183753-A2, OS-NOV-
2001 ]
~.. 3 . ._...
AAE07514 Human PYRIN-1 protein - 2..680 238/729 (32%)e-100
Homo
Sapiens, 1034 aa. [W0200161005-224..944362/729 (49%)
A2, 23-AUG-2001 ]
ABG03924 Novel human diagnostic 2..680 228/741 (30%)7e-78
protein
#3915 - Homo sapiens, 952 178..908334/741 (44%)
aa.
[WO200175067-A2, 11-OCT-
2001 ]
In a BLAST search of public sequence databases, the NOV3a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 3D.
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Table 3D. Public BLASTP
Results for NOV3a
Protein NOV3a Identities/
Accession Protein/Organism/Length Residues/SimilaritiesExpect
for
Number Match the Matched Value
ResiduesPortion
CAD19388 SEQUENCE 15 FROM PATENT 1..681 539/745 (72%)0.0
W00183753 - Homo Sapiens 116..859587/745 (78%)
(Human), 875 aa.
CAD 193 SEQUENCE 3 FROM PATENT 1..510 469/517 (90%)0.0
84
W00183753 - Homo Sapiens 116..631479/517 (91%)
(Human), 631 as (fragment).
CAD19386 SEQUENCE 7 FROM PATENT ~ 2..681247/750 (32%)e-104
W00183753 - Homo Sapiens 48..792 3811750 (49%)
(Human), 858 as (fragment).
Q96MN2 CDNA FLJ32126 FIS, CLONE 2..681 247/750 (32%)e-104
PEBLM2000112, WEAKLY ~ 381/750 (49%)
80..824
SIMILAR TO HOMO SAPIENS
NUCLEOTIDE-BINDING SITE
PROTEIN 1 MRNA - Homo sapiens
(Human), 919 aa.
Q96MN2 NACHT-, LRR- and PYD-containing2..681 247/750 (32%)e-104
protein 4 (PAAD and NACHT- 155..899381/750 (49%)
containing protein 2) (PYR1N-
containing APAF1-like protein
4)
(Ribonuclease inhibitor
2) - Homo
_ .. .... Sapiens (Human), 994. aa... _...
Example 4.
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 4A.
Table 4A. NOV4 Sequence Analysis
SEQ ID NO: 7 X782 by
NOV4a, ;TCTCAAGGGATAATCACTAAATTCTGCCGAAAGGACTGAGGAACGGTGCCTGGAAAAG
CG100851-O2 GGCAAGAATATCACGGCATGGGCATGAGTAGCTTGAAACTGCTGAAGTATGTCCTGTT
DNA Sequence ~TTTCTTCAACTTGCTCTTTTGGATCTGTGGCTGCTGCATTTTGGGCTTTGGGATCTAC
[[CTGCTGATCCACAACAACTTCGGAGTGCTCTTCCATAACCTCCCCTCCCTCACGCTGG
GCAATGTGTTTGTCATCGTGGGCTCTATCAAGGAAAACAAGTGTCTGCTTATGTCGTT
CTTCATCCTGC'Z'GCTGATTATCCTCCTTGCTGAGGTGACCTTGGCCATCCTGCTCTTT
GTATATGAACAGAAGCTGAATGAGTATGTGGCTAAGGGTCTGACCGACAGCATCCACC
GTTACCACTCAGACAATAGCACCAAGGCAGCGTGGGACTCCATCCAGTCATTTCTGCA
GTGTTGTGGTATAAATGGCACGAGTGATGGGACCAGTGGCCCACCAGCATCTTGCCCC
TCAGATCGAAAAGTGGAGGGTTGCTATGCGAAAGCAAGACTGTGGTTTCATTCCAATT
TCCTGTATATCGGAATCATCACCATCTGTGTATGTGTGATTGAGGTGTTGGGGATGTC
CTTTGCACTGACCCTGAACTGCCAGATTGACAAAACCAGCCAGACCATAGGGCTATGA
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TCTGCAGTAGTTCTGTGGTGAAGAGACTTGTTTCATCTCCTGGAAATGCAAAACCATT
~TATAGCATGAGCCCTACATGATCATCAG
ORF Start: ATG at 76 ORF Stop: TGA at 694
SEQ ID NO: 8 206 as MW at 22888.8kD
NOV4a, MGMSSLKLLKYVLFFFNLLFWTCGCCILGFGIYLLIHNNFGVLFHNLPSLTLGNVFVI
CG100851-O2 VGSIKENKCLLMSFFILLLIILLAEVTLAILLFVYEQKLNEYVAKGLTDSIHRYHSDN
PPOteln Se uenCe STKAAWDSIQSFLQCCGINGTSDGTSGPPASCPSDRKVEGCYAKARLWFHSNFLYIGI
q ITICVCVIEVLGMSFALTLNCQIDKTSQTIGL
Further analysis of the NOV4a protein yielded the following properties shown
in Table 4B.
Table 4B. Protein Sequence Properties NOV4a
PSort 0.6400 probability located in plasma membrane; 0.4600 probability
located in
analysis: Golgi body; 0.3700 probability located in endoplasmic reticulum
(membrane);
0.1000 probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 32 and 33
analysis:
A search of the NOV4a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 4C.
Table 4C. Geneseq Results for NOV4a
NOV4a Identities/
~ ~
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent ~ for
Identifier~ #, Date] Match the Matched Value
Residues Region
AAY96I41 Human haematopoietic CD53 I ..206 205/2I9 (93%)e-I
- 15
Homo sapiens, 219 aa. 1..219 205/219 (93%)
[US6111093-A, 29-AUG-2000]
AAB58136 s Lung cancer associated 1..206 205/219 (93%)e-1
polypeptide I
S
sequence SEQ ID 474 - Homo13..231 205/219 (93%)
sapiens, 231 aa. [W0200055180-
A2, 21-SEP-2000]
AAW89152 Human CD53 antigen - Homo 1..206 205/219 (93%)e-115
sapiens, 219 aa. [IJS5849898-A,1..219 205/219 (93%)
15- ~
DEC-1998]
AAW80455 Human CD53 antigen - Homo 1..206 205/219 (93%)e-1
i IS
Sapiens, 219 aa. [US5830731-A,1..219 205/219 (93%)
03-'
NOV-1998]
AAR91446 . Human CD53 antigen - 1..206 2051219 (93%)e-115
Homo '
sapiens, 219 aa. [LTS5506126-A,1..219 205/219 (93%)
09-
APR-1996]. . _ . . .. ...._ . _..
~. .
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In a BLAST search of public sequence databases, the NOV4a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 4D.
Table 4D. Public BLASTP Results for NOV4a
Protein NOV4a Identities/
AccessionProtein/Organism/Length Residues/SimilaritiesExpect
for
Number Match the Matched Value
Residues Portion
P19397 ~ Leukocyte surface antigen1..206 205/219 (93%)e-115
CD53
(Cell surface glycoprotein1..219 205/219 (93%)
CD53) -
Homo Sapiens (Human), 219
aa.
AAH21310 CD53 ANTIGEN - Mus musculus1..206 168/219 (76%)6e-95
(Mouse), 219 aa. 1..219 183/219 (82%)
Q61451 Leukocyte surface antigen 2..206 167/218 (76%)2e-94
CD53
(Cell surface glycoprotein1..218 182/218 (82%)
CD53) -
Mus musculus (Mouse), 218
aa.
A39574 leukocyte antigen OX-44 1..206 164/219 (74%~7e-94
- rat, 219
aa. 1..219 183/219 (82%)
P24485 Leukocyte surface antigen 2..206 163/218 (74%)3e-93
CD53
(Cell surface glycoprotein1..218 182/218 (82%)
CD53)
(Leukocyte antigen MRC
OX-44) -
- ' Rattus norvegicus (Rat),~~-
218 aa~x :.:..,..~."-..~
PFam analysis predicts that the NOV4a protein contains the domains shown in
the Table
4E.
Table 4E. Domain Analysis of NOV4a
Identities/
Pfam Domain NOV4a Match Region Similarities Expect Value
for the Matched Region
transmembrane4 10..36 17/27 (63%) 4.4e-08
27/27 (100%)
transmembrane4 58..197 52/202 (26%) 1.2e-44
_._ __ _ .____ ~.. _ _ _._ __~.. _. _~ _ _....._120/202'(59%) n.___. -
Example 5.
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 5A.
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Table SA. NOVS Sequence Analysis
SEQ ID NO: 9 1719 by
NOVSa, ATGCGGACGCCGGTGGTGATGACGCTGGGCATGGTGTTGGCGCCCTGCGGGCTCCTGC
CG101068-OlTCAACCTGACCGGCACCCTGGCGCCCGGCTGGCGGCTGGTGAAGGGCTTCCTGAACCA
DNA SeClilenCeGCCAGTGGACGTGGAGTTGTACCAGGGCCTGTGGGACATGTGTCGCGAGCAGAGCAGC
CGCGAGCGCGAGTGCGGCCAGACGGACCAGTGGGGCTACTTCGAGGCCCAGCCCGTGC
TGGTGGCGCGGGCACTCATGGTCACCTCGCTGGCCGCCACGGTCCTGGGGCTTCTGCT
GGCGTCGCTGGGCGTGCGCTGCTGGCAGGACGAGCCCAACTTCGTGCTGGCAGGGCTC
TCGGGCGTCGTGCTCTTCGTCGCTGGCCTCCTCGGCCTCATCCCGGTGTCCTGGTACA
GGTCAGCTACAGCCTGGTCCTGGGCTACCTGGGCAGCTGCCTCCTGCTGCTGGGCGGC
TTCTCGCTGGCGCTCAGCTTCGCGCCCTGGTGCGACGAGCGTTGTCGCCGCCGCCGCA
AGGGACCCTCCGCCGGGCCTCGCCGCAGCAGCGTCAGCACCATCCAAGTGGAGTGGCC
CGAGCCCGACCTGGCGCCCGCCATCAAGTACTACAGCGACGGCCAGCACCGACCGCCG
CCTGCCCAGCACCGCAAGCCCAAGCCCAAGCCCAAGGTCGGCTTCCCCATGCCGCGGC
CGCGGCCCAAGGCCTACACCAACTCGGTGGACGTCCTCGACGGGGAGGGGTGGGAGTC
CCAGGACGCTCCCTCGTGCAGCACCCACCCCTGCGACAGCTCGCTGCCCTGCGACTCC
GACCTCTAGACGCTTGTAGAGCCTGGGGGGCGCCGGGTGGCAAAGGACTCACCCCCGC
ACAGGCCCGCCTGGCTTCGAGTTGGAACCCGGACACTTGCCCCTCACTGGTGTGGATG
GAAATCTGCCTTTCGTGGGACCAAACAGGACTCCTTGGACGATTAGTTCAGGTTGGGT
TTGGTTTTCTTCTTAAAGAGTTTAGTTTTCCTCTCCAGAGGGATCAGGGTCCTCTTAG
GGAGTGACGGGCTTTTCATATATTTTTGCTGAAGAATATATGGAAAGGGTGGCATTTG
CGTCACGTGGACCAGGGACAGTGCTGAAATCAGCAGTGCTCAGAAACAATTTAACATG
TTGAAACGACAATATTCTAAAATACTGATGAATCTTGCATCAATATAATTATTGGGTT
TTTTTTCTTTTTCCTGCTGTATAACTCCTTGCCATGCAAACTCTCAAGAGGCCAATAT
ATTCCTGGCCATGTTTGAATGAGCCTCTTAAAATAAACTTAGAGCCATGCAAATGCCA
GCAGCTTAATGGATTTCATGGAATGAAATACCGTGATTAACTCATAGCTACATATCAT
TGCATAAATGGGATTTATCTTTTTTCTCACTTATTTTTGCGGTGAAAGTCGAGGGCAT
GCAAGAGTTTCTCTTCCAGAAGCCAAGAGGAGAACAAAGGTCCTAATGCTGTACTATT
CCACCCTTTGGACGCCTCATCCAGGACGCAGAGGACTCTAGGTTTAACATTTTGTACA
AAATGGAACCTGTTAATCATATTAAAGCACATATGTATATATCTTTTATTTATAAATA
AAATTTTAAAACAATAGTTTCAGTATAGCCACAAAAA
ORF Start: ATG at 1 ORF Stop: TAG at 877
SEQ ID NO: 10 292 as MW at 31914.SkD
NOVSa, MRTPVVMTLGMVLAPCGLLLNLTGTLAPGWRLVKGFLNQPVDVELYQGLWDMCREQSS
CG101068-Ol RERECGQTDQWGYFEAQPVLVARALMVTSLAATVLGLLLASLGVRCWQDEPNFVLAGL
PTOteln SeCluenCe SGVVLFVAGLLGLTPVSWYNfiFLGDRDVLPAPASPVTVQVSYSLVLGYLGSCLLLLGG
FSLALSFAPWCDERCRRRRKGPSAGPRRSSVSTIQVEWPEPDLAPAIKYYSDGQHRPP
PAQHRKPKPKPKVGFPMPRPRPKAYTNSVDVLDGEGWESQDAPSCSTHPCDSSLPCDS
Further analysis of the NOVSa protein yielded the following properties shown
in Table SB.
Table 5B. Protein Sequence Properties NOVSa
PSort 0.6400 probability located in plasma membrane; 0.4600 probability
located in
analysis: ~ Golgi body; 0.3700 probability located in endoplasmic reticulum
(membrane);
0.1000 probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 28 and 29
analysis:
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A search of the NOVSa protein against the Geneseq dafabase; a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table SC.
Table SC. Geneseq Results for NOVSa
NOVSa Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent for
Identifier'; #, Date] Match the Matched Value
Residues Region
:
AAB64401 Amino acid sequence of 9..206 74/206 (35%)1e-16
human
intracellular signalling 9..209 101/206 (48%)
molecule
INTRA33 - Homo sapiens,
217 aa.
[WO200077040-A2, 21-DEC-2000]
AAG75467 Human colon cancer antigen6..187 59/188 (31%)2e-13
protein
SEQ ID N0:6231 - Homo Sapiens,7..192 92/188 (48%)
210 aa. [W0200122920-A2,
OS-
~R-Zoo 1 J ~
__
ABB50278 Claudin 4 ovarian tumour 6..187 59/188 (31%)2e-13
marker
protein, SEQ ID N0:45 - 6..191 92/188 (48%)
Homo
Sapiens, 209 aa. [WO200175177-A2,
11-OCT-2001]
AAB43133 6..187 59/188 (31%)2e-13
Human
ORFX
ORF2897
polypeptide sequence SEQ 6..191 92/I88 (48%)
ID
N0:5794 - Homo Sapiens,
209 aa.
[WO200058473-A2, OS-OCT-2000]
ABB50396 Human secreted protein 9..187 59/185 (3I%)Ze-I3
encoded by ~
gene 96 SEQ ID N0:344 - 1..183 91/185 (48%)
Homo
Sapiens, 202 aa. [WO200162891-A2,
30-AUG-2001
In a BLAST search of public sequence databases, the NOVSa protein was found to
have
homology to the proteins-shown in the BLASTP data in Table SD.
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Table SD Public BLASTP
Results for NOVSa
Protein NOVSa Identities/
Accession Protein/Organism/Length Residues/ SimilaritiesExpect
for
Number Match the Matched Value
Residues Portion
Q96B33 SIMILAR TO RIKEN CDNA 28..292 252/267 (94%)e-147
2310014808 GENE - Homo 2..268 254!267 (94%)
sapiens (Human), 268
as
(fragment).
Q9D7D7 2310014B08RIK PROTEIN 1..292 230/296 (77%)e-135
(RIKEN CDNA 2310014808 1..296 248/296 (83%)
GENE) - Mus musculus
(Mouse),
296 aa.
095484 Claudin 9 - Homo Sapiens9..206 74!206 (35%)4 16~
(Human), 217 aa. 9..209 101/206 (48%)
Q9ZOS7 Claudin-9 - Mus musculus9..206 71/206 (34%)1e-14
(Mouse), 217 aa. ~ 9..209 991206 (47%)
Q98SR2 CLAUDIN-3 - Gallus gallus10..206 64/202 (31%)!e-13
(Chicken), 214 aa. 9..207 99/202 (48%)
PFam analysis predicts that the NOVSa protein contains the domains shown in
the Table
SE.
Table 5E. Domain Analysis of NOVSa
Identities/
Pfam Domain ~ NOVSa Match Region Similarities Expect Value
fox the Matched Region
PMP22 Claudin 3..177 40/194 (21%) 0.00018
108/194 (56%)
_._ ......__ . . .... ....... .. ..._.....~ ........... _._... .........._ ..
..................... ............... ....... ._... ... _...
................... ..................... . ... .....~~........ .... ....
..._.. .
Example 6.
The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 6A.
Table 6A. NOV6 Sequence Analysis
SEQ ID NO: 11 X2369 by
NOV6a, CGGCCGGAGCGCCGAGGCCCGGCCATGGCCACCACCAGCACCACGGGCTCCACCCTGC
CG101231-Ol TGCAGCCCCTCAGCAACGCCGTGCAGCTGCCCATCGACCAGGTCAACTTTGTAGTGTG
DNA Sequence CCAACTCTTTGCCTTGCTAGCAGCCATTTGGTTTCGAACTTATCTACATTCAAGCAAA
ACTAGCTCTTTTATAAGACATGTAGTTGCTACCCTTTTGGGCCTTTATCTTGCACTTT
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TTTGCTTTGGATGGTATGCCTTACACTTTCTTGTACAAAGTGGAATTTCCTACTGTAT
CATGATCATCATAGGAGTGGAGAACATGCACAATTACTGCTTTGTGTTTGCTCTGGGA
TACCTCACAGTGTGCCAAGTTACTCGAGTCTATATCTTTGACTATGGACAATATTCTG
CTGATTTTTCAGGCCCAATGATGATCATTACTCAGAAGATCACTAGTTTGGCTTGCGA
AATTCATGATGGGATGTTTCGGAAGGATGAAGAACTGACTTCCTCACAGAGGGATTTA
GCTGTAAGGCGCATGCCAAGCTTACTGGAGTATTTGAGTTACAACTGTAACTTCATGG
GGATCCTGGCAGGCCCACTTTGCTCTTACAAAGACTACATTACTTTCATTGAAGGCAG
ATCATACCATATCACACAATCTGGTGAAAATGGAAAAGAAGAGACACAGTATGAAAGA
ACAGAGCCATCTCCAAATAGTGCGGTTGTTCAGAAGCTCTTAGTTTGTGGGCTGTCCT
TGTTATTTCACTTGACCATCTGTACAACATTACCTGTGGAGTACAACATTGATGAGCA
TTTTCAAGCTACAGCTTCGTGGCCAACAAAGATTATCTATCTGTATATCTCTCTTTTG
GCTGCCAGACCCAAATACTATTTTGCATGGACGCTAGCTGATGCCATTAATAATGCTG
CAGGCTTTGGTTTCAGAGGGTATGACGAAAATGGAGCAGCTCGCTGGGACTTAATTTC
CAATTTGAGAATTCAACAAATAGAGATGTCAACAAGTTTCAAGATGTTTCTTGATAAT
TGGAATATTCAGACAGCTCTTTGGCTCAAAAGGGTGTGTTATGAACGAACCTCCTTCA
GTCCAACTATCCAGACGTTCATTCTCTCTGCCATTTGGCACGGGGTATACCCAGGATA
TTATCTAACGTTTCTAACAGGGGTGTTAATGACATTAGCAGCAAGAGCTGTAAGAAAT
AACTTTAGACATTATTTCATTGAACCTTCCCAACTGAAATTATTTTATGATGTTATAA
CATGGATAGTAACTCAAGTAGCAATAAGTTACACAGTTGTGCCATTTGTGCTTCTTTC
TATAAAACCATCACTCACGTTTTACAGCTCCTGGTATTATTGCCTGCACATTCTTGGT
ATCTTAGTATTATTGTTGTTGCCAGTAAAAAAAACTCAAAGAAGAAAGAATACACATG
AAAACATTCAGCTCTCACAATCCAAAAAGTTTGATGAAGGAGAAAATTCTTTGGGACA
GAACAGTTTTTCTACAACAAACAATGTTTGCAATCAGAATCAAGAAATAGCCTCGAGA
CATTCATCACTAAAGCAGTGATCGGGAAGGCTCTGAGGGCTGTTTTTTTTTTTTGATG
TTAACAGAAACCAATCTTAGCACCTTTTCAAGGGGTTTGAGTTTGTTGGAAAAGCAGT
TAACTGGGGGGAAATGGACAGTTATAGATAAGGAATTTCCTGTACACCAGATTGGAAA
TGGAGTGAAACAAGCCCTCCCATGCCATGTCCCCGTGGGCCACGCCTTATGTAAGAAT
ATTTCCATATTTCAGTGGGCACTCCCAACCTCAGCACTTGTCCGTAGGGTCACACGCG
TGCCCTGTTGCTGAATGTATGTTGCGTATCCCAAGGCACTGAAGAGGTGGAAAAATAA
TCGTGTCAATCTGGATGATAGAGAGAAATTAACTTTTCCAAATGAATGTCTTGCCTTA
AACCCTCTATTTCCTAAAATATTGTTCCTAAATGGTATTTTCAAGTGTAATATTGTGA
GAACGCTACTGCAGTAGTTGATGTTGTGTGCTGTAAAGGATTTTAGGAGGAATTTGAA
ACAGGATATTTAAGAGTGTGGATATTTTTAAAATGCAATAAACATCTCAGTATTTGAA
GGGTTTTCTTAAAGTATGTCAAATGACTACAATCCATAGTGAAACTGTAAACAGTAAT
GGACGCCAAATTATAGGTAGCTGATTTTGCTGGAGAGTTTAATTACCTTGTGCAGTCA
AAGAGCGCTTCCAGAAGGAATCTCTTAAAACATAATGAGAGGTTTGGTAATGTGATAT
TTTAAGCTTACTCTTTTTCTTAAAAGAGAGAGGTGACGAAGGAAGGCAG
~ORF Start: ATG at 25 ORF Stop: TGA at 1585
SEQ ID NO: 12 ~ 520 as MW at 59480.OkD
NOVC7a, MATTSTTGSTLLQPLSNAVQLPIDQVNFWCQLFALLAAIWFRTYLHSSKTSSFIRHV
CG101231-O1 VATLLGLYLALFCFGWYALHFLVQSGISYCIMIIIGVENMHNYCFVFALGYLTVCQVT
Protein SeCllleriCeR~IFDYGQYSADFSGPMMIITQKITSLACEIHDGMFRKDEELTSSQRDLAVRRMPSL
LEYLSYNCNFMGILAGPLCSYKDYITFIEGRSYHITQSGENGKEETQYERTEPSPNSA
VVQKLLVCGLSLLFHLTICTTLPVEYNIDEHFQATASWPTKIIYLYISLLAARPKYYF
AWTLADAINNAAGFGFRGYDENGAARWDLISNLRIQQIEMSTSFKMFLDNWNIQTALW
LKRVCYERTSFSPTIQTFILSAIWHGVYPGYYLTFLTGVLMTLAARAVRNNFRHYFIE
PSQLKLFYDVITWIVTQVAISYTVVPFVLLSIKPSLTFYSSWYYCLHILGILVLLLLP
VKKTQRRKNTHENIQLSQSKKFDEGENSLGQNSFSTTNNVCNQNQEIASRHSSLKQ
SEQ ID NO: 13 2270 by
NOVE)b, CGGCCGGAGCGCCGAGGCCCGGCCATGGCCACCACCAGCACCACGGGCTCCACCCTGC
CG101231-O2 TGCAGCCCCTCAGCAACGCCGTGCAGCTGCCCATCGACCAGGTCAACTTTGTAGTGTG
DNA S8 11812C8CCAACTCTTTGCCTTGCTAGCAGCCATTTGGTTTCGAACTTATCTACATTCAAGCAAA
ACTAGCTCTTTTATAAGACATGTAGTTGCTACCCTTTTGGGCCTTTATCTTGCACTTT
TTTGCTTTGGATGGTATGCCTTACACTTTCTTGTACAAAGTGGAATTTCCTACTGTAT
CATGATCATCATAGGAGTGGAGAACATGCAGCCAATGATGATCATTACTCAGAAGATC
ACTAGTTTGGCTTGCGAAATTCATGATGGGATGTTTCGGAAGGATGAAGAACTGACTT
CCTCACAGAGGGATTTAGCTGTAAGGCGCATGCCAAGCTTACTGGAGTATTTGAGTTA
CAACTGTAACTTCATGGGGATCCTGGCAGGCCCACTTTGCTCTTACAAAGACTACATT
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ACTTTCATTGAAGGCAGATCATACCATATCACACAATCTGGTGAAAATGGAAAAGAAG
AGACACAGTATGAAAGAACAGAGCCATCTCCAAATAGTGCGGTTGTTCAGAAGCTCTT
AGTTTGTGGGCTGTCCTTGTTATTTCACTTGACCATCTGTACAACATTACCTGTGGAG
TACAACATTGATGAGCATTTTCAAGCTACAGCTTCGTGGCCAACAAAGATTATCTATC
TGTATATCTCTCTTTTGGCTGCCAGACCCAAATACTATTTTGCATGGACGCTAGCTGA
TGCCATTAATAATGCTGCAGGCTTTGGTTTCAGAGGGTATGACGAAAATGGAGCAGCT
CGCTGGGACTTAATTTCCAATTTGAGAATTCAACAAATAGAGATGTCAACAAGTTTCA
AGATGTTTCTTGATAATTGGAATATTCAGACAGCTCTTTGGCTCAAAAGGGTGTGTTA
TGAACGAACCTCCTTCAGTCCAACTATCCAGACGTTCATTCTCTCTGCCATTTGGCAC
GGGGTATACCCAGGATATTATCTAACGTTTCTAACAGGGGTGTTAATGACATTAGCAG
CAAGAGCTGTAAGAAATAACTTTAGACATTATTTCATTGAACCTTCCCAACTGAAATT
ATTTTATGATGTTATAACATGGATAGTAACTCAAGTAGCAATAAGTTACACAGTTGTG
CCATTTGTGCTTCTTTCTATAAAACCATCACTCACGTTTTACAGCTCCTGGTATTATT
GCCTGCACATTCTTGGTATCTTAGTATTATTGTTGTTGCCAGT~~AAAAAAACTCAAAG
AAGAAAGAATACACATGAAAACATTCAGCTCTCACAATCCAAAAAGTTTGATGAAGGA
GAAAATTCTTTGGGACAGAACAGTTTTTCTACAACAAACAATGTTTGCAATCAGAATC
AAGAAATAGCCTCGAGACATTCATCACTAAAGCAGTGATCGGGAAGGCTCTGAGGGCT
GTTTTTTTTTTTTGATGTTAACAGAAACCAATCTTAGCACCTTTTCAAGGGGTTTGAG
TTTGTTGGAAAAGCAGTTAACTGGGGGGAAATGGACAGTTATAGATAAGGAATTTCCT
GTACACCAGATTGGAAATGGAGTGAAACAAGCCCTCCCATGCCATGTCCCCGTGGGCC
ACGCCTTATGTAAGAATATTTCCATATTTCAGTGGGCACTCCCAACCTCAGCACTTGT
CCGTAGGGTCACACGCGTGCCCTGTTGCTGAATGTATGTTGCGTATCCCAAGGCACTG
AAGAGGTGGAAAAATAATCGTGTCAATCTGGATGATAGAGAGAAATTAACTTTTCCAA
ATGAATGTCTTGCCTTAAACCCTCTATTTCCTAAAATATTGTTCCTAAATGGTATTTT
CAAGTGTAATATTGTGAGAACGCTACTGCAGTAGTTGATGTTGTGTGCTGTAAAGGAT
TTTAGGAGGAATTTGAAACAGGATATTTAAGAGTGTGGATATTTTTAAAATGCAATAA
ACATCTCAGTATTTGAAGGGTTTTCTTAAAGTATGTCAAATGACTACAATCCATAGTG
AAACTGTAAACAGTAATGGACGCCAAATTATAGGTAGCTGATTTTGCTGGAGAGTTTA
ATTACCTTGTGCAGTCAAAGAGCGCTTCCAGAAGGAATCTCTTAAAACATAATGAGAG
GTTTGGTAATGTGATATTTTAAGCTTACTCTTTTTCTTAAAAGAGAGAGGTGACGAAG
GAAGGCAG
ORF Start: ATG at 25 ORF Stop: TGA at 1486
SEQ ID NO: 14 487 as MW at 55677.7kD
NOV6b, MATTSTTGSTLLQPLSNAVQLPIDQVNFVVCQLFALLAAIWFRTYLHSSKTSSFIRHV
CG101231-O2 VATLLGLYLALFCFGWYALHFLVQSGISYCIMIIIGVENMQPMMIITQKITSLACEIH
PTOte2n SequenceDGMFRKDEELTSSQRDLAVRRMPSLLEYLSYNCNFMGILAGPLCSYKDYITFIEGRSY
HITQSGENGKEETQYERTEPSPNSAWQKLLVCGLSLLFHLTICTTLPVEYNIDEHFQ
ATASWPTKIIYLYISLLAARPKYYFAWTLADAINNAAGFGFRGYDENGAARWDLISNL
RIQQIEMSTSFKMFLDNWNIQTALWLKRVCYERTSFSPTIQTFILSAIWHGVYPGYYL
TFLTGVLMTLAARAVRNNFRHYFIEPSQLKLFYDVITWIVTQVAISYTVVPFVLLSIK
PSLTFYSSWYYCLHILGILVLLLLPVKKTQRRKNTHENIQLSQSKKFDEGENSLGQNS
FSTTNNVCNQNQEIASRHSSLKQ
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 6B.
Table 6B. Comparison of NOV6a against NOV6b.
NOV6a Residues/ ~ Identities/
Protein Sequence Match Residues Similarities for the Matched Region
NOV6b ~ 1..520 = 474/520 (91%)
. 1..487 4741520 (91%)
Further analysis .of the NOV6a protein yielded the following properties shown
in Table 6C.
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Table 6C. Protein Sequence Properties NOV6a
PSort ' 0.6000 probability located in plasma membrane; 0.4000 probability
located in
analysis: GoIgi body; 0.3406 probability located in mitochondria)
intermembrane space;
0.3384 probability located in mitochondria) inner membrane
SignalP , Cleavage site between residues 44 and 45
analysis:
A search of the NOV6a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 6D.
Table 6D. Geneseq Results for NOV6a
NOV6a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent for
Identifier#, Date] Match the Matched Value
Residues Region
AAG81345 Human AFP protein sequence98..520 419/423 (99%)0.0
SEQ
ID N0:208 - Homo Sapiens, 1..423 421/423 (99%)
423 aa.
[W0200129221-A2, 26-APR-2001]
v
AAB93797 ~ Human protein sequence 102..520 416/419 (99%)0.0
SEQ ID
N0:13560 - Homo Sapiens, 14..432 419/419 (99%)
432 aa.
[EP 1074617-A2, 07-FEB-2001
]
AAM93974 ~ Human stomach cancer 102..520 416/419 (99%)0.0
expressed
polypeptide SEQ ID NO 17 14..432 419/419 (99%)
- Homo : .
Sapiens, 432 aa. [W0200109317-
Al, 08-FEB-2001]
ABG04835 Novel human diagnostic 50..297 243/248 (97%)e-143
protein
#4826 - Homo Sapiens, 371 23..270 246/248 (98%)
aa.
[WO200175067-A2, 11-OCT-2001]
ABG04835 Novel human diagnostic 50..297 243/248 (97%)e-143
protein
#4826 - Homo Sapiens, 371 23..270 2461248 (98%)
aa.
T~;~___....~[W0200175067-A2, 1.1-OCT-2001]......_._..__.~..~_~x,~,~.,~_
__. __" ~ _ . ._ ~_.w~x
In a BLAST search of public sequence databases, the NOV6a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 6E.
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Table 6E. Public BLASTP Results for NOV6a
Protein '~ NOV6a Identities/
Accession Protein/Organism/Length Residues/ Similarities Expect
for
Number Match the Matched Value
Residues Portion
~
AAH25429 SIMILAR TO RIKEN CDNA 1..520 451/520 (86%)0.0
2810049606 GENE - Mus 1..519 479/520 (91
%)
musculus (Mouse), 519
aa.
CAC38595 SEQUENCE 207 FROM 98..520 419/423 (99%)0.0
PATENT W00129221 - Homo ~ 1..423 421/423 (99%)
Sapiens (Human), 423
aa.
AAH25020 RIKEN CDNA 2810049606 ~ 1..520 422/520 (81%)0.0
GENE - Mus musculus (Mouse),1..487 449/520 (86%)
487 aa.
Q9CZ73 2810049G06RIK PROTEIN 1..520 421/520 (80%)0.0
-
Mus musculus (Mouse), 1..487 448/520 (85%)
487 aa.
Q96KY4 SIMILAR TO RIKEN CDNA ~ 171..520348/350 (99%)0.0
2810049606 GENE - Homo 1..350 350/350 (99%)
Sapiens (Human), 350 ~
aa.
t
PFam analysis predicts that the NOV6a protein contains the domains shown in
the Table
6F.
Table 6F. Domain Analysis of NOV6a
Identities/
Pfam Domain NOV6a Match Region Similarities Expect Value
for the Matched Region
Adeno_Penton_B 204..222 8/20 (40%) 0.54
17/20 (85%)
MBOAT 148..442 108/334 (32%) ~ 4.1e-89
225/334 (67%)
_ .. _ . .. _... .
Example 7.
The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide
sequences axe
shown in Table 7A.
Table 7A. NOV7 Sequence Analysis
__~~" ,.~~Q ID.NO.~.15 ... . ... w,~ 537 by ,~ . ..~ ,..:-
NOV7a, ATTAGCAACGGCTCATGATGAACTCAATCAAAGGGGGCTTGACCAGCATCTCAGGTCT
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CG101362-O1 ACTCTATGTTTTCCAGTGCCCATCAGTGCCAAGGGTATTGATTAGCCTATCCGGGACA
DNA Sequence ~GAGAGAAGAAAGAGTGAGACACCACCACTAAAAGGGCTGCAGGTGGATACCGCCTC
CCTCAAGCTGGAAAAAGATTAGAAAGATGGTGAAAACAGGAAGACCTTCCTCATCCCA
CTATCAGGAAGATGAGGAAAGAGATCAGGAGGATCACAGGTGGAGAGGAGAAGAGGAC
CATGCTCGATCCTCTCTGGTAATAGGCCTGAGATTCCCTCTCGTACTGGGTGATACAC
ATCTGCTCCCAGTGTTCCATCCTCCAGGCTTCGGGCGCTTCTTGCAGAGGCCCAGGTC
ACTCCATGTGGCCACAAAGAGAACCAGCATCCAGCAGCCAAGGTTCGCCATAATGACT
GCTCTGCCTCGGTCGTGAGGAGAGGAGAAGCTCGCGGCGCCGCGGCTGTCAGCGACTG
GCTCGGAGGACAGGC
ORF StarE: ATG at 201 ORF Stop:_TGA at 4_80___
SEQ ID N0~16. a 93 as ~MW at 10769.1kD
NOV7a, MVKTGRPSSSHYQEDEERDQEDHRWRGEEDHARSSLVIGLRFPLVLGDTHLLPVFHPP
CG101362-Ol GFGRFLQRPRSLHVATKRTSIQQPRFAIMTALPRS
Protein Sequence
Further analysis of the NOV7a protein yielded the following properties shown
in Table 7B.
Table 7B. Protein Sequence Properties NOV7a
PSort ~ 0.6400 probability located in microbody (peroxisome); 0.4500
probability
analysis: ~ located in cytoplasm; 0.2288 probability located in lysosome
(lumen); 0.1000
probability located in mitochondrial matrix space
SignalP No Known Signal Sequence Predicted
~alysis: ~ .. _.. _ . _. ~.~u.-~,~..,~- ~.....~~~,:~. W _..~_ _W~.~;._~:,~"...
.. ..._...._.
A search of the NOV7a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 7C.
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Table 7C.'Geneseq Results
for NOV7a
NOV7a Identities/
Geneseq Protein/Organism/Length Residues/ SimilaritiesExpect
[Patent #,
IdentifierDate] Match for the Value
Residues Matched
Region
AAY19678 SEQ ID NO 396 from W099222431..30 24/30 (46%)0.94
-
Homo Sapiens, 133 aa. [W09922243-63..91 19/30 (62%)
Al, 06-MAY-1999]
AAB92467 Human protein sequence SEQ 2..90 24/90 (26%)1.2
ID
N0:10527 - Homo Sapiens, 318..398 41/90 (44%)
563 aa.
[EP1074617-A2, 07-FEB-2001]
AAU16292 Human novel secreted protein,2..90 24/90 (26%)1.2
Seq ID
1245 - Homo sapiens, 564 319..399 41/90 (44%)
aa.
[W0200155322-A2, 02-AUG-2001]
___ _______-____ -__
_ _
ABB50224 Human transcription factor 2..90 24/90 (26%)1.2
TRFX-75
- Homo sapiens, 596 aa. 351..431 41/90 (44%)
[WO200172777-A2, 04-OCT-2001]
AAM33060 Peptide #7097 encoded by 5..30 13/26 (50%)1.6
probe for
measuring placental gene 1..25 18/26 (69%)
expression -
Homo Sapiens, 49 aa.
[W0200157272-A2, 09-AUG-2001]
In a BLAST search of public sequence databases, the NOV7a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 7D.
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Table 7D. Public BLASTP
Results for NOV7a
~_~
~ 7a Identities/
Protein ' Similarities
Residues/ Expect
Accession Protein/Organism/Length for the
Match Value
Number Matched
Residues Portion
Q9D3A0 6330414C15RIK PROTEIN - ~ 1..30 14/30 (46%)2.2
Mus
musculus (Mouse), 150 aa. 51..79 19/30 (62%)
Q9Y269 Protein HSPC020 - Homo 1..30 14/30 (46%); 2.2
Sapiens
(Human), and, 121 aa. 51..79 19/30 (62%)
. ~ .
Q9UKD0 _ _ 24/90 (26%)2.9
DNA BINDING PROTEIN P96PIF2..90
(GLUCOCORTICOID 318..398 41/90 (44%)
MODULATORY ELEMENT
BINDING PROTEIN 1 ) - Homo
Sapiens (Human), 563 aa.
Q9NWH1 HYPOTHETICAL 61.4 KDA 2..90 24/90 (26%)2.9
PROTEIN - Homo Sapiens 318..398 41/90 (44%)
(Human),
563 aa.
Q9Y692 GLUCOCORTICOID 2..90 24/90 (26%)3.8
MODULATORY ELEMENT 328..408 40190 (43%)
BINDING PROTEIN-1 - Homo
sapiens (Human), 573 aa.
Examine 8.
The NOV8 clone was analyzed, and the nucleotide and.encoded polypeptide
sequences are
shown in Table 8A.
Table 8A. NOV8 Sequence Analysis
SEQ ID NO: 17 3653 by
NOV8a, CGGGATGCCCGGCTTGCTGAATTGGATCACGGGGGCAGCCCTGCCCCTCACCGCGTCT
CG101458-Ol GATGTTACCTCCTGTGTCAGCGGTTATGCCCTGGGCCTAACTGCCTCCCTCACCTATG
DNA SBqueriCe GCAACCTGGAAGCCCAGCCCTTCCAGGGTCTCTTCGTGTACCCCCTGGATGAGTGCAC
CACGGTGATCGGCTTTGAGGCAGTCATTGCCGACCGTGTCGTGACAGTACAGATCAAG
GACAAAGCCAAGCTGGAGAGCGGCCACTTCGATGCCTCCCATGTTCGATCCCCAACAG
GGATTTGGAGCGGATCCTGTTCGTGGCCAACCTGGGGACCATTGCCCCCATGGAGAAT
GGGTCCTTCTGCCTGCTGTCTGTGCCCCAACCGTGCCCCAGTTCTGCACCAAGAGCAC
TGGCACCTCCAACCAACAGGCCCAGGGGAAAGACAGGCACTGCTTCGGTGCCTGGGCC
CCGGGCTCCTGGAATAAGTTGTGCCTGGCGACTCTCCTGAACACCGAAGTGTCCAACC
CCATGGAGTATGAGTTCAACTTCCAGCTGGAGATCCGTGGGCCATGTCTGCTCGCAGG
TGTGGAGAGTCCCACTCATGAGATTCGTGCCGACGCCGCCCCATCTGCCCGCTCGGCC
TCATCCACCCCAGCGAGCCCCATATGCCCCATGTCCTGATAGAGAAAGGGGACATGAC
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CCTGGGAGAGTTTGACCAGCACTTGAAGGGAAGAACAGATTTCATTAAAGGGATGAAG
TTCCCCACCACTCCGTCATCATGCTCAACTTCTGTCCCGACCTCCAGTCAGTCCAGCC
GTGCCTGAGAAAGGCCCACGGGGAGTTCATCTTCCTCATTGACAGGAGCAGCAGCATG
AGCGGGATCAGCATGCACCGAGTCAAGGATGCCATGTTGGTGGCCCTTAAGAGCCTCA
TGCCAGCCTGCCTCTTCAATATCATTGGGTTTGGATCCACATTTAAGAGCCTTTTTCC
TTCCAGCCAGACCTACAGTGAGGACAGCTTGGCCATGGCTTGTGATGACATCCAGAGA
ATGAAGGCCGACATGGGTGGGACCAACATCCTTTCCCCTCTCAAGTGGGTCATCAGGC
AGCCAGTGCACCGAGGCCACCCGCGGCTCCTCTTCGTGATCACAGATGGCGCTGTCAA
CAACACAGGGAAGGTGCTGGAGCTGGTGCGAAATCACGCCTTCTCCACCAGGTGCTAT
AGCTTTGGAATTGGACCCAACGTCTGCCACAGACTGGTGAAAGGACTGGCATCTGTGT
CCGAGGGCAGTGCTGAGCTCCTGATGGAGGGGGAGCGGCTGCAACCCAAGATGGTCAA
ATCCTTGAAGAAGGCCATGGCCCCAGTCCTGAGCGATGTGACTGTGGAGTGGATCTTC
CCTGAGACCACTGAGGTCCTGGTCTCACCCGTCAGCGCCAGCTCCCTCTTCCCTGGAG
AACGGCTGGTGGGGTATGGCATTGTATGTGATGCTTCTTTGCACATCTCCAATCCCAG
ATCTGACAAGAGGCGCCGGTACAGCATGCTGCACTCTCAGGAGTCTGGCAGCTCTGTC
TTCTACCACTCTCAGGATGACGGACCCGGGCTGGAAGGTGGAGACTGTGCCAAGAACT
CGGGGGCACCCTTCATCCTAGGGCAGGCCAAAAATGCCCGGCTAGCCAGCGGAGACTC
TACCACCAAGCACGGTCTGAACCTCTCTCAGCGACGGAGGGCATACAGCACCAACCAG
ATCACCAATCACAAGCCCCTCCCAAGAGCCACCATGGCAAGTGACCCCATGCCAGCTG
CCAAGAGATACCCACTGCGGAAAGCCAGGCTGCAGGACCTCACCAACCAGACCAGCCT
GGATGTCCAGCGGTGGCAGATTGATTTGCAGGTATTGCTGAACAGTGGTCAGGACCTG
GCTGCCAGCCCTTCCTGCCCTGGGGCCAGGAGACCCAGGCCTGGAGCCCTGTGAGAGA
GCGGACTTCTGACAGCCGAAGCCCTGGAGATCTGCCCGCAGAGCCGTCCCACCATCCC
TCTGCCTTCGAGACAGAGACGTCCTCGGACTGGGACCCCCCAGCCGAGTCCCAGGAGC
GAGCCAGTCCCAGCAGGCCCGCCACCCCGGCCCCGGTGCTGGGCAAGGCCCTGGTCAA
AGGCCTGCACGACAGCCAACGCCTGCAGTGGGAGGTGAGCTTCGAGCTGGGGACCCCT
GGACCGGAGCGGGGCGGCGCGCAGGATGCCGACCTATGGAGCGAGACCTTCCACCACC
TGGCGGCCCGCGCCATCATCCGCGACTTCGAGCAGCTGGCGGAGCGCGAGGGCGAGAT
CGAGCAGGGTTCCAACCGCCGCTACCAAGTGAGCGCCTTGCACACCAGCAAGGCCTGC
TTAGCAAATACACAGCCTTCGTGCCTGTGGACGTGAGCAAGAGCCGGTACC
TGCCCACCGTGGTGGAGTACCCCAACTCTGGTCGTATGCTTGGCTCTCGGGCCCTGGC
GATTCGGCACCAGGAAATGGTAAATTTCAGGTCCTAGACATGGAGGCAAGTCCCACTG
CTCTCTTCAGCGAGGCCAGGTCCCCCGGCCGCGAGAAGCACGGTGCTTCTGAAGGTCC
CCAGCGCAGCCTGGCTACAAATACTCTTTCTTCCATGAAGGCCTCAGAGAATCTCTTT
GGATCCAGGCTAAATCTCAACAAGTCCAGGCTACTGACGCGAGCAGCCAAGGGCTTCC
TGAGCAAGCCACTGATCAAAGCTGTGGAGTCGACCTCCGGGAACCAGAGCTTCGACTA
CATACCTCTGGTGTCTCTGCAGCTGGCCTCCGGAGCCTTCCTGCTCAACGAAGCCTTC
TGTGAGGCCACGCACATCCCCATGGAGAAGCTCAAGTGGACGTCCCCCTTCACCTGCC
ATCGAGTGTCCCTCACCACCCGCCCGTCTGAGTCCAAGACCCCGAGTCCCCAGCTGTG
CACCAGCTCCCCGCCTAGGCACCCGTCCTGTGACAGCTTCTCCCTGGAGCCTCTGGCC
AAGGGCAAGCTGGGCCTGGAGCCGAGGGCAGTGGTGGAGCACACTGGGAAGCTGTGGG
CCACGGTGGTGGGGCTGGCATGGCTGGAGCACAGTTCGGCCTCCTACTTCACTGAGTG
GGAGTTGGTGGCTGCCAAGGCCAACTCATGGCTGGAGCAGCAGGAAGTACCCGAGGGC
CGCACGCAGGGCACACTCAAGGCCGCTGCCCGCCAGCTGTTTGTGCTTCTGCGGCACT
GGGATGAGAATCTCGAGTTCAATATGCTCTGCTATAACCCGAATTATGTGTAGTTGA
ORF Start: ATG at 5 ~ORF Stop: TAG at 3647_
SEQ ID NO: 18 1214 as MW at 1331 lB.OkD
NOVBa, MPGLLNWITGAALPLTASDVTSCVSGYALGLTASLTYGNLEAQPFQGLFWPLDECTT
CG101458-O1 VIGFEAVIADRWTVQIKDKAKLESGHFDASHVRSPTVTGKETRRAAAGPGKVTLDED
Protein S8 Lt~rlCe LERILFVANLGTIAPMENVTIFISTSSELPTLPSGAVRVLLPAVCAPTVPQFCTKSTG
TSNQQAQGKDRHCFGAWAPGSWNKLCLATLLNTEVSNPMEYEFNFQLEIRGPCLLAGV
ESPTHEIRADAAPSARSAKSIIITLANKHTFDRPVEILIHPSEPHMPHVLTEKGDMTL
GEFDQHLKGRTDFIKGMKKKSRAERKTEIIRKRLHKDIPHHSVIMLNFCPDLQSVQPC
LRKAHGEFIFLIDRSSSMSGISMHRVKDAMLVALKSLMPACLFNIIGFGSTFKSLFPS
SQTYSEDSLAMACDDIQRMKADMGGTNILSPLKWVIRQPVHRGHPRLLFVITDGAVNN
TGKVLELVRNHAFSTRCYSFGIGPNVCHRLVKGLASVSEGSAELLMEGERLQPKMVKS
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LKKAMAPVLSDVTVEWIFPETTEVLVSPVSASSLFPGERLVGYGIVCDASLHISNPRS
DKRRRYSMLHSQESGSSVFYHSQDDGPGLEGGDCAKNSGAPFILGQAKNARLASGDST
TKHGLNLSQRRRAYSTNQITNHKPLPRATMASDPMPAAKRYPLRKARLQDLTNQTSLD
VQRWQIDLQVLLNSGQDLNQGPKLRGPGARRPSLLPQGCQPFLPWGQETQAWSPVRER
TSDSRSPGDLPAEPSHHPSAFETETSSDWDPPAESQERASPSRPATPAPVLGKALVKG
LHDSQRLQWEVSFELGTPGPERGGAQDADLWSETFHHLAARAIIRDFEQLAEREGEIE
QGSNRRYQVSALHTSKACNIISKYTAFVPVDVSKSRYLPTVVEYPNSGRMLGSRALAQ
QWRGTSSGFGRPQTMLGEDSAPGNGKFQVLDMEASPTALFSEARSPGREKHGASEGPQ
RSLATNTLSSMKASENLFGSRLNLNKSRLLTRAAKGFLSKPLIKAVESTSGNQSFDYI
PLVSLQLASGAFLLNEAFCEATHIPMEKLKWTSPFTCHRVSLTTRPSESKTPSPQLCT
SSPPRHPSCDSFSLEPLAKGKLGLEPRAVVEHTGKLWATVVGLAWLEHSSASYFTEWE
LVAAKANSWLEQQEVPEGRTQGTLKAAARQLFVLLRHWDENLEFNMLCYNPNYV
Further analysis of the NOVBa protein yielded the following properties shown
in Table 8B.
Table 8B. Protein Sequence Properties NOVBa"
PSort 0.8700 probability located in nucleus; 0.8500 probability located in
analysis: endoplasmic reticulum (membrane); 0.7900 probability located in
plasma
membrane; 0.3325 probability located in microbody (peroxisome)
SignalP Cleavage site between residues 19 and 20
analysis:
A search of the NOV 8a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 8C.
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~. . ~ ... . Table 8C. _........
Geneseq .Results for NOVBa
. ~... .
NOVBa Identities/
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent ~ for ~
Identifier#, Datej Match the Matched Value
i
Residues Region
AAB82047 Human mast cell surface 13..565 168/565 (29%)2e-59
antigen - ~
Homo Sapiens, 786 aa. 15:.500 269/565 (46%)
[JP2001025388-A, 30-JAN-2001]
AAY82530 Human neurotransmitter 1034..121182/194(42%) 1e-32
associated
protein sequence SEQ ID 16..207 105/194 (53%)
N0:6 - 3
Homo Sapiens, 210 aa.
[W0200012685-A2, 09-MAR-
2000] _
AAU33242 Novel human secreted protein36..568 120/537 (22%)4e-23
~
#3?33 - Homo Sapiens, 1730650..1096214/537 (39%)
aa. '
[W0200179449-A2, 25-OCT-2001]
AAB51022 Human minor vault protein 36..568 120/537 (22%)6e-23
p193 - ~
Homo Sapiens, 1724 aa. 644..1090214/537 (39%)
[US6156879-A, OS-DEC-2000]
.
~.
AAY54373 cDNA sequence encoding 36..568 120/537 (22%)6e-23
the ~ ~ ~
human minor vault protein 644..1090214/537 (39%)
p193 -
Homo sa iens 1724 aa.
p
[W09962547-Al, 09-DEC-1999] t
In a BLAST search of public sequence databases, the NOVBa protein was found to
nave
homology to the proteins shown in the BLASTP data in Table 8D.
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Table 8D. Public BLASTP
Results for NOVBa
NOVBa ~ Identities/
Protein
Residues/ SimilaritiesExpect
AccessionProtein/Organism/Length for
'
Match the Matched Value
Number
Residues Portion
Q9CUE8 4931403E03RIK PROTEIN 1..1208 883/1218 0.0
- Mus ~ (72%)
musculus (Mouse), 1209 1..1209 1012/1218
as (82%)
', (fragment).
Q96M7I CDNA FLJ32784 FIS, CLONE588..953 362/369 (98%)0.0
~
TESTI2002245 - Homo Sapiens1..367 362/369 (98%)
(Human), 424 aa. ~
:
Q9BVH8 HYPOTHETICAL 106.2 KDA 274..1211 31111047 e-106
~ (29%)
PROTEIN - Homo Sapiens 32..998 467/1047
~ (43%)
(Human), 1001 as (fragment).
075668 DJ745E8.1 (BREAST CANCER~ 417..564148/148 (100%)4e-80
SUPPRESSOR CANDIDATE 1..148 148/148 (100%)
1
(BCSC-1) LIKE) - Homo
sapiens
(Human), 148 as (fragment).
Q9CTV9 5830475I06RII~ PROTEIN 13..565 165/567 (29%)
- Mus ~ Se-57
musculus (Mouse), 565 15..500 259/567 (45%)
as
~fragment).
_._. .. __...._ ~ .. .. . ..... ...
~- .-y_...~
PFam analysis predicts that the NOVBa protein contains the domains shown in
the Table
8E.
Table 8E. Domain Analysis of NOVBa
Identities/
Pfam Domain NOVBa Match Region Similarities Expect Value
for the Matched Region
wwa 355 523 ~ 37/203 (18%) 0.021
107/203 (53°1°)
Example 9.
The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 9A.
Table 9A. NOV9 Sequence Analysis
SEQ ID NO: 19 868 by
NOV9a, CGTTTTCTTCTACAATGTCTGAAGAAGTGACCTACGCGACACTCACATTTCAGGATTC
TGCTGGAGCAAGGAATAACCGAGATGGAAATAACCTAAGAAAAAGAGGTCATCCAGCT
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CG101475-Ol CCATCTCCCATTTGGCGTCATGCTGCTCTGGGTCTGGTAACTCTTTGCCTGATGTTGC
DNA Sequence TGATTGGGCTGGTGACATTGGGGATGATGTGTTTGCAGATATCTAATGACATTAACTC
AGATTCAGAGAAATTGAGTCAACTTCAGAAAACCATCCAACAGCAGCAGGATAACTTA
TCCCAGCAACTGGGCAACTCCAACAACTTGTCCATGGAGGAGGAATTTCTCAAGTCAC
AGATCTCCAGTGTACTGAAGAGGCAGGAACAAATGGCCATCAAACTGTGCCAAGAGCT
AATCATTCATTTTTCAGACCACAGATG'T'AATCCATGTCCTAAGATGTGGCAATGGTAC
CAAAATAGTTGCTACTATTTTACAACAAATGAGGAGAAAACCTGGGCTAACAGTAGAA
AGGACTGCATAGACAAGAACTCCACCC'I'AGTGAAGATAGACAGTTTGGAAGAAAAGGA
TTTTCTTATGTCACAGCCATTACTCATGTTTTCGTTCTTTTGGCTGGGATTATCATGG
GACTCCTCTGGCAGAAGTTGGTTCTGGGAAGATGGCTCTGTTCCCTCTCCATCCTTGA
GTAC'Z'ARAGAACTTGACCAGATCAATGGATCCAAAGGATGTGCTTATTTTCAAAAAGG
AAATATTTATATTTCTCGCTGTAGTGCTGAAATTTTTTGGATTTGCGAGAAGACAGCT
GCCCCAGTGAAGACTGAGGA'T'TTGGATTAGTATGCTTCTTCCAAATTCTCCAAGAA
_.
..
-
~
0~
..
0~ Start: ATG at
l s
Stop. TAG at 840
SEQ ID NO: 20 X275 as MW at 31470.4kD
NOV9a, MSEEVTYATLTFQDSAGARNNRDGNNLRKRGHPAPSPIWRHAALGLVTLCLMLLTGLV
CG101475-Ol TLGMMCLQISNDINSDSEKLSQLQKTIQQQQDNLSQQLGNSNNLSMEEEFLKSQISSV
Protein SeqLleriCeL~'QEQMAIICLCQELIIHFSDHRCNPCPKMWQWYQNSCYYFTTNEEKTWANSRKDCID
KNSTLVKIDSLEEKDFLMSQPLLMFSFFWLGLSWDSSGRSWFWEDGSVPSPSLSTKEL
DQINGSKGCAYFQKGNIYISRCSAEIFWICEKTAAPVKTEDLD
SEQ ID NO: 21 819 by
NOV9b, ACACTCACATTTCAGGATTCTGCTGGAGCAAGGAATAACCGAGATGGAAATAACCTAA
CGIOI47S-O2 G~GAGGGCATCCAGCTCCATCTCCCATTTGGCGTCATGCTGCTCTGGGTCTGGT
'DNA SeqLlenCe~CTCTTTGCCTGATGTTGCTGATTGGGCTGGTGACGTTGGGGATGATGTTTTTGCAG
ATATCTAATGACATTAACTCAGATTCAGAGAAATTGAGTCAACTTCAGAAAACCATCC
AACAGCAGCAGGATAACTTATCCCAGCAACTGGGCAACTCCAACAACTTGTCCATGGA
GGAGGAATTTCTCAAGTCACAGATCTCCAGTGTACTGAAGAGGCAGGAACAAATGGCC
ATCAAACTGTGCCAAGAGCTAATCATTCATACTTCAGACCACAGATGTAATCCATGTC
CTAAGATGTGGCAATGGTACCAAAATAGTTGCTACTATTTTACAACAAATGAGGAGAA
AACCTGGGCTAACAGTAGAAAGGACTGCATAGACAAGAACTCCACCCTAGTGAAGATA
GACAGTTTGGAAGAAAAGGATTTTCTTATGTCACAGCCATTACTCATGTTTTCGTTCT
TTTGGCTGGGATTATCATGGGACTCCTCTGGCAGAAGTTGGTTCTGGGAAGATGGCTC
TGTTCCCTCTCCATCCTTATTTAGTACTAAAGAACTTGACCAGATCAATGGATCCAAA
GGATGTGCTTATTTTCAAAAAGGAAATATTTATATTTCTCGCTGTAGTGCTGAAATTT
TTTGGATTTGCGAGAAGACAGCTGCCCCAGTGAAGACTGAGGATTTGGATTAGAAGGG
CGATTCC
ORF Start: at 1 ORF Stop: TAG at 80S
SEQ ID NO: 22 ~ 268 aa. ~MW at 30704 SkD
NOV9b, TLTFQDSAGARNNRDGNNLRKRGHPAPSPIWRHAALGLVTLCLMLLIGLVTLGMMFLQ
CG101475-02 ISNDINSDSEKLSQLQKTIQQQQDNLSQQLGNSNNLSMEEEFLKSQISSVLKRQEQMA
Protein SeqLlenCeIKLCQELIIHTSDHRCNPCPKMWQWYQNSCYYFTTNEEKTWANSRKDCIDKNSTLVKI
DSLEEKDFLMSQPLLMFSFFWLGLSWDSSGRSWFWEDGSVPSPSLFSTKELDQINGSK
GCAYFQKGNIYISRCSAEIFWICEKTAAPVKTEDLD
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 9B.
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Table 9B. Comparison of NOV9a against NOV9b.
Protein Sequence NOV9a Residues/ ~ Identities/
Match Residues Similarities for the Matched Region
NOV9b 9..275 241/268 (89%)
1..268 ' 241/268 (89%)
Further analysis of the NOV9a protein yielded the following properties shown
in Table 9C.
. J~~_~ ........_
Table 9C. Protein Sequence Properties NOV9a~
PSort 0.7900 probability located in plasma membrane; 0.3000 probability
located in
analysis: Golgi body; 0.2000 probability located in endoplasmic reticulum
(membrane);
0.1000 probability located in mitochondria) inner membrane
SignalP Cleavage site between residues 62 and 63
analysis:
A search of the NOV9a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 9D.
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Table 9D. Geneseq Results for NOV9a
NOV9a Identities!
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent for
Identifier#, Date] Match the Matched Value
Residues Region
AAU29320 Human PRO polypeptide 1..227 224/227 (98%)e-131
sequence
#297 - Homo Sapiens, 232 1..227 225/227 (98%)
aa.
[W0200168848-A2, 20-SEP-2001]
AAM79324 Human protein SEQ ID NO 1..270 91/270 (33%)3e-37
2970 -
Homo Sapiens, 289 aa. 25..280 147/270 (53%)
[W0200157190-A2, 09-AUG-
2001]
ABB11776 Human macrophage Ag homologue,1..270 91/270 (33%)3e-37
SEQ ID N0:2146 - Homo 25..280 147/270 (53%)
Sapiens,
289 aa. [W0200157188-A2,
09-
AUG-2001 ]
AAM78340 Human protein SEQ ID NO 1..270 88/270 (32%)!e-35
1002 -
Homo sapiens, 265 aa. 1..256 147/270 (53%)
[WO200157190-A2, 09-AUG-
2001 ]
AAY02283 Secreted protein clone 1..270 88/270 (32%)!e-35
br342_11
polypeptide sequence - 1..256 147/270 (53%)
Homo
Sapiens, 265 aa. [W09918127-Al,
15-APR-1999]
In a BLAST search of public sequence databases, the NOV9a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 9E.
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Table 9E. Public BLASTP
Results for NOV9a
Protein NOV9a Identities/
AccessionProtein/OrganismlLength Residues!SimilaritiesExpect
' for
Number Match the Matched Value
Residues Portion
'
Q9D403 4933425B16RIK PROTEIN - 1..275 197/276 (71%)e-113
Mus ~
musculus (Mouse), 275 aa. 1..275 227/276 (81
~ %)
AAL95693 C-TYPE LECTIN PROTEIN 1..270 88/270 (32%)2e-35
CLL-1 - Homo Sapiens (Human),1..256 147/270 (53%)
~
265 aa.
5
..... .. ............. ................ .... .... ........
.. ......... .... . ........ ..... ... ... . .
.... ... ~.. ... ... .....
Q9NZH3 C-TYPE LECTIN-LIKE 28..274 83/249 (33%)Se-33
~
RECEPTOR-1 - Homo sapiens 36..269 131/249 (52%)
(Human), 280 aa.
Q9~TA8 LECTIN-LIKE OXIDIZED LDL 36..272 79/247 (31%)Y~Se-27
3 ~
RECEPTOR - Oryctolagus 36..278 124/247 (49%)
cuniculus (Rabbit), 278 3
aa.
P78380 LECTIN-LIKE OXIDIZED LDL 36..266 79/245 (32%)3e-24
~
RECEPTOR - Homo Sapiens 32..268 124/245 (50%)
(Human), 273 aa.
PFam analysis predicts that the NOV9a protein contains the domains shown in
the Table
9F.
Table 9I'. Domain Analysis of NOV9a
Identities/
Pfam Domain NOV9a Match Region ~ Similarities ' Expect Value
for the Matched Region
Iectin_c 161..264 29/125 (23%) ~ 7.4e-06
61/125 (49%)
_..... ....
Example 10,
The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 1 OA.
Table 10A. NOV10 Sequence Analysis
SEQ ID NO: 23 516 by
NOVIOa, CACTGCGCATGCTATTTGGGCGCCCACCTCAGTGCACATGTTCACTGGGCGTCTTCTA
CG101772-Ol CTCTACCCCTTCGCCCTCGTGGGGGTGTGAGGGTCGCGTTCCTGCTGTCTGGACTTTT
DNA Sequence TCTGTCCCACTGAGACGCAATGTATCGATAACAAAACTTTTTATCTGCACACACACAC
ACACACACACACACCCCTGGTTCCAGGAGCCCGGTGATGAGGAGCCTCAGCAAGAGGA
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ACCACCAACTGAAAGTCGGGATCCTGCACCTGGTCAGGAGAGAGAAGAAGATCAGGGT
GCAGCTGAGACTCAATGCCTGACCTGGAAGCTGATCTCCAGGAGCTGTCTCAGTCAAA
GACTGGGGGTGAATGTGGAAATGAAGATTCTGCCAAAATCAGAACAATTTAAAATGCC
AGAAGGAGGTATGCTATCCATTATTATGTGCTTTCTGTTTTCCACAATATTATACTTT
TGATAATAAAAGAGAACATTACTATCCCTTTAAAATCAGAGTTCAAATGCAG
ORF Start: ATG at 9 ORF Stop: TGA at 465
SEQ ID NO: 24 152 as MW at 17265.6kD
NOVIOa, MLFGRPPQCTCSLGVFYSTPSPSWGCEGRVPAVWTFSVPLRRNVSITKLFICTHTHTH
CG101772-Ol THPWFQEPGDEEPQQEEPPTESRDPAPGQEREEDQGAAETQCLTWKLISRSCLSQRLG
~UNVEMKILPKSEQFKMPEGGMLSIIMCFLFSTILYF
Protein Sequence
Further analysis of the NOV 10a protein yielded the following properties shown
in Table
10B.
Table 10B. Protein Sequence Properties NOVlOa
PSort 0.9190 probability located in plasma membrane; 0.2000 probability
located in
analysis: lysosome (membrane); 0.1021 probability located in microbody
(peroxisome);
0.1000 probability located in endoplasmic reticulum (membrane)
SignalP No Known Signal Sequence Predicted
analysis:
A search of the NOV 1 Oa protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table l OC.
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Table 10C. Geneseq Results
for NOVlOa
NOVlOa Identities)
'
Geneseq ProteinlOrganism/Length Residues/SimilaritiesExpect
[Patent '
Identifier#, Date] Match for the Value
Residues Matched
Region
AAM39588 Human polypeptide SEQ ID 65..136 51/78 (65%)4e-20
NO
2733 - Homo Sapiens, 111 28..105 56/78 (71%)
aa.
[W0200153312-Al, 26-JUL-2001]
AAM41374 Human polypeptide SEQ ID 65..135 52/77 (67%)2e-19
NO
6305 - Homo sapiens, 106 29..105 57/77 (73%)
aa.
[W0200153312-Al, 26-JUL-2001]
ABG05297 Novel human diagnostic 65..136 48/78 (61%)8e-19
protein
#5288 - Homo Sapiens, 112 29..106 56178 (71
aa. %)
[W0200175067-A2, 11-OCT-2001]
ABG05297 Novel human diagnostic 65..136 48/78 (61%)8e-19
protein
#5288 - Homo Sapiens, 112 29..106 56/78 (71
aa. %)
[W0200175067-A2, 11-OCT-2001]
ABG27048 Novel human diagnostic 64..135 45/78 (57%)5e-15
protein
#27039 - Homo Sapiens, 70..147 53/78 (67%)
249 aa. ~
[W0200175067 A2, 1 l -OCT
2001 ]
.. . .... . _ ....
. . . . .
. ... ...
.. ._..
~ .
In a BLAST search of public sequence databases, the NOV 10a protein was found
to have
homology to the proteins shown in the BLASTP data in Table )OD.
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Table !OD Public BLASTP Results for NOVlOa ~~
Protein . NOVlOa Identities/
Residues/ Similarities for ; Expect
Accession Protein/Organism/Length Match the Matched ~ Value
Number
Residues Portion
Q8WTP9 RAGE-3 PROTEIN - Homo 65..136 51/78 (65%) ~ 1e-19
Sapiens (Human)~111 aa. 28..105 56/78 (71%)
Q8WYS9 ! HYPOTHETICAL 12.3-KDAy y 65..136 51/78 (65%) ~ 1e-19
PROTEIN -Homo sapiens ~ 28..105 56178 (71%)
' (Human), 111 aa.
Q9HD64 , G antigen family D 2 protein 1..136 59/149 (39%) ~ 3e-18
(RAGE-1) - Homo Sapiens 1..140 76/149 (50%)
(Human), 146 aa.
Q8WWM1 ' RAGE-5 PROTEIN - Homo 65..136 45/78 (57%) 9e-15
sapiens (Human), 108 aa. 25..102 53/78 (67%)
Q96GT9 ~ SIMILAR TO G ANTIGEN 8 65..136 39/78 (50%) ~ 7e-13
~ (XAGE-2 PROTEIN) - Homo 28..105. 53/78 (67%)
sapiens (Human), 111 aa.
Example 11.
The NOV 11 clone waS analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 1 1A.
Table 11A. NOVll Sequence Analysis
SEQ ID NO: 25 709 by
NOVlla, CGGCCGGTTTTGGTAGGCCCGGGCCGCCGCCAGGCCTCCGCCTGAGCCCGCACCCGCC
CG102532-O1ATGGACAACTACGCAGATCTTTCGGATACCGAGCTGACCACCTTGCTGCGCCGGTACA
DNA SequeriCeACATCCCGCACGGGCCTGTAGTAGGATCAACTCGTAGGCTTTACGAGAAGAAGATCTT
CGAGTACGAGACCCAGAGGCGGCGGCTCTCGCCCCCCAGCTCGTCCGCCGCCTCCTCT
TATAGCTTCTCTGACTTGAATTCGACTAGAGGGGATGCAGATATGTATGATCTTCCCA
AGAAAGAGGACGCTTTACTCTACCAGAGCAAGGGCTACAATGACGATCTTTTGTCTTC
TTCTGAAGAGGAGTGCAAGGATAGGGAACGCCCCATGTACGGCCGGGACAGTGCCTAC
CAGAGCATCACGCACTACCGCCCTGTTTCAGCCTCCAGGAGCTCCCTGGACCTGTCCT
ATTATCCTACTTCCTCCTCCACCTCTTTTATGTCCTCCTCATCATCTTCCTCTTCATG
GCTCACCCGCCGTGCCATCCGGCCTGAAAACCGTGCTCCTGGGGCTGGGCTGGGCCAG
GATCGCCAGGTCCCGCTCTGGGGCCAGCTGCTGCTTTTCCTGGTCTTTGTGATCGTCC
TCTTCTTCATTTACCACTTCATGCAGGCTGAAGAAGGCAACCCCTTCTGACTGCAGCC
AAGCTAATTCCGG
ORF Start: ATG at 59 ORF Stop: TGA at 686
SEQ ID NO: 26 209 as MW at 23844.1kD
,.,...,.~:.,.~..., ~.,.~..,~,....~.~.... .~,.w....-.,..~.~........J
NOVlla, MDNYADLSDTELTTLLRRYNIPHGPWGSTRRLYEKKIFEYETQRRRLSPPSSSAASS
CG102532-O1YSFSDLNSTRGDADMYDLPKKEDALLYQSKGYNDDLLSSSEEECKDRERPMYGRDSAY
QSITHYRPVSASRSSLDLSYYPTSSSTSFMSSSSSSSSWLTRRAIRPENRAPGAGLGQ
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PrOteln SeqllenCe DRQVPLWGQLLLFLVFVIVLFFIYHFMQAEEGNPF
Further analysis of the NOV 11 a protein yielded the following properties
shown in Table
11B.
Table 11B. Protein Sequence Properties NOVlla
PSort 0.8500 probability located in endoplasmic reticulum (membrane); 0.6000
analysis: probability located in nucleus; 0.4400 probability located in plasma
membrane; 0.2323 probability located in microbody (peroxisome)
SignalP No Known Signal Sequence Predicted
analysis:
A search of the NOV 11 a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 11 C. . .
Table 11C. Geneseq Results for NOVlla
NOVlla Identities/ 1
~
Geneseq ~ Protein/Organism/LengthResidues/SimilaritiesExpect
for
Identifier(Patent #, Date) Match the Matched ~ Value
Residues Region rc
AAY41294 ~ Human emerin sequence 1..209 209/254 (82%)~ e-112
(EMD HU) - Homo Sapiens, 1..254 209/254 (82%)
254
aa. [W09954468-Al, 28-OCT-~
1999] ~ rt
AAG02346 ~ Human secreted protein,1..51 51/51 (100%)2e-23
SEQ ID
NO: 6427 - Homo Sapiens, 1..51 51 /51 (
51 aa. 100%)
[EP 1033401-A2, 06-SEP-2000]
AAY41297 ~ Human thymopoietin gamma6..209 60/231 (25%)7e-10
~
sequence - Homo Sapiens, 114..333 107/231 (45%)
345 aa.
[WO9954468-A1, 28-OCT-1999]
AAR93188 ~ Thymopoietin-gamma - 6..209 60/231 (2S%)~ 7e-10
Homo
Sapiens, 345 aa. [W09609526-Al,114..333 107/231 (45%)
28-MAR-1996]
AAR76499 Human thymopoietin-gamma 6..209 60/231 (25%)~ 7e-10
-
Homo Sapiens, 345 aa. 114..333 107/231 (45%)
~ [W09517205-A1, 29-JUN-1995]
In a BLAST search of public sequence databases, the NOV 11 a protein was found
to have
homology to the proteins shown in the BLASTP data in Table I 1 D.
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~.. ~.... . . . ~_. _..~
.. ....... . .. ~_...
__, .
Table 11D. Public BLASTP
Results for NOVlla
Protein NOVlla Identities/
Accession Protein/Organism/Length Residues/ Similarities~ Expect
~ for
Number Match the Matched Value
Residues Portion
P50402 ' Emerin - Homo Sapiens 1..209 209/254 (82%)e-111
(Human), 254 aa. 1..254 209/254 (82%)
Q63190 Emerin - Rattus norvegicus1..209 162/256 (63%)1e-81
~ ~
(Rat), 260 aa. 1..256 182/256 (70%)
~
008579 Emerin - Mus musculus 1..209 162/255 (63%)1e-81
(Mouse), 259 aa. ~ 1..255 182/255 (70%)
Q61032 THYMOPOIETIN GAMMA - 6..209 66/231 (28%)2e-l
l
Mus musculus (Mouse), 112..331 106/231 (45%)
342 aa. ~ ~
AAC25390 THYMOPOIETIN GAMMA - 6..209 60/231 (25%). 2e-09
~
Homo sapiens (Human), 114..333 107/231 (45%)
345 aa. s,
.. ... . _ . h _ ~ ._
PFam analysis predicts that the NOV 11 a protein contains the domains shown in
the Table
11E.
Table 11E. Domain Analysis of NOVlla
Identities/
Pfam Domain NOVlla Match Region' Similarities Expect Value
for the Matched Region
LEM 1..44 22/47 (47%) 4.4e-24
43/47 (91 %)
Example 12.
The NOV 12 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 12A.
Table 12A. NOV12 Sequence Analysis
SEQ ID N0: 27 2812 by
NOVl2a, CATTGAGTCGGCTTTTCTACTGCTTCGGCTAGGGTACCTTGTGACCATGTCTTCCAAG
CG102575-Ol ~G~TAGAAAGCGGTTGAACCAAAGCGCGGAAAATGGTTCGTCCTTGCCCTCTGCTG
DNA SBqLlenCe CTTCCTCTTGTGCGGAGGCACGGGCTCCTTCTGCTGGATCAGACTTCGCGGCAACCTC
CGGGACTCTGACGGTGACCAACTTATTAGAAAAGGGTAAAATTCCTAAAACATTCCAG
AATTCCCTTATTCATCTTGGACTCAACACTATGAAGTCTGCAAATATATGTATAGGTC
GACCAGTGTTGCTTACTAGTTTGAACGGAAAGCAAGAGGTATATACAGCCTGGCCTAT
GGCAGGATTTCCTGGAGGCAAGGTCGGCCTGAGTGAAATGGCACAGAAAAATGTGGGT
GTGAGGCCTGGTGATGCCATCCAGGTCCAGCCTCTTGTGGGTGCTGTGCTACAGGCTG
AGGAAATGGATGTGGCACTGAGTGACAAAGATATGGAAATTAATGAAGAAGAACTGAC
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TGGTTGTATCCTGAGAAAACTAGATGGCAAGATTGTTTTACCAGGCAACTTTCTGTAT
TGTACATTCTATGGACGACCGTACAAGCTGCAAGTATTGCGAGTGAAAGGGGCAGATG
GCATGATATTGGGAGGGCCTCAGAGTGACTCTGACACTGATGCCCAAAGAATGGCCTT
TGAACAGTCCAGCATGGAAACCAGTAGCCTGGAGTTATCCTTACAGCTAAGCCAGTTA
GATCTGGAGGATACCCAGATCCCAACATCAAGAAGTACTCCTTATAAACCAATTGATG
ACAGAATTACAAATAAAGCCAGTGATGTTTTGCTGGATGTTACACAGAGCCCTGGAGA
TGGCAGTGGACTTATGCTAGAGGAAGTCACAGGTCTTAAATGTAATTTTGAATCTGCC
AGAGAAGGAAATGAGCAACTTACTGAAGAAGAGAGACTGCTAAAGTTCAGCATAGGAG
CAAAGTGCAATACTGATACTTTTTATTTTATTTCTTCAACAACAAGAGTCAATTTTAC
AGAGATTGATAAAAATTCAAAAGAGCAAGACAGTGATGTTAAAAGTAACTATGACCAT
GATAGAGGATTAAGTAGCCAGCTGAA.AGCAATTAGAGAAATAATTGAATTGCCCCTCA
AAATTCCTGCCCCTAGAGGATTGTTACTTTATGGTCCTCCATGTACTGGAAAAACAAT
GATCGCCAGGGCTGTTGCTAATGAATTTGGAGCCTATGTTTCTGTAATTAATGGTCCT
GAAATTATAAGCAAGTTCTATGGTGAGACTGAAGCAAAGTTACGTCAGATATTTGCTG
AAGCCACTCTAAGACACCCATCAATTATTTTTATTGATGAGCTGGATGCACTTTGTCC
GAAAAGAGAGGGGGCCCAGAATGAAGTGGAAAAAAGAGTTGTGGCTTCACTCTTAACA
CTGATGGATGGCATTGGTTCAGAAGTAAGTGAAGGACAAGTGTTGGTTCTTGGGGCCA
CAAATCGCCCTCATGCCTTGGATGCTGCTCTCCGAAGACCTGGGCGATTTGATAAAGA'
GATTGAGATTGGAGTTCCCAATGCTCAGGACCGGCTAGATATTCTCCAGAAACTGCTT'
CGAAGGGTACCCCATTTGCTCACTGAGGCTGAGCTGCTGCAGCTGGCAAATAGTGCTC
ATGGATACGTTGGAGCAGACTTGAAAGTCTTGTGTAATGAAGCAGGTCTCTGTGCCTT'
GCGGAGAATCCTGAAAAAACAGCCTAACCTCCCTGATGTCAAGGTGGCTGGACTGGTG''
AAGATTACTCTGAAGGATTTCTTGCAGGCAATGAATGATATCAGACCCAGTGCCATGA!
GGGAAATAGCAATTGATGTCCCAAATGTAAGTTATGATGATGTTGGTGGAGTTAGAAAi
GCAAATGGCCCAAATCAGAGAGCTTGTTGAGCTTCCACTACGCCATCCTCAACTTTTC
AAATCTATTGGTATTCCTGCCCCTAGAGGATTGTTACTTTATGGTCCTCCATGTACTG
GAAAAACAATGATCGCCAGGGCTGTTGCTAATGAATTTGGAGCCTATGTTTCTGTAAT
TAATGGTCCTGAAATTATAAGCAAGTATGTTGGTGAGAGTGAACGTGCTGTGCGACAA
GTTTTTCAACGAGCCAAGAACTCAGCACCATCAATTATTTTTATTGATGAGCTGGATG
CACTTTGTCCGAAAAGAGAGGGGGCCCAGAATGAAGTGGAAAAAAGAGTTGTGGCTTC
ACTCTTAACACTGATGGATGGCATTGGTTCAGTAAGTATAGTGTTGGTTCTTGGGGCC
ACAAATCGCCCTCATGCCTTGGATGCTGCTCTCCGAAGACCTGGGCGATTTGATAAAG
TCGAAGGGTACCCCATTTGCTCACTGAGGCTGAGCTGCTGCAGCTGGCAAATAGTGCT
CATGGATACGTTGGAGCAGACTTGAAAGTCTTGTGTAATGAAGCAGGTGAGTGTGGTT
TGCTATGGGACATTCAAGCCAATCTCATCATGAAAAGACATTTCACTCAGGCCTTGAG
CACTGTGACACCTAGAATTCCTGAGTCATTGAGACGTTTTTATGAAGATTATCAAGAG
AAGAGTGGGCTGCATACACTCTGAGAAAATATATATATTCAAGATGCTGAAAATCCTT
TCCAGAGAAAATTGTTTCTTTTTAAAATTTTTGAGAGTGTTAAAAAAAATTTTACTAG
GCAAAATGTTTGAAGTATGTTCAGTAGA
ORF Start: ATG at 47 ORF Stop: TGA at 2690
SEQ ID NO: 28 881 as MW at 96419.SkD
NOVl2a, MSSKKNRKRLNQSAENGSSLPSAASSCAEARAPSAGSDFAATSGTLWTNLLEKGKIP
CG1O2S7S-O1 KTFQNSLIHLGLNTMKSANICIGRPVLLTSLNGKQEVYTAWPMAGFPGGKVGLSEMAQ
Frotein Se lleriCe ~G~PGDAIQVQPLVGAVLQAEEMDVALSDKDMEINEEELTGCILRKLDGKIVLPG,
NFLYCTFYGRPYKLQVLRVKGADGMILGGPQSDSDTDAQRMAFEQSSMETSSLELSLQ',
LSQLDLEDTQIPTSRSTPYKPIDDRITNKASDVLLDVTQSPGDGSGLMLEEVTGLKCN':
FESAREGNEQLTEEERLLKFSIGAKCNTDTFYFISSTTRVNFTEIDKNSKEQDSDVKSI
NYDHDRGLSSQLKAIREIIELPLKIPAPRGLLLYGPPCTGKTMIARAVANEFGAYVSV:
INGPEIISKFYGETEAKLRQIFAEATLRHPSIIFIDELDALCPKREGAQNEVEKRWA'
SLLTLMDGIGSEVSEGQVLVLGATNRPHALDAALRRPGRFDKEIEIGVPNAQDRLDIL)
QKLLRRVPHLLTEAELLQLANSAHGWGADLKVLCNEAGLCALRRILKKQPNLPDVKV
AGLVKITLKDFLQAMNDIRPSAMREIAIDVPNVSYDDVGGVRKQMAQIRELVELPLRH
PQLFKSIGIPAPRGLLLYGPPCTGKTMIARAVANEFGAYVSVINGPEIISKWGESER
SIIFIDELDALCPKREGAQNEVEKRWASLLTLMDGIGSVSIVL
LRRPGRFDKEIEIGVPNAQDRLDILQKLLRRVPHLLTEAELLQL
LCNEAGECGLLWDIQANLIMKRHFTQALSTVTPRIPESLRRFYE
DYQEKSGLHTL
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~ SEQ ID NO: 29 2789 by
fNOVl2b, CAGAGTTCGCCCTTCATTGAGTCGGCTTTTCTACTGCTTCGGCTAGGGTACCTTGTGA
CG102S7S-02 _CCATGTCTTCCAAGAAGAATAGAAAGCGGTTGAACCAAAGCGCGGAAAATGGTTCGTC
DNA SeCILIeriCeCTTGCCCTCTGCTGCTTCCTCTTGTGTGGAGGCACGGGCTCCTTCTGCTGGATCAGAC
E TTCGCGGCAACCTCCGGGACTCTGACGGTGACCAACTTATTAGAAAAGGTAGATGACA'
AAATTCCTAAAACATTCCAGAATTCCCTTATTCATCTTGGACTCAACACTATGAAGTC
TGCAAATATATGTATAGGTCGACCAGTGTTGCTTACTAGTTTGAACGGAAAGCAAGAGI
GTGTATACAGCCTGGCCTATGGCAGGATTTCCTGGAGGCAAGGTCGGCCTGAGTGAAA',
TGGCACAGAAAAATGTGGGTGTGAGGCCTGGTGATGCCATCCAGGTCCAGCCTCTTGT''
GGGTGCTGTGCTACAGGCTGAGGAAATGGATGTGGCACTGAGTGACAAAGATATGGAA
ATTAATGAAGAAGAACTGACTGGTTGTATCCTGAGAAAACTAGATGGCAAGATTGTTT
TACCAGGCAACTTTCTGTATTGTACATTCTATGGACGACCGTACAAGCTGCAAGTATT
GCGAGTGAAAGGGGCAGATGGCATGATATTGGGAGGGCCTCAGAGTGACTCTGACACT
GATGCCCAAAGAATGGCCTTTGAACAGTCCAGCATGGAAACCAGTAGCCTGGAGTTAT
CCTTACAGCTAAGCCAGTTAGATCTGGAGGATACCCAGATCCCAACATCAAGAAGTAC
TCCTTATAAACCAATTGATGACAGAATTACAAATAAAGCCAGTGATGTTTTGCTGGAT
GTTACACAGAGCCCTGGAGATGGCAGTGGACTTATGCTAGAGGAAGTCACAGGTCTTA
AATGTAATTTTGAATCTGCCAGAGAAGGAAATGAGCAACTTACTGAAGAAGAGAGACT
GCTAAAGTTCAGCATAGGAGCAAAGTGCAATACTGATACTTTTTATTTTATTTCTTCA
ACAACAAGAGTCAATTTTACAGAGATTGATAAAAATTCAAAAGAGCAAGACAACCAAT
TTAAAGTAACTTATGACATGATAGGAGGATTAAGTAGCCAGCTGAAAGCAATTAGAGA
AATAATTGAATTGCCCCTCAAACAGCCTGAGCTTTTCAAGAGTTATGGAATTCCTGCC
CCTAGAGGAGTGTTACTTTATGGTCCTCCAGGTACTGGAAAAACAATGATCGCCAGGG
CTGTTGCTAATGAAGTTGGAGCCTATGTTTCTGTAATTAATGGTCCTGAAATTATAAG
CAAATTCTATGGTGAGACTGAAGCAAAGTTACGTCAGATATTTGCTGAAGCCACTCTA
CGACACCCATCAATTATTTTTATTGATGAGCTGGATGCACTTTGTCCGAAAAGAGAGG
GGGCCCAGAATGAAGTGGAA.AAAAGAGTTGTGGCTTCACTCTTAACACTGATGGATGG
CATTGGTTCAGAAGTAAGTGAAGGACAAGTGTTGGTTCTTGGGGCCACAAATCGCCCT
CATGCCTTGGATGCTGCTCTCCGAAGACCTGGGCGATTTGATAAAGAGATTGAGATTG
GAGTTCCCAATGCTCAGGACCGGCTAGATATTCTCCAGAAACTGCTTCGAAGGGTACC
CCATTTGCTCACTGAGGCTGAGCTGCTGCAGCTGGCAAATAGTGCTCATGGATACGTT
GGAGCAGACTTGAAAGTCTTGTGTAATGAAGCAGGTCTCTGTGCCTTGCGGAGAATCC
TGA11AAAACAGCCTAACCTCCCTGATGTCAAGGTGGCTGGACTGGTGAAGATTACTCT
GAAGGATTTCTTGCAGGCAATGAATGATATCAGACCCAGTGCCATGAGGGAAATAGCA
ATTGATGTCCCAAATGTATCCTGGTCAGATATAGGAGGACTGGAAAGTATCAAACTGA
AGTTGGAACAGGCTGTGGAATGGCCCTTAAAACATCCAGAGTCTTTCATTCGAATGGG
TATTCAGCCACCTAAAGGAGTTCTTCTCTATGGGCCACCTGGGTGCTCTAAAACAATG
ATAGCAAAGGCTTTGGCCAATGAGAGTGGACTGAATTTTCTAGCTATAAAGGGGCCTG
AATTAATGAATAAATATGTTGGTGAATCTGAAAGAGCAGTTAGAGAGACCTTCCGAAA
AGCAAGAGCAGTGGCGCCTTCCATTATTTTCTTTGATGAACTGGATGCCTTAGCAGTT
GAAAGGGGCAGTTCTTTAGGTGCTGGGAATGTAGCCGATCGTGTTTTGGCTCAGCTCT
TAACAGAAATGGATGGGATTGAACAGCTAAAGGATGTGACCATTTTGGCAGCTACTAA
CCGTCCAGATAGGATAGACAAGGCTTTGATGCGGCCTGGAAGAATTGATAGAATCATC
TATGTGCCTTTACCGGATGCAGCAACAAGAAGGGAAATATTTAAGCTGCAGTTTCACT
CCATGCCTGTCAGTAATGAAGTTGACCTGGATGAACTCATCCTTCAAACCGACGCATA
~CTCAGGAGCAGAGATTGTAGCTGTCTGCAGAGAGGCAGCTCTTCTGGCTCTGGAAGAA
GACATTCAAGCCAATCTCATCATGAAAACACATmTCACTCACGCCmm(iACzC'At'mCmrA
CACCTAGAATTCCTGAGTCATTGAGACGTTTTTATGAAGATTATCAAGAGAAGAGTGG
GCTGCATACACTCTGAGAAAATATATATATTCAAGATGCTGAAAATCCTTTCCAGAGA
ORF Start: ATG at 61 ORF Stop: TGA at 2740
SEQ ID NO: 30 X893 as MW at 97931.2kD
'NOVl2b, MSSKKNRKRLNQSAENGSSLPSAASSCVEARAPSAGSDFAATSGTLTVTNLLEKVDDK
CGIOZS7S-O2 IPKTFQNSLIHLGLNTMKSANICIGRPVLLTSLNGKQEVYTAWPMAGFPGGKVGLSEM
PTOtelri S8CILIeriCe AQ~GVRPGDAIQVQPLVGAVLQAEEMDVALSDKDMEINEEELTGCILRKLDGKIVL
PGNFLYCTFYGRPYKLQVLRVKGADGMTLGGPQSDSDTDAQRMAFEQSSMETSSLELS
LQLSQLDLEDTQIPTSRSTPYKPIDDRITNKASDVLLDVTQSPGDGSGLMLEEVTGLK
CNFESAREGNEQLTEEERLLKFSIGAKCNTDTFYFISSTTRVNFTEIDKNSKEQDNQF
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KVTYDMIGGLSSQLKAIREIIELPLKQPELFKSYGIPAPRGVLLYGPPGTGKTMIARA
VANEVGAYVSVINGPEIISKFYGETEAKLRQIFAEATLRHPSIIFIDELDALCPKREG
AQNEVEKRVVASLLTLMDGIGSEVSEGQVLVLGATNRPHALDAALRRPGRFDKEIEIG
VPNAQDRLDILQKLLRRVPHLLTEAELLQLANSAHGYVGADLKVLCNEAGLCALRRIL
KKQPNLPDVKVAGLVKITLKDFLQAMNDIRPSAMREIAIDVPNVSWSDIGGLESIKLK
LEQAVEWPLKHPESFIRMGIQPPKGVLLYGPPGCSKTMTAKALANESGLNFLAIKGPE
LMNKYVGESERAVRETFRKARAVAPSIIFFDELDALAVERGSSLGAGNVADRVLAQLL
TEMDGIEQLKDVTILAATNRPDRIDKALMRPGRIDRIIYVPLPDAATRREIFKLQFHS
MPVSNEVDLDELILQTDAYSGAEIVAVCREAALLALEEDIQANLIMKRHFTQALSTVT
PRIPESLRRFYEDYQEKSGLHTL
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 12B.
Table 12B. Comparison of NOVl2a against NOVl2b.
Protein Sequence NOVl2a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOV 12b 1..881 724/895 (80%)
~.... _...~ . ....... . ..... . ....... ......... .._... 1..893, .._.._. .....
.. . ~.. .. . ~... .64/895. (84%).
Further analysis of the NOV 12a protein yielded the following properties shown
in Table
12C.
Table 12C. Protein Sequence Properties NOVl2a
PSort 0.7000 probability located in plasma membrane; 0.3000 probability
located in
analysis: microbody (peroxisome); 0.2000 probability located in endoplasmic
reticulum
(membrane); 0.1000 probability located in mitochondrial inner membrane
SignalP No Known Signal Sequence Predicted
analysis:
A search of the NOV 12a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 12D.
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Table 12D. Geneseq Results for NOVl2a
f
NOVl2a Identities/
~
Geneseq Protein/Organism/Length Residues!SimilaritiesExpect
[Patent ~ for
Identifier#, Date] Match the Matched Value
~
Residues Region
~
AAU17209 Novel signal transduction 261..823 442/575 (76%)0.0
pathway ~
protein, Seq ID 774 - Homo2..574 481/575 (82%)
Sapiens, ~
574 aa. [W0200154733-A1, [
02-
AUG-2001 ]
AAB59399 Protein tyrosine phosphatase337..848 229/527 (43%)e-120
related
sequence - Unidentified, 190..711 340/527 (64%)
806 aa.
[W0200075339-A1, 14-DEC-2000]
AAE09327 Human intracellular regulatory337..848 229/527 (43%)e-120
molecule, VCP - Homo sapiens,190..711 340/527 (64%)
806 ~
aa. [US6274312-B1, 14-AUG-2001]
AAB05879 Human transitional endoplasmic337..848 229/527 (43%)e-120
~
reticulum ATPase protein 190..711 340/527 (64%)
sequence j
- Homo Sapiens, 806 aa. 1
[W0200034470-A1, 15-JI1N-2000]
. . .. . .... . .. ... . .. ... .. . .. . . .. . ..
..,... .. .. . .. ;f ..
. ..
ABB59038 Drosophila melanogaster 322..844 228/540 (42%)e-117
[
polypeptide SEQ ID NO 3906170..704 342/540 (63%)
- [
Drosophila melanogaster, a
801 aa.
[W0200171042-A2, 27-SEP-2001]
In a BLAST search of public sequence databases, the NOV 12a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 12E.
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Table 12E. Public BLASTP Results for NOVl2a
Protein NOVl2a Identities/
Accession Protein/Organism/Length Residues/Similarities Expect
for :
~
Match the Matched Value
Number
Residues Portion
AAM00262 SPERMATOGENESIS 1..881 7451895 (83%)0.0
ASSOCIATED FACTOR - Homo I ..893 7851895 (87%)
sapiens (Human), 893 aa.
Q9Z2K7 SPAF - Mus musculus (Mouse),1..881 6401895 (71 0.0
%)
892 aa. 1..892 721/895 (80%)
Q9CXZ7 2510048F20RIK PROTEIN 1..881 640/896 (71%)X0.0
- Mus
musculus (Mouse), 893 1..893 721/896 (80%)
aa.
Q8ZYN4 AAA FAMILY ATPASE, 356..876 265!537 (49%)e-136
'
POSSIBLE CELL DIVISION 184..714 358!537 (66%)
CONTROL PROTEIN CDC48
-
Pyrobaculum aerophilum,
731 aa.
Q58556 Cell division cycle protein309..855 271/578 (46%)e-136
48 ~
homolog MJ1156 - Methanococcus127..697 378!578 (64%)
jannaschii, 903 aa.
PFam analysis predicts that the NOV 12a protein contains the domains shown in
the Table
12F.
Table 12F. Domain Analysis of NOVl2a
Identities)
Pfam Domain NOVl2a Match Region ' Similarities . Expect Value
for the Matched Region
AAA 378..566 9S/217 (44%) 3.3e-75
1651217 (76%)
AAA 652..837 98/217 (45%) 2e-77
165/217 (76%)
Example 13.
The NOV 13 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 13A.
Table 13A. NOVl3 Sequence Analysis
SEQ ID NO. 31 420 by
NOVl3a, TGCAGAAGGTGACCCTGGGCCTGCTTGTGTTCCTGGCAGGCTTTCCTGTCCTGGACGC
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CG102615-Ol CAATGACCTAGAAGATAA.AAACAGTCCTTTCTACTATGACTGGCACAGCCTCCAGGTT
DNA Sequence GGCGGGCTCATCTGCGCTGGGGTTCTGTGCGCCATGGGCATCATCATCGTCATGAGTG
CAAAATGCAAATGCAAGTTTGGCCAGAAGTCCGGTCACCATCCAGGGGAGACTCCACC
TCTCATCACCCCAGGCTCAGCCCAAAGCTGATGAGGACAGACCAGCTGAAATTGGGTG
GAGGACCGTTCTCTGTCCCCAGGTCCTGTCTCTGCACAGAAACTTGAACTCCAGGATG
GAATTCTTCCTCCTCTGCTGGGACTCCTTTGCATGGCAGGGCCTCATCTCACCTCTCG
CAAGAGGGTCTCTT
ORF Start: at 3 ORF Stop: TGA at 261
SEQ ID NO: 32 86 as MW at 9131.6kD
NOVl3a, QKVTLGLLVFLAGFPVLDANDLEDKNSPFYYDWHSLQVGGLICAGVLCAMGIIIVMSA
CG102615-O1 KCKCKFGQKSGHHPGETPPLITPGSAQS
Protein Sequence
~SEQ ID NO: 33 462 by
NOVl3b, TCAGCCTGGTGAACCACACAGAGGCTGGGGCGAGGAGGATACCATCTGTCAGTCTTGG
CG102615-04 CTGGATGACATCATGGGAAGGGGGTATAGTGGGGCCTTGCAGGCCAGAGGTGGCTTGG
DNA Se uenCe AGGAGCCCCTGGAAAGAGGCTTAAGAGGCCAGCGCTCTGACATGCAGAAGGTGACCCT
q
GGGCCTGCTTGTGTTCCTGGCAGGCTTTCCTGTCCTGGACGCCAATGACCTAGAAGAT
AAAAACAGTCCTTTCTACTATGACTGGCACAGCCTCCAGGTTGGCGGGCTCATCTGCG
CTGGGGTTCTGTGCGCCATGGGCATCATCATCGTCATGAGTGCAAAATGCAAATGCAA
GTTTGGCCAGAAGTCCGGTCACCATCCAGGGGAGACTCCACCTCTCATCACCCCAGGC
TCAGCCCAAAGCTGATGAGGACAGACCAGCTGAAATTGGGTGGAGGACCGTTCTCT
ORF Start: ATG at 71 ORF Stop: TGA at 419
SEQ ID NO: 34 116 as BMW at 12362.2kD
i '
NOVl3b, MGRGYSGALQARGGLEEPLERGLRGQRSDMQKVTLGL,LVFLAGFPVLDANDLEDKNSP
CG102615-04 F~~SLQVGGLICAGVLCAMGIIIVMSAKCKCKFGQKSGHHPGETPPLITPGSAQS
Protein Sequence
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 13B.
Table 13B. Comparison of NOVl3a against NOVl3b.
Protein Sequence NOVl3a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOVl3b 1..86 86/86 (100%)
...,~~~... ~ _~.. ...."~" 31 1.16 _~T:.~~~.~.....86/86 (100%) _... . ...
Further analysis of the NOVl3a protein yielded the following properties shown
in Table
13C.
Table 13C. Protein Sequence Properties NOVl3a
PSort 0.4600 probability located in plasma membrane; 0.2000 probability
located in
analysis: lysosome (membrane); 0.1000 probability located in endoplasmic
reticulum
(membrane); 0.1000 probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 20 and 21
analysis:
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A search of the NOV 13 a protein against the Geneseq database, a proprietary
dafa'~ase that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 13D.
Table 13D. Geneseq Results for NOVl3a
NOV.l3a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
(Patent for
Identifier#, Date] Match the Matched Value
ResiduesRegion
AAM23962 Human EST encoded protein 1..86 86/86 (100%)7e-47
SEQ
ID NO: 1487 - Homo Sapiens,2..87 86/86 (100%)
87 aa.
~W0200154477-A2, 02-AUG-2001]
_.~~ .. ...
AAW92959 1..86 86/86 (1.00%)7e-47
Human
MAT-8
protein
- Homo
Sapiens, 87 aa. [W09905276-Al,2..87 86/86 (100%)
04-FEB-1999]
~",,~.. _._. . .
AAY48304 Human prostate cancer-associated1..86 86/86 (100%)7e-47
protein 1 - Homo sapiens, 2..87 86/86 (100%)
87 aa.
[DE19811194-Al, 16-SEP-1999]
.. . .
AAR90990 Human Mat-8 polypeptide 1..86 86/86 (100%)7e-47
- Homo ~
sapiens, 87 aa. [WO9605322-A1,2..87 86/86 (100%)
22-FEB-1996]
AAB53415 Human colon cancer antigen 1..86 86/112 (76%)2e-42
protein
sequence SEQ ID N0:955 - 39..150 86/112 (76%)
Homo
Sapiens, 150 aa. [W0200055351-A1, t
..
21-SEP-2000]
In a BLAST search of public sequence databases, the NOV 13 a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 13E.
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Table 13E. Public BLASTP Results for NOVl3a
NOVl3a Identities/
'
Protein Similarities1
Residues/ f Expect
AccessionProtein/Organism/Length Match for the ~ Value
Number Residues Matched
!
Portion
Q14802 FXYD domain-containing ion I..86 86/86 (100%)2e-46
transport
regulator 3 precursor (Chloride2..87 86/86 (100%)
conductance inducer protein
Mat-8)
(Mammary tumor 8 kDa protein)
(Phospholernman-Like) - Homo
Sapiens
(Human), 87 aa.
Q61835 FXYD domain-containing ion 1..86 63/86 (73%)2e-33
transport
regulator 3 precursor (Chloride2..87 72/86 (83%)
conductance inducer protein
Mat-8)
(Mammary tumor 8 kDa protein)
(Phospholemman-like) - Mus
musculus
(Mouse}, 88 aa.
;.
....
097797 FXYD domain-containing ion 2..84 60/83 (72%)~ 8e-32
transport
regulator 3 precursor (Chloride3..85 68/83 (81%)f
conductance inducer protein
Mat-8)
(Mammary tumor 8 kDa protein)
- Sus
scrofa (Pig), 88 aa.
Q9D2W0 FXYD domain-containing ion I ..86 45/86 (S2%)4e-21
transport
regulator 4 precursor (Channel2..87 59/86 (68%)
inducing factor) (CHIF) -
Mus
musculus (Mouse), 88 aa.
Q631 I FXYD domain-containing ion .' 3..86 44/84 (52%)7e-20
3 transport
regulator 4 precursor (Channel4..87 55/84 (65%)
3
inducing factor) (CHIF)
(Corticosteroid-induced protein)
-
LRattus norvegicus (Rat),
87 as
PFam analysis predicts that the NOV 13a protein contains the domains shown in
the Table
13F.
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Table 13F. Domain Analysis of NOVl3a
Identities!
Pfam Domain NOVl3a Match Similarities Expect
Region . for the Matched Value
Region
ATP1G1_PLM_MAT8 19..74 27/57 (47%) 2.7e-35
55/57 (96%)
Example 14.
The NOV 14 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 14A.
Table 14A. NOV14
SEQ ID NO: 35 1638 by
NOVl4a, ~TTATCTAATATGTTTGTTTTAGCTACATCTTTATCAAGCCAAGTGAATCCTGACTGGC
CG102646-Ol G~CATATATCATGCTTGCAGTATATTTTCTAATTTTACTTGTTATTGGATATTATGG
DNA Se LlenCe TTATAAGCAAGCAACCGGAGATTTAAGTGAATATATGCTTGGCGAAAGAAATATTGGT
q CCATATGTCACTGCCTTATCTGCCGGAGCTTCAGATATGAGCGGTTGGATGATTATGG
GATTACCTGGAGAAGTTTATACTACAGGTTTATCAGCAGCATGGTTAGCTATTGGGTT
AACTATCGGAGCTTATGTTAACTACATACTTGTAGCACCAAGACTTCGTGTGTACACT
GAAAAAGCCAATGACTCAATTACATTGCCTAATTACTTTACACATCGTCTTAATGATA
ATTCCAATATTATTAAAATTATCTCTGGTGGTATCATTGTTGTATTTTTTACACTCTA
TACTCATTCAGGTATGGTATCAGGTGGTAAATTATTTGATAGTGCTTTTGGTTTAGAC
TATCATATTGGACTTATTTTAATCTCTGTCATTGTAATTTTATATACTTTTTTTGGTG
GCTATTTAGCAGTGTCGTTAACTGACTTTTTCCAAGGGGTTGTCATGTTAATTGCGAT
GGTTATGGTACCTATTGTAGCCATGATGCAGCTCGGAGGTATGGATGCTTTTTCACAA
GCAGCAACATTAAAACCTACTAATTTAGATTTATTTAAAGGAACAACTATTATAGGCA
TCATTTCATTCTTTGCTTGGGGATTAGGCTATTTTGGCCAGCCTCATATCATTGTACG
ATTTATGTCTATCAAATCCGTACGACAATTAAAAACGTCTAGAAGATTTGGTATTAGT
TGGATGGCTATTAGTTTAATCGGTGCAGTATGTGTTGGATTAATTGGCATTTCGTTTG
TACAAGATAAAGGTGTTGAATTAAAAGATCCAGAAACACTATTTATTTTAATGGGACA
CATCCTCTTGTAGGTGGGTTCCTACTTGCAGCCATTTTGGCAGCAATT
TTTCTTCCCAATTACTTGTGACTTCAAGTTCACTTACAGAAGATTTTT
GGGTCGATTATCTGTTGTAGTCGTTGCGATTATCTCCATCCTCATTGCATGGACGCCA
AATGACACTATCTTAAATCTTGTTGGTAACGCTTGGGCTGGATTCGGTGCAGCATTTG
GTCCACTGGTATTATTATCTCTCTATTCGAAAGGTTTAAGTCGTACTGGAGCTATTTC
TGGAATGTTATCAGGAGCAATTGTCGTCATTCTTTGGATTGTGTTTGTTAAACCATTA
GGAGCATATAATGATTTCTTTAATTTATATGAAATTATTCCTGGTTTCTTAACAAGTC
TTATTGTGACATATGTAGTGAGTCTTGTAACTAAAAAGCCAGATCTCAATGTTCAAAA
AGATTTAGAAGACGTCAAACGTATTGTAAAAGGACAATAAATTAATAATATTCAACGA
TGCTTAATGTCAATATTATTTCAATTAGTGCATTACTCTTATAATATGAAACACAAAT
AAATTTTTATACAT
ORF Start: ATG at 10 ORF Stop: TAA at 1546
SEQ ID NO: 36 512 as ~MW at 55813.4kD
NOVl4a, MFVLATSLSSQVNPDWRTYIMLAVYFLILLVIGYYGYKQATGDLSEYMLGERNIGPYV
CG102646-O1 T~'SAGASDMSGWMIMGLPGEVYTTGLSAAWLAIGLTIGAYVNYILVAPRLRVYTEKA
NDSITLPNYFTHRLNDNSNIIKIISGGIIVVFFTLYTHSGMVSGGKLFDSAFGLDYHI
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Protein SeqLtenCe GLILISVIVTLYTFFGGYLAVSLTDFFQGVVMLIAM~JM~IPhVAMMQLGGMDAFSQAAT
LKPTNLDLFKGTTIIGIISFFAWGLGYFGQPHITVRFMSTKSVRQLKTSRRFGISWMA
ISLIGAVCVGLIGISFVQDKGVELKDPETLFILMGQILFHPLVGGFLLAAILAAIMST
ISSQLLVTSSSLTEDFYKLIRGEEAAKQHKKEFLLVGRLSWWAIISILIAWTPNDT
ILNLVGNAWAGFGAAFGPLVLLSLYSKGLSRTGAISGMLSGAIWILWIVFVKPLGAY
NDFFNLYEIIPGFLTSLIVTYWSLVTKKPDLNVQKDLEDVKRIVKGQ
Further analysis of the NOV 14a protein yielded the following properties shown
in Table
14B.
Table 14B. Protein Sequence Properties NOVl4a
PSort 0.8200 probability located in plasma membrane; 0.4600 probability
located in
analysis: Golgi body; 0.3700 probability located in endoplasmic reticulum
(membrane);
0.1000 probability located in endoplasmic reticulum (lumen)
SignaIP ~ Cleavage site between residues 37 and 38
analysis:
A search of the NOV 14a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table I4C.
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Table 14C. Geneseq Results
for NOVl4a
NOVl4a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent for
Identifier#, Date] Match the Matched Value
Residues Region
AAB767S7 ~ Co~bacteriurn glutamicum20..506 233/502 (46%)e-127
MCT
protein SEQ ID N0:496 - 9..499 339/502 (67%)
E Corynebacterium glutamicum,
524
~ aa. [W0200100805-A2,
04-JAN- j
2001 )
AAG93195 C glutamicum protein fragment20..506 2331502 (46%)e-127
SEQ
' ID NO: 6949 - Corynebacterium9_.499 339/502 (67%)
glutamicum, 524 aa. [EPl
108790-
A2, 20-JLTN-2001
z
AAW20806 ~ H. pylori transporter 64..506 2081450 (46%)e-I
protein, I2
09ap20802orf27 - Helicobacter5..445 306/450 (67%)
~
~ pylori, 446 aa. [W09640893-Al,
19-DEC-1996]
AAG82S96 ~ S. epidermidis open reading266..510 171/245 (69%)1e-94
frame
protein sequence SEQ ID 163..407 208I245 (84%)
NO:2286 -
Staphylococcus epidermidis,
408 aa.
[WO200I34809-A2, I7-MAY-
2001 J
_.
AAB96626 ~ Putative P. abyssi permease24..508 174/503 (34%)4e-83
#22 -
Pyrococcus abyssi, 537 I I ..507275/503 (S4%)
aa.
a [FR2792651-A1, 27-OCT-2000]
In a BLAST search of public sequence databases, the NOV 14a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 14D.
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Table 14D. Public BLASTP Results for NOVl4a
Protein NOVl4a Identities/ ~
Residues/Similarities Expect
AccessionProtein/Organism/Length for '
Match the Matched Value
Number
ResiduesPortion
'
Q99SY5 HIGH AFFINITY PROLINE 1..510 378/510 (74%)0.0
PERMEASE - Staphylococcus I ..510 443/5I0 (86%)
aureus (strain Mu50 / ATCC
~ 700699), and, 512 aa.
030986 HIGH AFFINITY PROLINE ~ 1..494 366/494 (74%)0.0
PERMEASE - Staphylococcus 1..493 431/494 (87%)
aureus, 497 aa.
Q53584 PROLINE PERMEASE ~ 1..494 366/494 (74%)0.0
HOMOLOG - Staphylococcus 1..493 430/494 (86%)
~
aureus, 497 aa.
006493 Osmoregulated proline transporter20..494 268/478 (56%)e-I58
~
_ 7..473 371/478 (77%)
(Sodium/proline symporter)
- ~
Bacillus subtilis, 492 aa.
P94392 HOMOLOGUE OF PROLINE ~ 54..504 243/452 (53%)e-142
PERMEASE OF E. COLI - Bacillus1..442 336/452 (73%)
~
subtilis, 449 aa.
~ .
PFaxn analysis predicts that the NOV 14a protein contains the domains shown in
the Table
14E.
Table 14E. Domain Analysis of NOVl4a
Identities/
Pfam Domain NOVl4a Match Region Similarities Expect Value
for the Matched Region
SSF 47..447 134/449 (30%) 5.7e-121
318/449 (71 %)
Example 15.
The NOV 1 S clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 15A.
Table 15A. NOV15 Seque_nce_Analysis
..-...__.._.......--.,~,~......_..~SEQ..ID~~NO: 37 .'~°"'~'~...,..- l
146 bp "'""..~..~....~......-_.~_..r....~......._~.~..,-~.............",..~..
NOVISa, ~CTAGCTCGACAGCTTCCCGGCGGCTGCGCGATGGACAGCCCCGAGGTGACCTTCACTC
TCGCCTATCTGGTGTTCGCCGTGTGCTTCGTGTTCACGCCCAACGAGTTCCACGCGGC
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CG1O287S-O1
GGGGCTCACGGTGCAGAACCTGCTGTCGGGCTGGC"T~GtG~~A~C'G2~'~GA~~~GCCG~CTTC~F
DNA SCCILteriCeGTGCCCTTCCACTTGCGCCGCACGGCCGCCACGCTGTTGTGCCACTCGCTGCTGCCGC
TCGGCTACTATGTGGGCATGTGCCTTGCGGCTTCAGAAAAGCGGCTCCACGCCCTCAG
CCAGGCCCCTGAGGCCTGGCGGCTCTTCCTGCTGCTGGCCGTGACCCTCCCCTCCATC
GCCTGCATCCTGATCTACTACTGGTCCCGTGACCGGTGGGCCTGCCACCCACTGGCGC
GCACCCTGGCCCTCTACGCCCTCCCACAGTCTGGCTGGCAGGCTGTTGCCTCCTCTGT
CAACACTGAGTTCCGGCGGATTGACAAGTTTGCCACCGGTGCACCAGGTGCCCGTGTG
ATTGTGACAGACACGTGGGTGATGAAGGTAACCACCTACCGAGTGCACGTGGCCCAGC
AGCAGGACGTGCACCTGACTGTGACGGAGTCTCGGCAGCATGAGCTCTCGCCAGACTC
GAACTTGCCCGTGCAGCTCCTCACCATCCGTGTGGCCAGCACCAACCCTGCTGTGCAG
GCCTTTGACATCAGGCTGAACTCCACTGAGTACGGGGAGCTCTGCGAGAAGCTCCGGG
CACCCATCCGCAGGGCAGCCCATGTGGTCATCCACCAGAGCCTGGGCGACCTGTTCCT
GGAGACATTTGCCTCCCTGGTAGAGGTCAACCCGGCCTACTCAGTGCCCAGCAGCCAG
GAGCTGGAGGCCTGCATAGGCTGCATGCAGACACGTGCCAGCGTGAAGCTGGTGAAGA
CCTGCCAGGAGGCAGCCACAGGCGAGTGCCAGCAGTGTTACTGCCGCCCCATGTGGTG
CCTCACCTGCATGGGCAAGTGGTTCGCCAGCCGCCAGGACCCCCTGCGCCCTGACACC
TGGCTGGCCAGCCGCGTGCCCTGCCCCACCTGCCGCGCACGCTTCTGCATCCTGGATG
TGTGCACCGTGCGCTGAGTGGGCTGGGGCCTTGAGGTGACTCTG
ORF Start: ATG at 31 ORF Stop: TGA at 1117
SEQ ID NO: 38 . 362 as MW at 40433.3kD
NOVISa, MDSPEVTFTLAYLVFAVCFVFTPNEFHAAGLTVQNLLSGWLGSEDAAFVPFHLRRTAA
CG102878-O1 TLLCHSLLPLGYYVGMCLAASEKRLHALSQAPEAWRLFLLLAVTLPSIACILIYXWSR
P1'Otelri DRWACHPLARTLALYALPQSGWQAVASSVNTEFRRIDKFATGAPGARVIVTDTWVMKV
Se LleriCe
TTYRVHVAQQQDVHLTVTESRQHELSPDSNLPVQLLTIRVASTNPAVQAFDIRLNSTE
YGELCEKLRAPIRRAAHWIHQSLGDLFLETFASLVEVNPAYSVPSSQELEACIGCMQ
TRASVKLVKTCQEAATGECQQCYCRPMWCLTCMGKWFASRQDPLRPDTWLASRVPCPT
CRARFCILDVCTVR
SEQ ID NO: 39 1115 by
NOVISb, TTCGCCCTTGGCTGCGCGATGGACAGCCCCGAGGTGACCTTCACTCTCGCCTATCTGG
CG102878-02 TGTTCGCCGTGTGCTTCGTGTTCACGCCCAACGAGTTCCACGCGGCGGGGCTCACGGT
DNA Se LleriCBGCAGAACCTGCTGTCGGGCTGGCTGGGCAGCGAGGACGCCGCCTTCGTGCCCTTCCAC
TTGCGCCGCACGGCCGCCACGCTGTTGTGCCACTCGCTGCTGCCGCTCGGCTACTACG
TGGGCATGTGCCTTGCGGCTTCAGAAAAGCGGCTCCACGCCCTCAGCCAGGCCCCTGA
GGCCTGGCGGCTCTTCCTGCTGCTGGCCGTGACCCTCCCCTCCATTGCCTGCATCCTG
ATCTACTACTGGTCCCGTGACCGGTGGGCCTGCCACCCACTGGCGCGCACCCTGGCCC
TCTACGCCCTCCCACAGTCTGGCTGGCAGGCTGTTGCCTCCTCTGTCAACACTGAGTT
CCGGCGGATTGACAAGTTTGCCACCGGTGCACCAGGTGCCCGTGTGATTGTGACAGAC
ACGTGGGTGATGAAGGTAACCACCTACCGAGTGCACGTGGCCCAGCAGCAGGACGTGC
ACCTGACTGTGACGGAGTCTCGGCAGCATGAGCTCTCGCCAGACTCGAACTTGCCCGT
GCAGCTCCTCACCATCCGTGTGGCCAGCACCAACCCTGCTGTGCAGGCCTTTGACATC
TGGCTGAACTCCACTGAGTACGGGGAGCTCTGCGAGAAGCTCCGGGCACCCATCCGCA
GGGCAGCCCATGTGGTCATCCACCAGAGCCTGGGCGACCTGTTCCTGGAGACATTTGC
CTCCCTGGTAGAGGTCAACCCGGCCTACTCAGTGCCCAGCAGCCAGGAGCTGGAGGCC
TGCATAGGCTGCATGCAGACACGTGCCAGCGTGAAGCTGGTGAAGACCTGCCAGGAGG
CAGCCACAGGCGAGTGCCAGCAGTGTTACTGCCGCCCCATGTGGTGCCTCACCTGCAT
GGGCAAGTGGTTCGCCAGCCGCCAGGACCCCCTGCGCCCTGACACCTGGCTGGCCAGC
CGCGTGCCCTGCCCCACCTGCCGCGCACGCTTCTGCATCCTGGATGTGTGCACCGTGC
GCTGATGTGGCGG
ORF Start: ATG at 19 ORF Stop: TGA at 110S
SEQ ID NO: 40 362 as MW at 40463.4kD
.
~
NOVISb, ....,..~..".-".y,.~.,.,
.
MDSPEVTFTLAYLVFAVCFVFTPNEFHAAGLTVQNLLSGWLGSEDAAFVPFHLRRTAA
CG102878-02 TLLCHSLLPLGYYVGMCLAASEKRLHALSQAPEAWRLFLLLAVTLPSIACILIYYWSR
PrOtelri SeClLl2riCeDRWACHPLARTLALYALPQSGWQAVASSVNTEFRRIDKFATGAPGARVIVTDTWVMKV
TTYRVHVAQQQDVHLTVTESRQHELSPDSNLPVQLLTIRVASTNPAVQAFDIWLNSTE
YGELCEKLRAPZRRAAHV(TIHQSLGDLFLETFASLVEVNPAYSVPSSQELEACIGCMQ
TRASVKLVKTCQEAATGECQQCYCRPMWCLTCMGKWFASRQDPLRPDTWLASRVPCPT
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CRARFCILDVCTVR
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 15B.
Table 15B. Comparison of NOVlSa against NOVlSb.
Protein Sequence' NOVlSa Residues/'3 Identities/
Match Residues Similarities for the Matched Region
NOVlSb 1..362 361/362 (99%)
1..362 361/362 (99%)
Further analysis of the NOV 15a protein yielded the following properties shown
in Table
15C.
Table 15C. Protein Sequence Properties NOVlSa
PSort ' 0.6760 probability located in plasma membrane; 0.1000 probability
located in
analysis: ° endoplasmic reticulum (membrane); 0.1000 probability
located in
endoplasmic reticulum (lumen); 0.1000 probability located in outside
SignalP Cleavage site between residues 29 and 30
analysis:
A search of the NOV 1 Sa protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 15D.
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Table 15D. Geneseq Results for NOVlSa
NOVlSa Identities/
Geneseq Protein/Organism/Length Residues!Similarities Expect
[Patent for
Identifier' #, Date] Match the Matched Value
ResiduesRegion
AAG81377 Human AFP protein sequence 1..362 360/362 (99%)0.0
SEQ
ID N0:272 - Homo Sapiens, 1..362 360/362 (99%)
362 aa.
[W0200129221-A2, 26-APR-2001
ABB69639 Drosophila melanogaster 1..358 122/389 (31%)7e-60
polypeptide SEQ ID NO 357091..383 200/389 (51%)
-
Drosophila melanogaster,
409 aa.
[W0200171042-A2, 27-SEP-2001]
AAG23427 Arabidopsis thaliana protein337..36213/26 (50%) 2.8
fragment SEQ ID NO: 26729 77..102 16/26 (61
- %)
Arabidopsis thaliana, 284
aa.
[EP1033405-A2, 06-SEP-2000]
AAG23426 Arabidopsis thaliana protein337..36213/26 (50%) 2.8
fragment SEQ ID NO: 26728 206..23116/26 (61
- %)
Arabidopsis thaliana, 413
aa.
[EP1033405-A2, 06-SEP-2000]
ABGl 1786~ Novel human diagnostic 285..35423/89 (25%) 3.6
protein
#11777 - Homo Sapiens, 198 54..141 37/89 (40%)
aa.
[W0200175067-A2, 11-OCT-2001)
In a BLAST search of public sequence databases, the NOV 15a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 15E.
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Table 15E. Public BLASTP Results for NOVlSa
~~~~~NOVlSa Identities/
Protein Residues/Similarities ' Expect
for
Accession Protein/Organism/Length Match the Matched Value
Number Residues Portion
CAC38627 SEQUENCE 271 FROM 1..362 361/362 (99%~0.0
PATENT W00129221 - Homo 1..362 361/362 (99%)
Sapiens (Human), 362
aa.
Q9DCF3 ?; .0610039G24RIK PROTEIN1..362 323/362 (89%)0.0
-
~ Mus musculus (Mouse), 1..362 341/362 (93%)
362 aa. ~
~..
Q96GP5 SIMILAR TO RIKEN CDNA 1..226 226/226 (I00%)' e-129
0610039624 GENE - Homo 1..226 226/226 (100%)
sapiens (Human), 232
aa.
Q9VN16 CG14646 PROTEIN - Drosophila1..358 122/389 (31%)2e-59
~
melanogaster (Fruit fly),1..383 200/389 (51
409 aa. , ~ %)
Q95TM4 LD39811P - Drosophila 20..358 116/370 (31%)1e-55
melanogaster (Fruit fly),4..367 190/370 (51%)
_........ 393 aa. .. . ~ ....... ...............
_..... _ _..~ ....... _...~ .. . . _ _.._....
.
Example 16.
The NOV16 clone Was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 16A.
Table 16A. NOV16 Sequence Analysis
SEQ ID NO: 41 X2765 by
NOVl6a, -
°~CTGGCGGCGTCGCATGGAGGGCTCTGGGGGCGGTGCGGGCGAGCGGGCGCCGCTGCTG
CG103459-Ol GGCGCGCGGCGGGCGGCGGCGGCCGCGGCGGCGGCTGGGGCGTTCGCGGGCCGGCGCG
DNA SequenCB CGGCGTGCGGGGCCGTGCTGCTGACGGAGCTGCTGGAGCGCGCCGCTTTCTACGGCAT
CACGTCCAACCTGGTGCTATTCCTGAACGGGGCGCCGTTCTGCTGGGAGGGCGCGCAG
GCCAGCGAGGCGCTGCTGCTCTTCATGGGCCTCACCTACCTGGGCTCGCCGTTCGGAG
~GCTGGCTGGCCGACGCGCGGCTGGGCCGGGCGCGCGCCATCCTGCTGAGCCTGGCGCT
CTACCTGCTGGGCATGCTGGCCTTCCCGCTGCTGGCCGCGCCCGCCACGCGAGCCGCG
CTCTGCGGTTCCGCGCGCCTGCTCAACTGCACGGCGCCTGGTCCCGACGCCGCCGCCC
GCTGCTGCTCACCGGCCACCTTCGCGGGGCTGGTGCTGGTGGGCCTGGGCGTGGCCAC
CGTCAAGGCCAACATCACGCCCTTCGGCGCCGACCAGGTTAAAGATCGAGGTCCGGAA
GCCACTAGGAGATTTTTTAATTGGTTTTATTGGAGCATTAACCTGGGAGCGATCCTGT
CGTTAGGTGGCATTGCCTATATTCAGCAGAACGTCAGCTTTGTCACTGGTTATGCGAT
CCCCACTGTCTGCGTCGGCCTTGCTTTTGTGGCCTTCCTCTGTGGCCAGAGCGTTTTC
ATCACCAAGCCTCCTGATGGCAGTGCCTTCACCGATATGTTCAAGATACTGACGTATT
CCTGCTGTTCCCAGAAGCGAAGTGGAGAGCGCCAGAGTAATGGTGAAGGCATTGGAGT
CTTTCAGCAATCTTCTAAACAAAGTCTGTTTGATTCATGTAAGATGTCTCATGGTGGG
CCATTTACAGAAGAGAAAGTGGAAGATGTGAAAGCTCTGGTCAAGATTGTCCCTGTTT
TCTTGGCTTTGATACCTTACTGGACAGTGTATTTCCAAATGCAGACAACATATGTTTT
ACAGAGTCTTCATTTGAGGATTCCAGAAATTTCAAATATTACAACCACTCCTCACACG
CTCCCTGCAGCCTGGCTGACCATGTTTGATGCTGTGCTCATCCTCCTGCTCATCCCTC
TGAAGGACAAACTGGTCGATCCCATTTTGAGAAGACATGGCCTGCTCCCATCCTCCCT
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GAAGAGGATCGCCGTGGGCATGTTCTTTGTCATGTGCTCAGCCTTTGCTGCAGGAATT
TTGGAGAGTAAAAGGCTGAACCTTGTTAAAGAGAA.AACCATTAATCAGACCATCGGCA
ACGTCGTCTACCATGCTGCCGATCTGTCGCTGTGGTGGCAGGTGCCGCAGTACTTGCT
GATTGGGATCAGCGAGATCTTTGCAAGTATCGCAGGCCTGGAATTTGCATACTCAGCT
GCCCCCAAGTCCATGCAGAGTGCCATAATGGGCTTGTTCTTTTTCTTCTCTGGCGTCG
GGTCGTTCGTGGGTTCTGGACTGCTGGCACTGGTGTCTATCAAAGCCATCGGATGGAT
GAGCAGTCACACAGACTTTGGTAATATTAACGGCTGCTATTTGAACTATTACTTTTTT
CTTCTGGCTGCTATTCAAGGAGCTACCCTCCTGCTTTTCCTCATTATTTCTGTGAAAT
ATGACCATCATCGAGACCATCAGCGATCAAGAGCCAATGGCGTGCCCACCAGCAGGAG
GGCCTGACCTTCCTGAGGCCATGTGCGGTTTCTGAGGCTGACATGTCAGTAACTGACT
GGGGTGCACTGAGAACAGGCAAGACTTTAAATTCCCATAAAATGTCTGACTTCACTGA
AACTTGCATGTTGCCTGGATTGATTTCTTCTTTCCCTCTATCCAAAGGAGCTTGGTAA
GTGCCTTACTGCAGCGTGTCTCCTGGCACGCTGGGCCCTCCGGGAGGAGAGCTGCAGA
TTTCGAGTATGTCGCTTGTCATTCAAGGTCTCTGTGAATCCTCTAGCTGGGTTCCCTT
TTTTACAGAAACTCACAAATGGAGATTGCAAAGTCTTGGGGAACTCCACGTGTTAGTT
GGCATCCCAGTTTCTTAAACAAATAGTATCACCTGCTTCCCATAGCCATATCTCACTG
T TTAATAAACTGTTACTTATATTTAAGAAAGTGAGGATTTTTTTTTT
TTAAAGATAAAAGCATGGTCAGATGCTGCAAGGATTTTACATAAATGCCATATTTATG
GTTTCCTTCCTGAGAACAATCTTGCTCTTGCCATGTTCTTTGATTTAGGCTGGTAGTA
AACACATTTCATCTGCTGCTTCAAA.AAGTACTTACTTTTTAAACCATCAACATTACTT
TTCTTTCTTAAGGCAAGGCATGCATAAGAGTCATTTGAGACCATGTGTCCCATCTCAA
GCCACAGAGCAACTCACGGGGTACTTCACACCTTACCTAGTCAGAGTGCTTATATATA
GCTTTATTTTGGTACGATTGAGACTAAAGACTGATCATGGTTGTATGTAAGGAAAACA
TTCTTTTGAACAGAAATAGTGTAATTAAA.AATAATTGAAAGTGTTAAATGTGAACTTG
AGCTGTTTGACCAGTCACATTTTTGTATTGTTACTGTACGTGTATCTGGGGCTTCTCC
GTTTGTTAATACTTTTTCTGTATTTGTTGCTGTATTTTTGGCATAACTTTATTATAAA
AAGCATCTCAAATGCG
ORF Start: ATG at 14 ORF Stop: TGA at 1745
~,. ,NO
~ 42
.
SEQ"IIO
~7~
aa~MW at
62004~6kD
. ~
.
16a, ~..
NO .~
.
y
~
"y.."r,~.,....
Z''iEGSGGGAGERAPLLGARRP~~AAAAAAGAFAGRRAACGAVLLTELLERAAFYGITSNL
V
CG103459-O1 VLFLNGAPFCWEGAQASEALLLFMGLTYLGSPFGGWLADARLGRARAILLSLALYLLG
PPOtelri SequenceM~FPLLAAPATRAALCGSARLLNCTAPGPDAAA12CCSPATFAGLVLVGLGVATVKAN
ITPFGADQVKDRGPEATRRFFNWFYWSINLGAILSLGGIAYIQQNVSFVTGYAIPTVC
VGLAFVAFLCGQSVFITKPPDGSAFTDMFKILTYSCCSQKRSGERQSNGEGIGVFQQS
SKQSLFDSCKMSHGGPFTEEKVEDVKALVKIVPVFLALIPYWTVYFQMQTTYVLQSLH
LRIPEISNITTTPHTLPAAWLTMFDAVLILLLIPLKDKLVDPILRRHGLLPSSLKRIA
VGMFFVMCSAFAAGILESKRLNLVKEKTINQTIGNVVYHA.ADLSLWWQVPQYLLIGIS
EIFASIAGLEFAYSAAPKSMQSAIMGLFFFFSGVGSFVGSGLLALVSIKAIGWMSSHT
DFGNINGCYLNYYFFLLAAIQGATLLLFLIISVKYDHHRDHQRSRANGVPTSRRA
Further analysis of the NOV 16a protein yielded the following properties shown
in Table
16B.
Table 16B. Protein Sequence Properties NOVl6a
PSort 0.6000 probability located in plasma membrane; 0.4000 probability
located in
analysis: Golgi body; 0.3000 probability located in endoplasmic reticulum
(membrane);
0.3000 probability located in microbody (peroxisome)
SignalP No Known Signal Sequence Predicted
analysis:
A search of the NOV 16a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 16C.
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Table 16C. Geneseq Results
for NOVl6a
NOVl6a ~ Identities/
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent for
Identifier#, Date] Match ~ the Matched Value
Residues~ Region
AAU12071 Human PHT1 variant protein1..577 577/577 (100%)0.0
from
Caco-2 cells - Homo Sapiens,1..577 577/577 (100%)
577
aa. [WO200I92468-A2, 06-DEC-
2001 ]
AAU12068 Human PHT1 protein isolated1..577 577/577 (100%)~ 0.0
from
Caco-2 cells - Homo Sapiens,1..577 ~ 577/577 (100%)
577
aa. [W0200192468-A2, 06-DEC-
2001 ]
AAU12070 Human PHT1 variant protein1..577 ~ 575/577 (99%)0.0
from
BeWo cells - Homo Sapiens,1..577 ~ 576/577 (99%)
577
aa. [W0200192468-A2, 06-DEC-
2001 ]
AAE16771 Human transporter and 1..577 ~ 576/577 (99%)0.0
ion channel-'
8 (TRICH-8) protein - 1..576 ~ 576/577 (99%)
Homo
Sapiens, 576 aa. [W0200192304-
A2, 06-DEC-2001]
AAB82821 Human proton/oligonucleotide22..577 ~ 555/556 (99%)0.0
transporter hPHTI polypeptide1..556 ~ 555/556 (99%)
-
Homo Sapiens, 556 aa.
[W0200160854-Al, 23-AUG-
2001
_. . . _ ~ _ . _. .. ~.. ~ . .. . _
.. _. _._
_. .
.
In a BLAST search of public sequence databases, the NOV 16a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 16D.
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Table 16D. Public BLASTP
Results for NOVl6a'
Protein , NOVl6a Identities)
AccessionProtein/Organism/Length Residues/ SimilaritiesExpect
for
Number Match the Matched Value
Residues Portion
009014 ~ PEPTIDE/HISTIDINE 9..576 500/578 (86%)0.0
TRANSPORTER - Rattus 3..571 531/578 (91%)
' norvegicus (Rat), 572
aa.
Q91 W98 SIMILAR TO PEPTIDE 9..576 496/578 (85%)0.0
TRANSPORTER 3 - Mus 3..573 531/578 (91%)
musculus (Mouse), 574
aa.
AAH28394 SIMILAR TO PEPTIDE 117..577 460/461 (99%)0.0
TRANSPORTER 3 - Homo 1..461 460/461 (99%)
sapiens (Human), 461
aa.
Q9P2X9 i PEPTIDE TRANSPORTER 9..558 289/570 (50%)e-152
3 - ~
Homo Sapiens (Human), 14..564 379/570 (65%)
581 aa.
Q9WU80 3 CAMP INDUCIBLE 1 8..567 279/577 (48%)e-144
~
PROTEIN - Mus musculus 6..570 366/577 (63%)
~
(Mouse), 578 aa.
PFam analysis predicts that the NOV 16a protein contains the domains shown in
the Table
16E.
Table 16E. Domain Analysis of NOVl6a
Identities/
Pfam Domain NOVl6a Match Region Similarities Expect Value
for the Matched Region
PTR2 ( 103..496 ~ 109/448 (24%) 6.7e-103
..._ _.... .. 310/448. (69%)y .. _. ~ ._ . ... . . ........
Example 17.
The NOV 17 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table I7A.
Table 17A. NOV17 Sequence Analysis
~SEQ ID NO: 43 1393 by
NOVI7a, CCCATGGAGGCTCCGGGACCCCGCGCCTTGCGGACTGCGCTCTGTGGCGGCTGTTGCT
CG104210-Ol GCCTCCTCCTATGTGCCCAGCTGGCTGTGGCTGGTAAAGGAGCTCGAGGCTTTGGGAG
DNA Sequence GGGAGCCCTGATCCGCCTGAATATCTGGCCGGCGGTCCAAGGGGCCTGCAAACAGCTG
GAGGTCTGTGAGCACTGCGTGGAGGGAGACAGAGCGCGCAATCTCTCCAGCTGCATGT
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GGGAGCAGTGCCGGCCAGAGGAGCCAGGTCACTGTGTGGCCCAATCTGAGGTGGTCAA
GGAAGGTTGCTCCATCTACAACCGCTCAGAGGCATGTCCAGCTGCTCACCACCACCCC
ACCTATGAACCGAAGACAGTCACAACAGGTAGCCCCCCAGTCCCTGAGGCCCACAGCC
CTGGATTTGACGGGGCCAGCTTTATCGGAGGTGTCGTGCTGGTGTTGAGCCTACAGGC
GGTGGCTTTCTTTGTGCTGCACTTCCTCAAGGCCAAGGACAGCACCTACCAGACGCTG
TGAGTACCTGGCCAGCAGCAAGTACCTGAGTCCCAGCTCACCTCCTGGTTCCTGCCCC
ACCGTTCCCCTTCAGTACCCAGGGTGCTGTCTTCTCCACTGGCAAGCCCTCAGGACGG
TGACAGCGTGCTCCATGTGAGCCACACCCCTTTTGTCTCCTCCAGTTGGGGTGTTTCC
TTTGTCAGATGTTGGCTGGGACCAGGACTCAGCCTGGGCCAGTCTAGGAGCCCAGCTG
AGCCCTCCTGTGTCTTTTCCCTTCATGCTGCCAGCAGGGAAGAGAACCAGTAGGTGCC
AGCCCAGCAACCTGTGGCCCGCGTTTCTGTGGCTGTGGGCAGGAGCTGGGCCTTGTGT
CTAGTTGGGTTTTGCTCTGAGAAGGGGAGCTGTGCTGAGGCCCTCTGTGTGCCGTGTG
TGCTGTGGGGCGGGTCGCCACAGCCTGTGTTAAAGTGTTTGCTCTTCCTCTGCTGCCT
CCTCTCGAGGCAGGGGGTCCTTGGCTGGCTGAGGCAGTGTCACCTTCCTGAGTGTCCT
CTTTGGCCTCTGCAGAATCTGACCCCTTTGGGCCTGGACTCCATCCTGAGGGGAAAGG
AGGATGCAGAGGGTGGCCTCTGGGCACCCTTGTGGGTAAGCGGGGGGCGGGGGCGGGA
AAAACTCTGGCCGCCAGTTTTTGGCTCCTGCGGGCACCAAGCAGGCTCAGTGTCTGAT
GCTTGACATCTCCTCCTGTCCTGGGCCTGGAACCTGCAGCTGAGAAAATCCCTCAACC
ACCTCGTCTCCTCCATCGCCCCTGCTGGGCCCCCCAGCCTGACAGTGGGTTGTATGCC
TGCCTCTTTCCACCAACTGGCCTGGGCACTGCCCCCAAATAAAGGAACTCTGCACTGC
A
ORF Start: ATG at 4 ORF Stop: TGA at 523
SEQ ID N0: 44 173 as ~MWat 18421.OkD
~.
w..~
NOVI7a, .
MEAPGPRALRTALCGGCCCLLLCAQLAVAGKGARGFGRGALIRLNIWPAVQGACKQLE
CG104210-Ol VCEHCVEGDRARNLSSCMWEQCRPEEPGHCVAQSEVVKEGCSIYNRSEACPAAHHHPT
P1'Otelri YEPKTVTTGSPPVPEAHSPGFDGASFIGGWLVLSLQAVAFFVLHFLKAKDSTYQTL
Sequence
SEQ ID NO: 4S X561 by
NOVl7b, CCCATGGAGGCTCCGGGACCCCGCGCCTTGCGGACTGCGCTCTGTGGCGGCTGTTGCT
CG104210-02 GCCTCCTCCTATGTGCCCAGCTGGCTGTGGCTGGTAAAGGAGCTCGAGGCTTTGGGAG
DNA SeqlleriCeGGGAGCCCTGA'T'CCGCCTGAATATCTGGCCGGCGGTCCAAGGGGCCTGCAAACAGCTG
GAGGTCTGTGAGCACTGCGTGGAGGGAGACAGAGCGCGCAATCTCTCCAGCTGCGTGT
GGGAGCAGTGCCGGCCAGAGGAGCCAGGACACTGTGTGGCCCAATCTGAGGTGGTCAA
GGAAGGTTGCTCCATCTACAACCGCTCAGAGGCATGTCCAGCTGCTCACCACCACCCC
ACCTATGAACCGAAGACAGTCACAACAGGGAGCCCCCCAGTCCCTGAGGCCCACAGCC
CTGGATTTGACGGGGCCAGCTTTATCGGAGGTGTCGTGCTGGTGTTGAGCCTACAGGC
GGTGGCTTTCTTTGTGCTGCACTTCCTCAAGGCCAAGGACAGCACCTACCAGACGCTG
TGAGTACCTGGCCAGCAGCAAGTACCTGAGTCCCAGCTC
ORF Start: ATG at 4 ORF Stop: TGA at S23
SEQ ID NO: 46 173 as MW at 18389.OkD
NOVI7b, MEAPGPRALRTALCGGCCCLLLCAQLAVAGKGARGFGRGALIRLNIWPAVQGACKQLE
CG104210-02 VCEHCVEGDRARNLSSCWEQCRPEEPGHCVAQSEWKEGCSIYNRSEACPAAHHHPT
PT'Otelri YEPKTVTTGSPPVPEAHSPGFDGASFTGGVVLVLSLQAVAFFVLHFLKAKI3STYQTL
Sequence
SEQ ID NO: 47 349 by
NOVI7C, _CACCGGATCCGGTAAAGGAGCTCGAGGCTTTGGGAGGGGAGCCCTGATCCGCCTGAAT
272249075 ATCTGGCCGGCGGTCCAAGGGGCCTGCAAACAGCTGGAGGTCTGTGAGCACTGCGTGG
DNA
AGGGAGACAGAGCGCGCAATCTCTCCAGCTGCATGTGGGAGCAGTGCCGGCCAGAGGA
Sequence
GCCAGGACACTGTGTGGCCCAATCTGAGGTGGTCAAGGAAGGTTGCTCCATCTACAAC
CGCTCAGAGGCATGTCCAGCTGCTCACCACCACCCCACCTATGAACCGAAGACAGTCA
CAACAGGGAGCCCCCCAGTCCCTGAGGCCCACAGCCCTGGATTTGACGGGGTCGACGG
C
ORF Start: at 2 ORF Stop: end of sequence
SEQ ID NO: 48 l I6 as MW at 12383.7kD
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NOV17C, TGSGKGARGFGRGALIRLNIWPAVQGACKQLEVCEHCVEGDRARNLSSCMWEQCRPEE
272249075 PGHCVAQSEWKEGCSIYNRSEACPAAHHHPTYEPKTVTTGSPPVPEAHSPGFDGVDG
Protein Sequence
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 17B.
Table 17B. Comparison of NOVl7a against NOVl7b and NOVl7c.
Protein Sequence NOVl7a Residues/ Identities/
Match Residues ~ Similarities for the Matched Region
NOV 17b 1..173 139/173 (80%)
1..173 140/173 (80%)
NOV 17c 41..139 99/99 (100%)
15..113 99/99 (100%)
Further analysis of the NOV 17a protein yielded the following properties shown
in Table
17C.
Table 17C. Protein Sequence Properties NOVl7a
PSort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400
analysis: probability located in plasma membrane; 0.4600 probability located
in Golgi
body; 0.1000 probability located in endoplasmic reticulum (lumen)
SignalP ~ Cleavage site between residues 30 and 31 ~~~
analysis:
S A search of the NOV 17a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 17D.
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Table 17D. Geneseq Results for NOVl7a
NOVl7a Identities/
Geneseq Protein/Organism/Length Residues!Similarities Expect
jPatent for
Identifier,'' #, Date] Match the Matched Value
ResiduesRegion
AAE03827Human gene 10 encoded secreted1..173 173/173 (100%)e-103
protein HBINS58, SEQ ID 1..173 173/173 (100%)
NO: 73
- Homo Sapiens, 173 aa.
[W0200136440-A1, 25-MAY-
2001]
AAE03852Human gene l0.encoded secreted1..160 159/160 (99%)5e-94
protein HBINS58, SEQ ID 1..160 159/160 (99%)
N0:98 -
Homo Sapiens, 210 aa.
[W0200136440-Al, 25-MAY-
2001
AAB58415Lung cancer associated polypeptide73..173 41/124 (33%) 8e-10
sequence SEQ ID 753 - Homo 95..214 56/124 (45%)
sapiens, 214 aa. [W0200055180-
A2, 21-SEP-2000)
AAG03771Human secreted protein, 73..173 38/124 (30%) 1e-07
SEQ ID
NO: 7852 - Homo Sapiens, 78..197 52/124 (41%)
197 aa.
[EP1033401-A2, 06-SEP-2000
_..
ABB65987: Drosophila melanogaster 116..17329160 (48%) 9e-05
~
polypeptide SEQ ID NO 24753127..18335/60 (58%)
-
Drosophila melanogaster,
183 aa.
[WO200171042-A2, 27-SEP-2001]
In a BLAST search of public sequence databases, the NOV 17a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 17E.
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Table 17E. Public BLASTP Results for NOVl7a
NOVl7a Identities/
Protein '
Residues/Similarities Expect
Accession Protein/Organism/Length for
Match the Matched Value
Number
Residues Portion
Q9D6W7 2310047NO1RII~ PROTEIN 1..173 1401173 (80%)1e-82
-
Mus musculus (Mouse), 1..17I 150/173 (85%)
172 aa.
Q9BPV0 CD164 ISOFORM DELTA 4 73..173 41/111 (36%) 6e-11
-
Homo sapiens (Human), ~ 561111 (49%) ~
184 aa. ~
78..184
Q9CVT7 CDI64 ANTIGEN - Mus 25..173 51/173 (29%) 2e-10
musculus (Mouse), 161 5..161 67/173 (38%)
as
(fragment).
Q9QX82 ENDOLYN PRECURSOR - 54..173 41/140 (29%) 4e-10
Rattus norvegicus (Rat),57..195 59/140 (41%)
I95 aa.
Q9Z317 MGC-24V - Mus musculus 54..173 44/I44 (30%) 7e-10
_.. .._~..._,..~Mouse), 197 aa.,~ 58..197 __rv58/144 ~..........~v
.. _ (39%)
Example 18.
The NOV 18 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table I 8A.
Table 18A. NOV18 Sequence Analysis
SEQ ID N049 788 by
NOVlBa, CTTTTGCCTTTATGCAACCAACATGGAGATTTTGTACCATGTCCTGTTCTTAGTGCTT
CG104251-Ol G~TGTCCTAACCTGAAGCTGAAGAAGCCGCCCTGGCTGCACATGCTGTCGGCCATGA
DNA Se ueriCBCTGTATGCTCTGGTGGTGGTGTCTTCCTCATTACCGGAGGAATCATTTATGATGTTAT
q
TGTTGAACCTCCAAGTGTTGGCTCTATGACTGATGAACATGGGCATCAGAGGCCAGTA
GCTTTCTTTGCCTATAGAGTAAATGGACAATATATTATGGAAGGACTTGCATCCAGCT
TCCTGTTTACAATGGGAGGTTTAGGTTTCATAATCCTGGACCAATTGAATGCACCAAA
TATCCCAAAACTCAATAGATTTCTTCTTCTATTCATTGGATTTGTCTGTGTTCTATTG
AGTATTTTCATGGCTAGAGTATTCATGAGAATGAAACTGCCGAGCTATCTGATGGGTT
AGAGTGCCTTTGAGAAGAAATCAGTGGATACTGGATTTTTTCTTGTCAATGAAGTTTT
AAAGGCTGTACCAATCCTCTAATATGAAATGTGGAAAAGAATGAAGAGCAGCAGTAAA
AGAAATATCTAGTGAAAAAACAGGAAGCGTATTGAAGCTTGGACTAGAATTTCTTCTT
GGTATTAAAGAGACAAGTTTATCACAGAATTTTTTTTCCTGCTGGCCTATTGCTATAC
CAATGATGTTGAGTGGCATTTTCTTTTTAGTTTTTCATTAAAATATATTCCATATCTA
CAACTATAATATCAAATAAAGTGATTATTTTTTA
ORF Start: ATG at 23 ORF Stop: TAG at 464
SEQ ID NO: 50 147 as MW at 16447.7kD
NOVIBa, MEILYHVLFLVLECPNLKLKKPPWLHMLSAMTVCSGGGVFLITGGIIYDVIVEPPSVG
CG104251-Ol SMTDEHGHQRPVAFFAYRVNGQYIMEGLASSFLFTMGGLGFIILDQLNAPNTPKLNRF
LLLFIGFVCVLLSIFMARVFMRMKLPSYLMG
Protein Sequence
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Further analysis of the NOV 18a protein yielded the following 'properties
shown m Table
18B.
Table 18B. Protein Sequence Properties NOVl8a
PSort 0.6400 probability located in plasma membrane; 0.4600 probability
located in
analysis: Golgi body; 0.3700 probability located in endoplasmic reticulum
(membrane);
0.1000 probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 42 and 43
analysis:
A search of the NOV 18a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 18C.
Table 18C. Geneseq Results for NOVlBa
NOVl8a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent ' for
Identifier#, Date) Match the Matched Value
ResiduesRegion
AAY53631 A bone marrow secreted protein1..147 133/149 (89%)1 e-69
=
designated BMS155 - Homo 1..149 135/149 (90%)
~
Sapiens, 149 aa. [W09933979-A2,
08-JUL-1999]
AAY53042 Human secreted protein clone1..147 133/I49 (89%)1e-69
~ ~
pu282_10 protein sequence 1..149 135/149 (90%)
SEQ ID ;
N0:90 - Homo Sapiens, 149
aa.
[W09957132-A1, 11-NOV-1999]
AAB12143 Hydrophobic domain protein 1..147 133/149 (89%)1e-69
~
isolated from WERI-RB cells1..149 135/149 (90%)
-
Homo sapiens, 149 aa. t
[WO200029448-A2, 25-MAY-
2000]
AAY59670 Secreted protein 108-005-5-0-F6-FL1..147 133/149 (89%)1e-69
- Homo Sapiens, 149 aa. 1..149 135/149 (90%)
[W09940189-A2, I2-AIIG-1999]
AAY60146 Human endometrium tumour 1..147 133/149 (89%)1 e-69
EST :
encoded protein 206 - Homo 23..171 135/149 (90%)
Sapiens, 171 aa. [DE19817948-A1,
2.1-~CT-1999]
_..........
In a BLAST search of public sequence databases, the NOV I 8a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 18D.
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Table 18D. Public BLASTP Results for NOVlBa
Protein NOVl8a Identities/
Residues/ SimilaritiesExpect
Accession Protein/Organism/Length for
~ Match the MatchedValue
Number '
Residues Portion
Q9NRP0 DC2 (HYDROPHOBIC PROTEIN 1..147 133/149 3e-69
(89%)
HSF-28) (HYPOTHETICAL 1..149 135/149
16.8 (90%)
I~IDA PROTEIN) - Homo
sapiens
(Human), 149 aa.
Q9P075 HSPC307 - Homo sapiens 1..147 133/149 3e-69
(89%)
(Human), 167 as (fragment).19..167 135/149
~ (90%)
Q9CPZ2 2310008MlORII~ PROTEIN 1..147 132/149 6e-69
(88%)
(RIKEN CDNA 2310008M10 1..149 135/149
(90%)
GENE) - Mus musculus (Mouse),
149 aa.
Q9P1R4 HDCMD45P - Homo Sapiens ~ 1..147 132/149 2e-68
~ (88%)
(Human), 160 as (fragment).~ 12..160 134/149
(89%)
AAH24224 SIMILAR TO DC2 PROTEIN 40..147 96/108 (88%)7e-50
- ~
Homo Sapiens (Human), 12..119 100/108
119 aa. (91%)
~
Examine 19.
The NOV 19 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 19A.
Table 19A. NOV19 Sequence Analysis
SEQ ID NO: 51 3761 by
NOVl9a, GGGCGCGCCGAGCCGGGCGCGGGGGCGCTGAACGGCGGAGCGGGAGCGGCCGGAGGAG
CG104934-O1 _CCATGGACTGCAGCCTCGTGCGGACGCTCGTGCACAGATACTGTGCAGGAGAAGAGAA
DNA Se uenCeTTGGGTGGACAGCAGGACCATCTACGTGGGACACAGGGAGCCACCTCCGGGCGCAGAG
q
GCCTACATCCCACAGAGATACCCAGACAACAGGATCGTCTCGTCCAAGTACACATTTT
GGAACTTTATACCCAAGAATTTATTTGAACAATTCAGAAGAGTAGCCAACTTTTATTT
CCTTATCATATTTCTGGTGCAGTTGATTATTGATACACCCACAAGTCCAGTGACAAGC
GGACTTCCACTCTTCTTTGTCATTACTGTGACGGCTATCAAACAGGGTTATGAAGACT
GGCTTCGACATAAAGCAGACAATGCCATGAACCAGTGTCCTGTTCATTTCATTCAGCA
CGGCAAGCTCGTTCGGAAACAAAGTCGAAAGCTGCGAGTTGGGGACATTGTCATGGTT
AAGGAGGACGAGACCTTTCCCTGCGACTTGATCTTCCTTTCCAGCAACCGGGGAGATG
GGACGTGCCACGTCACCACCGCCAGCTTGGATGGAGAATCCAGCCATAAAACGCATTA
CGCGGTCCAGGACACCAAAGGCTTCCACACAGAGGAGGATATCGGCGGACTTCACGCC
ACCATCGAGTGTGAGCAGCCCCAGCCCGACCTCTACAAGTTCGTGGGTCGCATCAACG
TTTACAGTGACCTGAATGACCCCGTGGTGAGGCCCTTAGGATCGGAAAACCTGCTGCT
TAGAGGAGCTACACTGAAGAACACTGAGAAAATCTTTGGTGTGGCTATTTACACGGGA
ATGGAAACCAAGATGGCATTAAATTATCAATCAAAATCTCAGAAGCGATCTGCCGTGG
AAA.AATCGATGAATGCGTTCCTCATTGTGTATCTCTGCATTCTGATCAGCAAAGCCCT
GATAAACACTGTGCTGAAATACATGTGGCAGAGTGAGCCCTTTCGGGATGAGCCGTGG
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TATAATCAGAAAACGGAGTCGGAAAGGCAGAGGAATCTGTTCCTCAAGGCATTCACGG
ACTTCCTGGCCTTCATGGTCCTCTTTAACTACATCATCCCTGTGTCCATGTACGTCAC
GGTCGAGATGCAGAAGTTCCTCGGCTCTTACTTCATCACCTGGGACGAAGACATGTTT
GACGAGGAGACTGGCGAGGGGCCTCTGGTGAACACGTCGGACCTCAATGAAGAGCTGG
GACAGGTGGAGTACATCTTCACAGACAAGACCGGCACCCTCACGGAAAACAACATGGA
GTTCAAGGAGTGCTGCATCGAAGGCCATGTCTACGTGCCCCACGTCATCTGCAACGGG
CAGGTCCTCCCAGAGTCGTCAGGAATCGACATGATTGACTCGTCCCCCAGCGTCAACG
GGAGGGAGCGCGAGGAGCTGTTTTTCCGGGCCCTCTGTCTCTGCCACACCGTCCAGGT
GAAAGACGATGACAGCGTAGACGGCCCCAGGAAATCGCCGGACGGGGGGAAATCCTGT
GTGTACATCTCATCCTCGCCCGACGAGGTGGCGCTGGTCGAAGGTGTCCAGAGACTTG
GCTTTACCTACCTAAGGCTGAAGGACAATTACATGGAGATATTAAACAGGGAGAACCA
CATCGAAAGGTTTGAATTGCTGGAAATTTTGAGTTTTGACTCAGTCAGAAGGAGAATG
AGTGTAATTGTAAAATCTGCTACAGGAGAAATTTATCTGTTTTGCAAAGGAGCAGATT
CTTCGATATTCCCCCGAGTGATAGAAGGCAAAGTTGACCAGATCCGAGCCAGAGTGGA
GCGTAACGCAGTGGAGGGGCTCCGAACTTTGTGTGTTGCTTATAAAAGGCTGATCCAA
GAAGAATATGAAGGCATTTGTAAGCTGCTGCAGGCTGCCAAAGTGGCCCTTCAAGATC
GAGAGAAA.AAGTTAGCAGAAGCCTATGAGCAAATAGAGAAAGATCTTACTCTGCTTGG
TGCTACAGCTGTTGAGGACCGGCTGCAGGAGAAAGCTGCAGACACCATCGAGGCCCTG
CAGAAGGCCGGGATCAAAGTCTGGGTTCTCACGGGAGACAAGATGGAGACGGCCGCGG
CCACGTGCTACGCCTGCAAGCTCTTCCGCAGGAACACGCAGCTGCTGGAGCTGACCAC
CAAGAGGATCGAGGAGCAGAGCCTGCACGACGTCCTGTTCGAGCTGAGCAAGACGGTC
CTGCGCCACAGCGGGAGCCTGACCAGAGACAACCTGTCCGGACTTTCAGCAGATATGC
AGGACTACGGTTTAATTATCGACGGAGCTGCACTGTCTCTGATAATGAAGCCTCGAGA
AGACGGGAGTTCCGGCAACTACAGGGAGCTCTTCCTGGAAATCTGCCGGAGCTGCAGC
GCGGTGCTCTGCTGCCGCATGGCGCCCTTGCAGAAGGCTCAGATTGTTAAATTAATCA
AATTTTCAAAAGAGCACCCAATCACGTTAGCAATTGGCGATGGTGCAAATGATGTCAG
CATGATTCTGGAAGCGCACGTGGGCATAGGTGTCATCGGCAAGGAAGGCCGCCAGGCT
TTCACGGGCATTTTTATTACATTAGGATCTCTGAGCTCGTGCAGTACTTCTTCTATAA
GAACGTCTGCTTCATCTTCCCTCAGTTTTTATACCAGTTCTTCTGTGGGTTTTCACAA
CAGACTTTGTACGACACCGCGTATCTGACCCTCTACAACATCAGCTTCACCTCCCTCC
CCATCCTCCTGTACAGCCTCATGGAGCAGCATGTTGGCATTGACGTGCTCAAGAGAGA
CCCGACCCTGTACAGGGACGTCGCCAAGAATGCCCTGCTGCGCTGGCGCGTGTTCATC
TACTGGACGCTCCTGGGACTGTTTGACGCACTGGTGTTCTTCTTTGGTGCTTATTTCG
TGTTTGAAAATACAACTGTGACAAGCAACGGGCAGATATTTGGAAACTGGACGTTTGG
AACGCTGGTATTCACCGTGATGGTGTTCACAGTTACACTAAAGCTTGCATTGGACACA
TCTTTTCGCTTCTCTGGGGAGGAGTGATCTGGCCGTTCCTCAACTACCAGAGGATGTA
CTACGTGTTCATCCAGATGCTGTCCAGCGGGCCCGCCTGGCTGGCCATCGTGCTGCTG
GTGACCATCAGCCTCCTTCCCGACGTCCTCAAGAAAGTCCTGTGCCGGCAGCTGTGGC
ATTCACCCCTCTTGCCTCTCTGCAGAGCCCAGGCTACCAGAGCACCTGTCCCTCGGCC
GCCTGGTACAGCTCCCACTCTCAGCAGGTGACACTCGCGGCCTGGAAGGAGAAGGTGT
CCACGGAGCCCCCACCCATCCTCGGCGGTTCCCATCACCACTGCAGTTCCATCCCAAG
TCACAGCTGCCCTAGGTCCCGTGTGGGAATGCTCGTGTGATGGATGGTCCTAAGCCTG
TGGAGACTGTGCACGTGCCTCTTCCTGGCCCCCAGCAGGCAAGGAGGGG
CCTTGCCCTCGAGCATGGCACCCTGGCCGCCTGGACCCAGCACTGTGGT
ORF Start: ATG at 61 ORF Stop: TGA at 3634
SEQ ID NO: 52 1191 as MW at 135846.OkD
NOVl9a, MDCSLVRTLVHRYCAGEENWVDSRTIYVGHREPPPGAEAYIPQRYPDNRIVSSKYTFW
CG104934-01 NFIPKNLFEQFRRVANFYFLIIFLVQLITDTPTSPVTSGLPLFFVITVTAIKQGYEDW
PPOtelri LRHKADNAMNQCPVHFIQHGKLVRKQSRKLRVGDIVMVKEDETFPCDLIFLSSNRGDG
Sequence
TCHVTTASLDGESSHKTHYAVQDTKGFHTEEDIGGLHATIECEQPQPDLYKFVGRTNV
YSDLNDPVVRPLGSENLLLRGATLKNTEKIFGVAIYTGMETKMALNYQSKSQKRSAVE
KSMNAFLIVYLCILISKALINTVLKYMWQSEPFRDEPWYNQKTESERQRNLFLKAFTD
FLAFMVLFNYIIPVSMYVTVEMQKFLGSYFITWDEDMFDEETGEGPLVNTSDLNEELG
QVEYIFTDKTGTLTENNMEFKECCIEGHVYVPHVICNGQVLPESSGIDMIDSSPSVNG
REREELFFRALCLCHTVQVKDDDSVDGPRKSPDGGKSCVYISSSPDEVALVEGVQRLG
FTYLRLKDNYMEILNRENHIERFELLEILSFDSVRRRMSVIVKSATGEIYLFCKGADS
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SIFPRVIEGKVDQIRARVERNAVEGLRTLCVAYKRLIQEEYEGICKLLQAAKVALQDR
EKKLAEAYEQIEKDLTLLGATAVEDRLQEKAADTIEALQKAGIKVWVLTGDKMETAAA
TCYACKLFRRNTQLLELTTKRIEEQSLHDVLFELSKTVLRHSGSLTRDNLSGLSADMQ
DYGLIIDGAALSLIMKPREDGSSGNYRELFLEICRSCSAVLCCRMAPLQKAQIVKLIK
FSKEHPITLAIGDGANDVSMILEAHVGIGVIGKEGRQAARNSDYAIPKFKHLKKMLLV
HGHFYYTRISELVQYFFYKNVCFIFPQFLYQFFCGFSQQTLYDTAYLTLYNISFTSLP
ILLYSLMEQHVGIDVLKRDPTLYRDVAKNALLRWRVFIYWTLLGLFDALVFFFGAYFV
FENTTVTSNGQIFGNWTFGTLVFTVMVFTVTLKLALDTHYWTWINHFVIWGSLLFYVV
FSLLWGGVIWPFLNYQRMYYVFIQMLSSGPAWLAIVLLVTISLLPDVLKKVLCRQLWP
TATERVQNGCAQPRDRDSEFTPLASLQSPGYQSTCPSAAWYSSHSQQVTLAAWKEKVS
TEPPPILGGSHHHCSSIPSHSCPRSRVGMLV
Further analysis of the NOV 19a protein yielded the following properties shown
in Table
19B.
YTabIeYl9B. Protein Sequence Properties NOVI9a
PSort 0.6000 probability located m plasma membrane, 0.4000 probability located
in
analysis: ~ Golgi body; 0.3000 probability located in endoplasmic reticulum
(membrane);
0.0300 probability located in mitochondria) inner membrane
SignaIP No Known Signal Sequence Predicted
analysis:
A search of the NOV 19a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 19C.
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Table 19C. Geneseq Results for NOVl9a
NOVl9a Identities/
Geneseq ProteinlOrganism/Length Residues/Similarities Expect
for
Identifier(Patent #, Date) Match the Matched Value
Residues Region
.
AAO14200 ~ Human transporter and 1..1191 1190/1192 0.0
ion (99%)
channel TRICH-17 - Homo 1..l 192 1191/1192
(99%)
Sapiens, 1192 aa. [W0200204520-
A2, 17-JAN-2002]
AAB42368 Human ORFX ORF2132 338..1109770/772 (99%)0.0
polypeptide sequence SEQ 1..772 772/772 (99%)
ID ~
N0:4264 - Homo sapiens,
797 aa.
[W0200058473-A2, OS-OCT- ' ,
.
2000]
AAG67546 Amino acid sequence of 22..1109 657/1119 (58%)0.0
a human
transporter protein - 18..1106 833/1119 (73%)
Homo
Sapiens, 1177 aa. [W0200164878-
A2, 07-SEP-2001
AA014203 Human transporter and 22..1109 583/1119 (52%)0.0
ion
channel TRICH-20 - Homo 18..1040 755/I 119
a (67%)
Sapiens, 1096 aa. [WO200204520-
A2, 17-JAN-2002]
AAM39290 Human polypeptide SEQ 370..1109424/771 (54%)0.0
ID NO '
2435 - Homo Sapiens, 815 1..744 544/771 (69%)
aa.
[W0200153312-Al, 26-JUL-
2001
In a BLAST search of public sequence databases, the NOV 19a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 19D.
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....Table , .
19D. . .....
a - . .
lic
BLA
. TP
Results
a ....
.
P b S
for
NOV19
~
Protein ~ NOVl9a Identities/
AccessionProtein/Organism/Length ' Residues/Similarities Expect
for
Number Match the Matched Value
s
Residues Portion
P98197 Potential phospholipid- ~ 1..l 1074/1195 (89%)0.0
189
transporting ATPase IH I..1185 1117/1195 (92%)
(EC
3.6.3.1 ) - Mus musculus
(Mouse),
1187 aa.
P98196 Potential phospholipid- 338..1109772/772 (100%)0.0
transporting ATPase IS ~ I..772 772/772 (100%)
(EC
3 .6.3.1 ) - Homo Sapiens
(Human),
797 as (fragment).
...... .~ m
,~.., 14..997 633/992 (63%) 0.0
Q8WX24
BB206I21.1
(ATPASE,
CLASS
VI, TYPE 11C ) - Homo 1..962 770/992 (76%)
sapienS
Human), 962.aa (fragment).. ..
Q9NOZ4 RING-FINGER BINDING ~ 22..1109574/1123 (51%)0.0
PROTEIN - Oryctolagus ~ 10..1036752/1123 (66%)
cuniculus (Rabbit), 1107
as
(fragment).
Q9Y2G3 Potential phospholipid- ~ 486..1109358/625 (57%) 0.0
transporting ATPase IR 1..601 462/625 (73%)
(EC
3.6.3.1 ) - Homo sapiens x
(Human),
672 as (fragment).
PFam analysis predicts that the NOV I 9a protein contains the domains shown in
the Table
19E.
Table 19E. Domain Analysis of NOVl9a
Identities/
Pfam Domain NOVl9a Match Region Similarities Expect Value
for the Matched Region
Hydrolase 408..846 46/448 (10%) 0.0058
_. __ ~~_ _ 258/448 (58%)
_. _.._ . .. _.. _... _ ._ _____ . __ ____ _ _ .. _... .__.
Example 20.
The NOV20 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 20A.
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Table 20A. NOV20 Sequence
SEQ ID NO: 532588 by
NOV2Oa, ~AGTTCCGAACAGAAGGCTGTGTATTCTCTGCCGCTTATTGTGGCCTCGACAGGCCATG
CG105463-O1 GTTACTTTGGCCACTGCCAGAGCAGCCTTGGCACTATGGAGGAGCCTAGGGCTACCCC
DNA Se tl0riC8 TCAGCTGTACTTGGGGCTGGTCCTGCAGTTGCTACCCAGGGTTATGGCAGCACTGCCT
GAAGGTGTGAGACCAAATTCGAATCCTTATGGTTTTCCATGGGAATTGGTGATATGTG
CAGCTGTCCTTGGATTTGTTGCTGTTCCCTTTTTTTTGTGGAGAAGTTTTAGATCGGT
TAGGAGTCGGCTTTATGTGGGAAGAGAGAAAGAGCTTGCTATAGCGCTTTCTGGACTA
ATTGAAGAAAAATGTAGACTACTTGAAAAATTTAGCCTTGTTCAAAAAGAGTATGAAG
GCTATGAAGTAGAGTCATCTTTAGAGGATGCCAGCTTTGAGAAGGAGGCAACAGAAGC
ACAAAGTCTGGAGGCAAACTGTGAAAAGCTGAACAGGTCCAATTCTGAACTGGAGCAT
GAAATACTCTGTCTAGAAAAGGGGATAAAAGAAGAGAAATCTAAACATTCTGAACAAG
ATGAGGTGATGGCAGATATTTCCAAAAAGATACAGTCTCTAGAAGATGAGTCAAAATC
CCTCAAATCACTACTAACTGAAGCCAAAATGACCTTCAAGGGATTTCAAATGAATGAA
GAAAA.ACTGGAGATAGGAATACAAGATGCTTCGAGTGAAAATTGTCAACTTCAGGAAA
GCCAGAAACAGCTTTTGCAAGAAGCTGAAGTATGGAAAGAACAAGTGAGTGAACTTAA
TAAACAGAAAATAACATTTGAAGACTCCAA.AGTACACGCAGAACAAGTTCTAAATGAT
AAAGAAAATCACATCGAGACTCTGACTGAACGCTTGCTAAAGATCAAAGATCAGGCTG
CTGTGCTGGAAGAAGACATAACGGATGATGGTAACTTGGAATTAGAAATGAACAGTGA
ATTGAAAGATGGTGCTTACTTAGATAATCCTCCAAAAGGAGCTTTGAAGAAACTGATT
CATGCTGCTAAGTTAAATGCTTCTTTAACAACCTTAGAAGGAGAAAGAAACCAATTTA
TATTCAGTTATCTGAAGTTGATAA.AACCAAGGAAGAGCTTAGAGAGCATATTAAAAAT
CTTCAGACGGAACAAGCATCTTTGCAGTCGGAA.AACACACATTTTGAAAGTGAGAATC
AGAAACTTCAACAGAAAGTTAATGACTGAGTTATATCAAGAAAATGAAATGAAACTCT
ACAGGAAATTAATAGTAGAGGAAAATAACCGGTTAGAGAAAGAGAAACTTTCTAAAGT
AGACGAAATGATCAGCCATGCCACTGAAGAGCTGGAGACCTGCAGAAAGCGAGCCAAA
GATCTTGAAGAAGAACTTGAGAGAACTATTCTTTTTTATCAAGGGAAGATTATATACC
ATGAGAAAAA.AGCACATGATAATTGTTTGGCAGCATGGACTGCTGAAAGAAACCTCAA
~TGATTTAAGGAAAGAAAATGCTCACAAAAGACAAAAATTAGCTGAAACAGAGTTTAAA
ATTAAACTTTTAGAAAAAGATCCTTATGCACTTGATGTTCCAAATACAGCATTTGGCA
GAGAGCATTCCTCATATGGTCCCTCACCATTGGGTCGGCCTTCATCTGAAACGAGAGC
TTTTCTCTATCTTCCGACTTTGTTGGAGGGTCCACTGAGACTCTCACCTTTGCTTCCA
GGGGGAGGAGGAAGAGACCCAAGAGGCCCAGGGAATCCTCTGGACCACCAGATTACCA
CACTGGGCCCCTGTCACCTCCGTGGGAACAGGACCGTAGGATGATGTTTC
GGACAATCATATCCTGATTCAGCTCTTCCTCCACAAAGGCAAGACAGATT
TAAAATGGATGGGTCAATGCCTTCAGAAATGGAATCCAGTGGAAATGATACCAAAGAT
AATCTTGGTAATTTAAATGTGGCTGATTCATCTCTCCCTGCTGGAAATGAAGTGAGTG
GCCCTGGCTTTGTTCCTCCACCTCTTGCTTCAATCAGAGGTCCATTGTTTCCAGTGGA
TACGAGGGGCCCGTTCATGAGAAGAGGACCTCCTTTCCCTCCACCTCCTCCAGGAACC
ATGTTTGGAGCTTCTCCAGATTATTTTCCACCAAGGGATGTCCCAGGTCCACCACGTG
CTCCATTTGCAATGAGAAATGTCTGTCCACCGAGGGGTTTTCCTCCTTACCTTCCCCC
AAGACCTGGATTTTGCCCCCACCCCCACCCCCACAGTGAGTTCCCTTTAGGGTTGAGT
CTGCCTTCAAATGAGCCTGCTGCTGAAGATCCAGAACCACGGCAAGAAACCTGATAAT
ATTTTTGCTGTCTTCAAAAGTCATTTTGACTATTCTCATTTTCAGTTGAAGTAACTGC
TGTTACTTCAGTGATTACACTTTTGCTCAAATTGAA
ORF Start ATG at 94 ORF Stop TGA at 2488
~.. ID N . _.. ...... . 7 . . ~ ..
3
SEQ O. 54 98 as MW at 90383.6kD
NOVZOa, MEEPRATPQLYLGLVLQLLPRVMAALPEGVRPNSNPYGFPWELVICAAVLGFVAVPFF
CG105463-O1 LWRSFRSVRSRLYVGREKELAIALSGLIEEKCRLLEKFSLVQKEYEGYEVESSLEDAS
Protein Se lleriCB FEKEATEAQSLEANCEKLNRSNSELEHEILCLEKGIKEEKSKHSEQDEVMADISKKIQ
SLEDESKSLKSLLTEAKMTFKGFQMNEEKLEIGIQDASSENCQLQESQKQLLQEAEVW
KEQVSELNKQKITFEDSKVHAEQVLNDKENHIETLTERLLKIKDQAAVLEEDITDDGN
LELEMNSELKDGAYLDNPPKGALKKLIHAAKLNASLTTLEGERNQFIFSYLKLIKPRK
150
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SLESILKIFRRNKHLCSRKTHILKVRIRNFNRKLMTELYQENEMKLYRKLIVEENNRL
EKEKLSKVDEMISHATEELETCRKRAKDLEEELERTILFYQGKIIYHEKKAHDNCLAA
WTAERNLNDLRKENAFiKRQKLAETEFKIKLLEKDPYALDVPNTAFGREHSSYGPSPLG
RPSSETRAFLYLPTLLEGPLRLSPLLPGGGGRDPRGPGNPLDHQITKERGESSCDRFT
DPHKAPSDTGPLSPPWEQDRRMMFPPPGQSYPDSALPPQRQDRFYSNSARRSGLAELR
SFNIPSLDKMDGSMPSEMESSGNDTKDNLGNLNVADSSLPAGNEVSGPGFVPPPLASI
RGPLFPVDTRGPFMRRGPPFPPPPPGTMFGASPDYFPPRDVPGPPRAPFAMRNVCPPR
GFPPYLPPRPGFCPHPHPHSEFPLGLSLPSNEPAAEDPEPRQET
SEQ ID NO: SS 2483 by
NOV2Ob, AGCTGGAATTCGCCCTTCTCGACAGGCCATGGTTACTTTGGCCACTGCCAGAGCAGCC
CG105463-02 TTGGCACTATGGAGGAGCCTAGGGCTACCCCTCAGCTGTACTTGGGGCTGGTCCTGCA
DNA Se tleriCeGTTGCTACCCAGGGTTATGGCAGCACTGCCTGAAGGTGTGAGACCAAATTCGAATCCT
TATGGTTTTCCATGGGAATTGGTGATATGTGCAGCTGTCCTTGGATTTGTTGCTGTTC
CCTTTTTTTTGTGGAGAAGTTTTAGATCGGTTAGGAGTCGGCTTTATGTGGGAAGAGA
GAAAGAGCTTGCTATAGCGCTTTCTGGACTAATTGAAGAAAAATGTAGACTACTTGAA
AAATTTAGCCTTGTTCAA.AAAGAGTATGAAGGCTATGAAGTAGAGTCATCTTTAGAGG
ATGCCAGCTTTGAGAAGGAGGCAACAGAAGCACAAAGTCTGGAGGCAAACTGTGAAAA
GCTGAACAGGTCCAATTCTGAACTGGAGCATGAAATACTCTGTCTAGAAAAGGGGATA
AAAGAAGAGAAATCTAAACATTCTGAACAAGATGAGGTGATGGCAGATATTTCCAAAA
AGATACAGTCTCTAGAAGATGAGTCAAAATCCCTCAAATCACTACTAACTGAAGCTAA
AATGACCTTCAAGGGATTTCAAATGAATGAAGAAAAACTGGAGATAGGAATACAAGAT
GCTTCGAGTGAAAATTGTCAACTTCAGGAAAGCCAGAAACAGCTTTTGCAAGAAGCTG
AAGTATGGAAAGAACAAGTGAGTGAACTTAATAAACAGAAAATAACATTTGAAGACTC
CAAAGTACACGCAGAACAAGTTCTAAATGATAAAGAAAATCACATCGAGACTCTGACT
GAACGCTTGCTAAAGATCAAAGATCAGGCTGCTGTGCTGGAAGAAGACATAACGGATG
ATGGTAACTTGGAATTAGAAATGAACAGTGAATTGAAAGATGGTGCTTACTTAGATAA
TCCTCCAAAAGGAGCTTTGAAGAAACTGATTCATGCTGCTAAGTTAAATGCTTCTTTA
ACAACCTTAGAAGGAGAAAGAAACCAATTTATATTCAGTTATCTGAAGTTGATAAAAC
CAAGGAAGAGCTTAGAGAGCATATTAAAAATCTTCAGACGGAACAAGCATCTTTGCAG
TCGGAAAACACACATTTTGAAAGTGAGAATCAGAAACTTCAACAGAAAGTTAATGACT
GAGTTATATCAAGAAAATGAAATGAAACTCTACAGGAAATTAATAGTAGAGGAAAATA
ACCGGTTAGAGAAAGAGAAACTTTCTAAAGTAGACGAAATGATCAGCCATGCCACTGA
AGAGCTGGAGACCTGCAGAAAGCGAGCCAAAGATCTTGAAGAAGAACTTGAGAGAACT
ATTCTTTTTTATCAAGGGAAGATTATATACCATGAGAAAAAAGCACATGATAATTGTT
TGGCAGCATGGACTGCTGAAAGAAACCTCAATGATTTAAGGAAAGAAAATGCTCACAA
AAGACAAAAATTAGCTGAAACAGAGTTTAAAATTAAACTTTTAGAAAAAGATCCTTAT
GCACTTGATGTTCCAAATACAGCATTTGGCAGAGAGCATTCCTCATATGGTCCCTCAC
CATTGGGTCGGCCTTCATCTGAAACGAGAGCTTTTCTCTATCTTCCGACTTTGTTGGA
GGGTCCACTGAGACTCTCACCTTTGCTTCCAGGGGGAGGAGGAAGAGGCCCAAGAGGC
CCAGGGAATCCTCTGGACCACCAGATTACCAAGGAAAGAGGAGAATCAAGCTGTGATA
GGTTTACTGATCCTCACAAGGCTCCTTCTGACACTGGGCCCCTGTCACCTCCGTGGGA
ACAGGACCGTAGGATGATGTTTCCTCCACCAGGACAATCATATCCTGATTCAGCTCTT
CCTCCACAAAGGCAAGACAGATTTTATTCTAATTCTGCTAGACGCTCTGGACTAGCAG
AACTCAGAAGTTTT.AATATACCTTCTTTGGATAAAATGGATGGGTCAATGCCTTCAGA
AATGGAATCCAGTGGAAATGATACCAAAGATAATCTTGGTAATTTAAATGTGGCTGAT
TCATCTCTCCCTGCTGGAAATGAAGTGAGTGGCCCTGGCTTTGTTCCTCCACCTCTTG
CTCCAATCAGAGGTCCGTTGTTTCCAGTGGATACGAGGGGCCCGTTCATGAGAAGAGG
ACCTCCTTTCCCTCCACCTCCTCCAGGAACCATGTTTGGAGCTTCTCCAGATTATTTT
CCACCAAGGGATGTCCCAGGTCTACCACGTGCTCCATTTGCAATGAGAAATGTCTGTC
CACCGAGGGGTTTTCCTCCTTACCTTCCCCCAAGACCTGGATTTTGCCCCCACCCCCA
CCCCCACATTCTGAAGATAGAGTGAGTTCCCTTTAGGGTTGAGTGCCTTCAAATGAGC
CTGCTGCTGAAGATCCAGAACCACGGCAAGAAACCTGATAATATTTT
ORF Start: ATG at 67 ORF Stop: TGA at 2401
SEQ ID NO: S6 778 as MW at 882SS.SkD
NOV2Ob, MEEPRATPQLYLGLVLQLLPRVMAALPEGVRPNSNPYGFPWELVICAAVLGFVAVPFF
CG105463-02 LWRSFRSVRSRLYVGREKELAIALSGLIEEKCRLLEKFSLVQKEYEGYEVESSLEDAS
P1'Otelri FEKEATEAQSLEANCEKLNRSNSELEHEILCLEKGIKEEKSKHSEQDEVMADTSKKIQ
SeCllleriCe
SLEDESKSLKSLLTEAKMTFKGFQMNEEKLEIGIQDASSENCQLQESQKQLLQEAEVW
1S1
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KEQVSELNKQKITFEDSKVHAEQVLNDKENHIETLTERLLKIKDQAAVLEEDITDDGN
LELEMNSELKDGAYLDNPPKGALKKLIHAAKLNASLTTLEGERNQFIFSYLKLIKPRK
SLESTLKIFRRNKHLCSRKTHT~LKVRIRNFNRKLMTELYQENEMKLYRKLIVEENNRL
EKEKLSKVDEMISHATEELETCRK.RAKDLEEELERTILFYQGKI IYHEKI~AIiDNCLA.A
WTAERNLNDLRKENAHKRQKLAETEFKIKLLEKDPYALDVPNTAFGREHSSYGPSPLG
RPSSETRAFLYLPTLLEGPLRLSPLLPGGGGRGPRGPGNPLDHQITKERGESSCDRFT
DPHKAPSDTGPLSPPWEQDRRMMFPPPGQSYPDSALPPQRQDRFYSNSARRSGLAELR
SFNIPSLDKMDGSMPSEMESSGNDTKDNLGNLNVADSSLPAGNEVSGPGFVPPPLAPI
RGPLFPVDTRGPFMRRGPPFPPPPPGTMFGASPDYFPPRDVPGLPRAPFAMRNVCPPR
GFPPYLPPRPGFCPHPHPHILKIE
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 20B.
Table 20B. Comparison of NOV20a against NOV20b.
Protein Sequence NOV20a Residues/ Identities/
Match Residues Similarities for the Matched Region
~~ .. ....~. .. . .
NOV20b ~ I..750 662/750 (88%)
1..750 662/750 (88%)
_... . _.... _.. . ..
Further analysis of the NOV20a protein yielded the following properties shown
in Table
20C.
Table 20C. Protein Sequence Properties NOV20a
PSort a 0.4600 probability located in plasma membrane; 0.1000 probability
located in
analysis: endoplasmic reticulum (membrane); 0.1000 probability located in
endoplasmic reticulum (lumen); 0.1000 probability located in outside
SignalP ; Cleavage site between residues 25 and 26
analysis:
A search of the NOV20a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 20D.
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Table 20D. Geneseq Results
for NOV20a
NOV20a Identities/
~
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent for
Identifier#, Datej Match the Matched Value
Residues~ Region
AAY77574 Human cytoskeletal protein1..798 ~63 8/812 0.0
(78%)
(HCYT) (clone 3768043) I ..806 683/812 (83%)
- Homo
Sapiens, 806 aa. [W0200006730-
A2, 10-FEB-2000]
ABG05280 Novel human diagnostic 1..797 639/814 (78%)0.0
protein ~
#5271 - Homo Sapiens, 881 59..867 ~ 684/814
aa. (83%)
x
[W0200175067-A2, II-OCT-2001]
ABG05280 Novel human diagnostic 1..797 639/814 (78%)0.0
protein
#5271 - Homo Sapiens, 881 59..867 ~ 684/814
aa. (83%)
[W0200175067-A2, 11-OCT-2001]
ABG20258 Novel human diagnostic 1..797 634/814 (77%)0.0
protein
#20249 - Homo sapiens, 59..867 681/814 (82%)
881 aa.
[W0200175067-A2, 11-OCT-2001] ~
ABG20258 Novel human diagnostic 1..797 ~ 634/814 0.0
protein (77%)
#20249 - Homo sapiens, 59..867 681/814 (82%)
881 aa.
[W02001.75067.,A2, 1l-OCT-2001]..... . .. ~ _.. .. . ...
_.. .
...
_~
In a BLAST search of public sequence databases, the NOV20a protein Was found
to have
homology to the proteins shown in the BLASTP data in Table 20E.
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Table 20E. Public BLASTP
Results for NOV20a
Protein NOV20a Identities/
Accession Protein/Organism/Length Residues/Similarities for
, Expect
Number Match the Matched Value
Residues Portion
015320 Meningioma-expressed antigen1..798 653/810 (80%) 0.0
6/11
(MEA6) (MEA11) - Homo Sapiens1..804 696/810 (85%)
(Human), 804 aa.
Q96SG9 BASOOG10.2 (NOVEL PROTEIN 1..798 616/805 (76%) 0.0
SIMILAR TO MENINGIOMA 15..816 670/805 (82%)
EXPRESSED ANTIGEN 6 (MEA6)
AND 11 (MEA 11 )) - Homo
Sapiens
(Human), 825 as (fragment).
~~~
Q96RT6 CTAGE-2 - Homo Sapiens 30..775 605/749 (80%) 0.0
(Human), o~
754 aa. 1..746 6421749 84/
( )~
095046 WUGSC:H_DJ0988G15.3 1..770 570/775 (73%) 0.0
PROTEIN (DJ1005H11.2) 1..775 633/775 (8I%)
(WUGSC:H DJ0988G15.3
PROTEIN) - Homo Sapiens
(Human), 777 aa.
AAH26864 SIMILAR TO MENINGIOMA 30..796 5201783 (66%) ~
~ 0.0
EXPRESSED ANTIGEN 6 1..778 600/783 (76%)
(COILED-COIL PROLINE-RICH)
-
Mus musculus (Mouse), 779
aa.
PFam analysis predicts that the NOV20a protein contains the domains shown in
the Table
20F.
Table 20F. Domain Analysis of NOV20a
Identities/
Pfam Domain NOV20a Match Region : Similarities Expect Value
for the Matched Region
No Significant Matches Found
Example 21.
The NOV21 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 21A.
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Table 21A. NOV21 Sequence Analysis
SEQ ID NO: 57 1269 by
NOV2la, AGCGGGGGGCGCTGCCTCGAGCCTCATGGCTGCCCCTGCTTCCGTCATGGGCCCACTC
CG105491-O1 GGGCCCTCTGCCCTGGGCCTTCTGCTGCTGCTCCTGGTGGTGGCCCCTCCCCGGGTCG
DNA Se uenCeCAGCATTGGTCCACAGACAGCCAGAGAACCAGGGAATCTCCCTAACTGGCAGCGTGGC
q
CTGTGGTCGGCCCAGCATGGAGGGGAAAATCCTGGGCGGCGTCCCTGCGCCCGAGAGG
AAGTGGCCGTGGCAGGTCAGCGTGCACTACGCAGGCCTCCACGTCTGCGGCGGCTCCA
TCCTCAATGAGTACTGGGTGCTGTCAGCTGCGCACTGCTTTCACAGGGACAAGAATAT
CAAAATCTATGACATGTACGTAGGCCTCGTAAACCTCAGGGTGGCCGGCAACCACACC
CAGTGGTATGAGGTGAACAGGGTGATCCTGCACCCCACATATGAGATGTACCACCCCA
TCGGAGGTGACGTGGCCCTGGTGCAGCTGAAGACCCGCATTGTGTTTTCTGAGTCCGT
GCTCCCGGTTTGCCTTGCAACTCCAGAAGTGAACCTTACCAGTGCCAATTGCTGGGCT
ACGGGATGGGGACTAGTCTCAAAACAAGGTGAGACCTCAGACGAGCTGCAGGAGGTGC
AGCTCCCGCTGATCCTGGAGCCCTGGTGCCACCTGCTCTACGGACACATGTCCTACAT
CATGCCCGACATGCTGTGTGCTGGGGACATCCTGAATGCTAAGACCGTGTGTGAGGGC
GACTCCGGGGGCCCACTTGTCTGTGAATTCAACCGCAGCTGGTTGCAGATTGGAATTG
TGAGCTGGGGCCGAGGCTGCTCCAACCCTCTGTACCCTGGAGTGTATGCCAGTGTTTC
CTATTTCTCAAAATGGATATGTGATAACATAGAAATCACGCCCACTCCTGCTCAGCCA
GCCCCTGCTCTCTCTCCAGCTCTGGGGCCCACTCTCAGCGTCCTAATGGCCATGCTGG
CTGGCTGGTCAGTGCTGTGAGGTCAGGATACCCACTCTAGGATTCTCATGGCTGCACA
CCCTGCCCCAGCCCAGCTGCCTCCAGACCCCTAAGCATCTCCTGTCCTGGCCTCTCTG
AAGCAGACAAGGGCCACCTATCCCGGGGGTGGATGCTGAGTCCAGGAGGTGATGAGCA
AGTGTACAAAAGAAAAAAGGGAAGGGGGAGAGGGGCTGGTCAGGGAGAACCCAGCTTG
GGCAGAGTGCACCTGAGATTTGATAAGATCATTAAATATTTACAAAGCAAA
ORF Start: ATG at 26 ORF Stop: TGA at 1004
SEQ ID NO: 58 326 as MW at 35323.8kD
NOV2la, MAAPASVMGPLGPSALGLLLLLLVVAPPRVAALVHRQPENQGISLTGSVACGRPSMEG
CG105491-O1 KILGGVPAPERKWPWQVSVHYAGLHVCGGSILNEYWVLSAAHCFHRDKNIKIYDMYVG
Protein SequenceI'~RVAGNHTQWYEVNRVILHPTYEMYHPIGGDVALVQLKTRIVFSESVLPVCLATP
EVNLTSANCWATGWGLVSKQGETSDELQEVQLPLILEPWCHLLYGHMSYIMPDMLCAG
DILNAKTVCEGDSGGPLVCEFNRSWLQIGIVSWGRGCSNPLYPGVYASVSYFSKWICD
NIEITPTPAQPAPALSPALGPTLSVLMAMLAGWSVL
Further analysis of the NOV21 a protein yielded the following properties shown
in Table
21B.
Table 215. Protein SequenceyProperties NOV2la
PSort 0.7900 probability located in plasma membrane; 0.3000 probability
located in
analysis: Golgi body; 0.2000 probability located in endoplasmic reticulum
(membrane);
0.1007 probability located in microbody (peroxisome)
SignalP ~ Cleavage site between residues 33 and 34
analysis:
.. .. ... . Y. ", .. .... . .. . .... ... . . .. . . ... ...... . . . .. . ..
.....
A search of the NOV21 a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 21 C.
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Table 21C. Geneseq Results
for NOV2la -~,-
NOV2la Identities/
Geneseq Protein/Organism/Length Residues/Similarities ~ Expect
[Patent ~ for
Identifier#, Date] Match the Matched Value
ResiduesRegion
AAU82747 Amino acid sequence of 1..326 3261326 (100%)0.0
novel ~
human protease #46 - Homo 1..326 326/326 (100%)
Sapiens, 326 aa. [W0200200860-
,
A2, 03-JAN-2002]
AAB73945 Human protease T - Homo 13..288 115/286 (40%)6e-53
Sapiens, ~
290 aa. [W0200116293-A2, 4..272 152/286 (S2%)
08-
MAR-2001 J
AAE03821 Human gene 4 encoded secreted13..288 115/286 (40%)6e-53
protein HWHIH 10, SEQ ID 4..272 152/286 (52%)
NO: 67 ~
Homo Sapiens, 290 aa.
[W0200136440-A1, 25-MAY-
2001 J
AAU12282 Human PRO4327 polypeptide 13..288 1151286 (40%)6e-53
sequence - Homo Sapiens, 4..272 152/286 (52%)
290 aa.
[W0200140466-A2, 07-JUN-2001]
a
AAY73388 HTRM clone 3376404 protein13..288 115/286 (40%)~6e-53
~
sequence - Homo Sapiens, 4..272 152/286 (52%)
290 aa.
[W09957144-A2, 11-NOV-1999
In a BLAST search of public sequence databases, the NOV21 a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 21 D.
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Table 21D. Public BLASTP Results for NOV2la
Protein NOV2la Identities/
Accession ~ Protein/Organism/Length Residues/ Similarities for Expect
Number ' Match the Matched Value
Residues Portion
Q9BQR3 ' Marapsin precursor (EC 3.4.21.-) - 13..288 1151286 (40%) 1e-52
Homo Sapiens (Human), 290 aa. 4..272 152/286 (52%)
Q9PVX7 ~ EPIDERMIS SPECIFIC SERINE ~ 50..314 98/275 (35%) Se-52
PROTEASE - Xenopus laevis 16..287 159/275 (57%)
(African clawed frog), 389 aa.
AAH24903 . RIKEN CDNA 2010001P08 51..326 114/288 (39%) 2e-51
GENE - Mus musculus (Mouse), 45..329 158/288 (54%)
331 aa.
Q91XC4 SIMILAR TO DISTAL ~ 51..288 106/247 (42%) 6e-51
INTESTINAL SERINE 28..272 138/247 (54%)
PROTEASE - Mus musculus
(Mouse), 310 aa.
Q9Q'YZ9 DISTAL INTESTINAL SERINE ~ 51..288 105/247 (42%) 7e-50
PROTEASE - Mus musculus 28..272 137/247 (54%)
(Mouse), 310 aa.
PFam analysis predicts that the NOV21 a protein contains the domains shown in
the Table
21E.
Table 21E. Domain Analysis of NOV2la
Identities/
Pfam Domain NOV2la Match Region Similarities Expect Value
for the Matched Region ..
trypsin 60..288 87/265 (33%) 5.3e-72
.. ~~.. _ 172/265 (65%) ' ..~~M-' . a
Examine 22.
The NOV22 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 22A.
Table 22A. NOV22 Sequence Analysis
SEQ ID NO: 59 4131 by
NOV22a, GTGCCGAGGATGGCCAGGCAGCCACCGCCGCCCTGGATCCATGCAGCCTTCCTCCTCT
CG105954-Ol GCCTCCTCAGTCTTGGCGGAGCCATCGAA.ATTCCTATGGTTCCAAGCATTCAGAATGA
'GCTGACGCAGCCGCCAACCATCACCAAGCAGTCAGCGAAGGATCACATCGTGGACCCC
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DNA SeClLleriCe CGTGATAACATCCTGATTGAGTGTGAAGCAAAAGGGAACCCTGCCCCCAGCTTCCACT
~GGACACGAAACAGCAGATTCTTCAACATCGCCAAGGACCCCCGGGTGTCCATGAGGAG
~GAGGTCTGGGACCCTGGTGATTGACTTCCGCAGTGGCGGGCGGCCGGAGGAATATGAG
GGGG.AATATCAGTGCTTCGCCCGCAACAAATTTGGCACGGCCCTGTCCAATAGGATCC
GCCTGCAGGTGTCTAAATCTCCTCTGTGGCCCAAGGAAAACCTAGACCCTGTCGTGGT
CCAAGAGGGCGCTCCTTTGACGCTCCAGTGCAACCCCCCGCCTGGACTTCCATCCCCG
GTCATCTTCTGGATGAGCAGCGCCATGGAGCCCATCACCCAAGACAAACGTGTCTCTC
AGGGCCATAACGGAGACCTATACTTCTCCAACGTGATGCTGCAGGACATGCAGACCGA
CTACAGTTGTAACGCCCGCTTCCACTTCACCCACACCATCCAGCAGAAGAACCCTTTC
ACCCTCAAGGTCCTCACCAGTAAGCCTTATAATGACTCGTCCTTAAGAAACCACCCTG
ACATGTACAGTGCCCGAGGAGTTGCAGAAAGAACACCAAGCTTCATGTATCCCCAGGG
CACCGCGAGCAGCCAGATGGTGCTTCGTGGCATGGACCTCCTGCTGGAATGCATCGCC
TCCGGGGTCCCAACACCAGACATCGCATGGTACAAGAAAGGTGGGGACCTCCCATCTG
ATAAGGCCAAGTTTGAGAACTTTAATAAGGCCCTGCGTATCACAAATGTCTCTGAGGA
AGACTCCGGGGAGTATTTCTGCCTGGCCTCCAACAAGATGGGCAGCATCCGGCACACG
ATCTCGGTGAGAGTAAAGGCTGCTCCCTACTGGCTGGACGAACCCAAGAACCTTATTC
TGGCTCCTGGCGAGGATGGGAGACTGGTGTGTCGAGCCAATGGAAACCCCAAACCCAC
TGTCCAGTGGATGGTGAATGGGGAACCTTTGCAAGCGGCACCACCTAACCCAAACCGT
GAGGTGGCCGGAGACACCATCATCTTCCGGGACACCCAGATCAGCAGCAGGGCTGTGT
ACCAGTGCAACACCTCCAACGAGCATGGCTACCTGCTGGCCAACGCCTTTGTCAGTGT
GCTGGATGTGCCGCCTCGGATGCTGTCGCCCCGGAACCAGCTCATTCGAGTGATTCTT
~TACAACCGGACGCGGCTGGACTGCCCTTTCTTTGGGTCTCCCATCCCCACACTGCGAT
iGGTTTAAGAATGGGCAAGGAAGCAACCTGGATGGTGGCAACTACCATGTTTATGAGAA
CGGCAGTCTGGAAATTAAGATGATCCGCAAAGAGGACCAGGGCATCTACACCTGTGTC
~GCCACCAACATCCTGGGCAAAGCTGAAAACCAAGTCCGCCTGGAGGTAAAAGACCCCA
CCAGGATCTACCGGATGCCCGAGGACCAGGTGGCCAGAAGGGGCACCACGGTGCAACT
~GGAGTGTCGGGTGAAGCACGACCCCTCCCTGAAACTCACCGTCTACTGGCTGAAGGAT
IGACGAGCCGCTCTATATTGGAAACAGGATGAAGAAGGAAGACGACTCCCTGACCATCT
~TTGGGGTGGCAGAGCGGGACCAGGGCAGTTACACGTGTGTCGCCAGCACCGAGCTAGA
iCCAAGACCTGGCCAAGGCCTACCTCACCGTGCTAGGACGGCCAGACCGGCCCCGGGAC
ICTGGAGCTGACCGACCTGGCCGAGAGGAGCGTGCGGCTGACCTGGATCCCCGGGGATG
CTAACAACAGCCCCATCACAGACTACGTCGTCCAGTTTGAAGAAGACCAGTTCCAACC
TGGGGTCTGGCATGACCATTCCAAGTACCCCGGCAGCGTTAACTCAGCCGTCCTCCGG'
'CTGTCCCCGTATGTCAACTACCAGTTCCGTGTCATTGCCATCAACGAGGTTGGGAGCA
GCCACCCCAGCCTCCCATCCGAGCGCTACCGAACCAGTGGAGCACCCCCCGAGTCCAA'
TCCTGGTGACGTGAAGGGAGAGGGGACCAGAAAGAACAACATGGAGATCACGTGGACG':
CCCATGAATGCCACCTCGGCCTTTGGCCCCAACCTGCGCTACATTGTCAAGTGGAGGC'
GGAGAGAGACTCGAGAGGCCTGGAACAACGTCACAGTGTGGGGCTCTCGCTACGTGGTi
GGGGCAGACCCCAGTCTACGTGCCCTATGAGATCCGAGTCCAGGCTGAAAATGACTTC
GGGAAGGGCCCTGAGCCAGAGTCCGTCATCGGTTACTCCGGAGAAGATTATCCCAGGG~':
CTGCGCCCACTGAAGTTAAAGTCCGAGTCATGAACAGCACAGCCATCAGCCTTCAGTG)
GAACCGCGTCTACTCCGACACGGTCCAGGGCCAGCTCAGAGAGTACCGAGCCTACTAC'
TGGAGGGAGAGCAGCTTGCTGAAGAACCTGTGGGTGTCTCAGAAGAGACAGCAAGCCA
GCTTCCCTGGTGACCGCCTCCGTGGCGTGGTGTCCCGCCTCTTCCCCTACAGTAACTA
CAAGCTGGAGATGGTTGTGGTCAATGGGAGAGGTGATGGGCCTCGCAGTGAGACCAAG
GAGTTCACCACCCCGGAAGGAGTACCCAGTGCCCCTAGGCGTTTCCGAGTCCGGCAGC
CCAACCTGGAGACAATCAACCTGGAATGGGATCATCCTGAGCATCCAAATGGGATCAT
GATTGGATACACTCTCAAATATGTGGCCTGTACGTTCTCCCCAGTTAACGGGACCAAA
GTAGGAAAGCAGATAGTGGAAAACTTCTCTCCCAATCAGACCAAGTTCACGGTGCAAA
GAACGGACCCCGTGTCACGCTACCGCTTTACCCTCAGCGCCAGGACGCAGGTGGGCTC
TGGGGAAGCCGTCACAGAGGAGTCACCAGCACCCCCGAATGAAGGTAGGTGCATGGCA
GCAGCCCCTGGGGTAAAACCCCCGACTACCGTGGGTGCGACGGGCGCTGTGAGCAGTA
CCGATGCTACTGCCATTGCTGCCACCACCGAAGCCACAACAGTCCCCATCATCCCAAC
TGTCGCACCTACCACCATGGCCACCACCACCACCGTCGCCACAACTACTACAACCACT
GCTGCCGCCACCACCACCACGGAGAGTCCTCCCACCACCACCTCCGGGACTAAGATAC
ACGAATCCGGTACTGCGCATCGCCCATGCTCCCCAGCCCCTGATGAGCAGTCCATATG
GAACGTCACGGTGCTCCCCAACAGTAAATGGGCCAACATCACCTGGAAGCACAATTTC
GGGCCCGGAACTGACTTTGTGGTTGAGTACATCGACAGTAACCATACGAAAAAAACTG
TCCCAGTTAAGGCCCAGGCTCAGCCTATACAGCTGACAGACCTCTATCCCGGGATGAC
ATACACGTTGCGGGTTTATTCCCGGGACAACGAGGGCATCAGCAATCATTCACGGGTT
TGCTTCCGGCCCTCCCCGCCAGCTTACACCAACAACCAAGCGGACATCGCCACCCAGG
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GCTGGTTCATTGGGCTTATGTGCGCCATCGCCCTCCTGGTGCTGATCCTGCTCATCGT
CTGTTTCATCAAGAGGAGTCGCGGCGGCAAGTACCCAGTACGAGAAAAGAAGGATGTT
CCCCTTGGCCCTGAAGACCCCAAGGAAGAGGATGGCTCATTTGACTATAGGTCTTTGG
CCAGTGATGAGGACAACAAGCCCCTGCAGGGCAGTCAGACATCTCTGGACGGCACCAT
CAAGCAGCAGGAGAGTGACGACAGCCTGGTGGACTATGGCGAGGGTGGCGAGGGTCAG
TTCAATGAAGACGGCTCCTTCATCGGCCAGTACACGGTCAAAAAGGACAAGGAGGAAA
CAGAGGGCAACGAAAGCTCAGAGGCCACGTCACCTGTCAATGCTATCTACTCTCTGGC
CTAACGGAGCCCA
ORF Start: ATG at 10 ORF Stop TAA at 41_20
SEQ ID NO: 60 ~~~1370 Yaa MW at 152752.SkD
NOV22a, ~QPPPPWIHAAFLLCLLSLGGAIEIPMVPSIQNELTQPPTITKQSAKDHIVDPRDN
CG105954-Ol ILIECEAKGNPAPSFHWTRNSRFFNIAKDPRVSMRRRSGTLVIDFRSGGRPEEYEGEY
PrOteln SequenceQCFARNKFGTALSNRIRLQVSKSPLWPKENLDPVWQEGAPLTLQCNPPPGLPSPVIF
WMSSAMEPITQDKRVSQGHNGDLYFSNVMLQDMQTDYSCNARFHFTHTIQQKNPFTLK
VLTSKPYNDSSLRNHPDMYSARGVAERTPSFMYPQGTASSQMVLRGMDLLLECIASGV
PTPDIAWYKKGGDLPSDKAKFENFNKALRITNVSEEDSGEYFCLASNKMGSIRHTISV
RVKAAPYWLDEPKNLTLAPGEDGRLVCRANGNPKPTVQWMVNGEPLQAAPPNPNREVA
GDTIIFRDTQISSRAVYQCNTSNEHGYLLANAFVSVLDVPPRMLSPRNQLIRVILYNR
TRLDCPFFGSPIPTLRWFKNGQGSNLDGGNYHVYENGSLEIKMIRKEDQGIYTCVATN
ILGKAENQVRLEVKDPTRTYRMPEDQVARRGTTVQLECRVKHDPSLKLTVYWLKDDEP
LYIGNRMKKEDDSLTIFGVAERDQGSYTCVASTELDQDLAKAYLTVLGRPDRPRDLEL
TDLAERSVRLTWIPGDANNSPITDYVVQFEEDQFQPGVWHDHSKYPGSVNSAVLRLSP
YVNYQFRVIAINEVGSSHPSLPSERYRTSGAPPESNPGDVKGEGTRKNNMEITWTPMN
ATSAFGPNLRYIVKWRRRETREAWNNVTVWGSRYVVGQTPVYVPYEIRVQAENDFGKG
PEPESVIGYSGEDYPRAAPTEVKVRVMNSTAISLQWNRVYSDTVQGQLREYRAYYWRE
SSLLKNLWVSQKRQQASFPGDRLRGWSRLFPYSNYKLEMWVNGRGDGPRSETKEFT
TPEGVPSAPRRFRVRQPNLETINLEWDHPEHPNGIMIGYTLKYVACTFSPVNGTKVGK
QIVENFSPNQTKFTVQRTDPVSRYRFTLSARTQVGSGEAVTEESPAPPNEGRCMAAAP
GVKPPTTVGATGAVSSTDATAIAATTEATTVPIIPTVAPTTMATTTTVATTTTTTAAA
TTTTESPPTTTSGTKIHESGTAHRPCSPAPDEQSTWNVTVLPNSKWANITWKHNFGPG
TDFVVEYIDSNHTKKTVPVKAQAQPIQLTDLYPGMTYTLRVYSRDNEGISNHSRVCFR
PSPPAYTNNQADIATQGWFIGLMCAIALLVLILLIVCFIKRSRGGKYPVREKKDVPLG
PEDPKEEDGSFDYRSLASDEDNKPLQGSQTSLDGTIKQQESDDSLVDYGEGGEGQFNE
DGSFIGQYTVKKDKEETEGNESSEATSPVNAIYSLA
Further analysis of the NOV22a protein yielded the following properties shown
in Table
22B.
"~.w.,..,..,~..~....,,...~.~..~~..~~..~,.".
~- ,~",y,~,~YTable 22B~qProtein SequencelProperties NOV22a
PSort ~~ 0.4600 probability located in plasma membrane; 0.1000 probability
located in
analysis: endoplasmic reticulum (membrane); 0.1000 probability located in
endoplasmic reticulum (lumen); 0.1000 probability located in outside
SignaIP ~ Cleavage site between residues 25 and 26
analysis:
A search of the NOV22a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 22C.
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Table 22C. Geneseq Results for NOV22a
__ _.__ ___. _ NOV22a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
for
Identifier[Patent #, Date] Match the Matched Value
Residues Region
AAM78714 Human protein SEQ ID NO 92..1050 928/959 (96%)0.0
1376 -
Homo sapiens, 937 aa. 1..937 933/959 (96%)
[W0200157190-A2, 09-AUG-
2001]
AAM787I ' Human protein SEQ ID 92..1050 928/974 (95%)0.0
NO I 377 - '
Homo Sapiens, 952 aa. 1..952 933/974 (95%)
[WO200157190-A2, 09-AUG-
2001]
AAW59994 Human neural cell adhesion15..1072 517/1075 0.0
(48%)
molecule splice variant 20..1082 708/1075
NrCAMvar (65%)
- Homo Sapiens, 1304 aa.
[WO9836062-Al, 20-AUG-1998]
AAU10650 ~ Chicken Nr-CAM protein 12..1054 508/1055 ' 0.0
sequence (48%)
'' - Gallus sp, 1268 aa. 12..1042 696/1055
[US6313265- (65%)
B1, 06-NOV-2001]
AAB90717 Human C0722_1 protein 15..1060 509/1058 ~ 0.0
sequence (48%)
SEQ ID 130 - Homo Sapiens,20..1047 701/1058
1192 (66%)
aa. [WO200119988-A1, 22-MAR-
2001
In a BLAST search of public sequence databases, the NOV22a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 22D.
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Table 22D. Public BLASTP Results for NOV22a
Protein NOV22a Identities/
Residues/ Expect
AccessionProtein/Organism/Length Similarities
for the
Number Matched PortionValue
Residues
042414 NEUROFASCIN PRECURSOR 13..13701026/1372 (74%)0.0
- Gallus gallus (Chicken),14..13691148/1372 (82%)
1369 '
aa.
Q91Z60 NEUROFASCIN 155 KDA I..I042 985/1042 (94%) 0.0
ISOFORM - Rattus norvegicus1..1031 1008/1042 (96%)
(Rat), 1174 aa.
Q9QVN5 NEUROFASCIN ISOFORM - 25..104296511019 (94%) 0.0
Rattus sp, 1151 aa. 1..1008 987/1019 (96%)
Q90924 i NEUROFASCIN PRECURSOR 13..1114844/1114 (75%) 0.0
- Gallus gallus (Chicken),14..1120949/1 114 (84%)
1272
aa.
094856 KIAA0756 PROTEIN - Homo 193..1050830/858 (96%) 0.0
Sapiens (Human), 836 1..836 833/858 (96%)
as
(fragment).
PFam analysis predicts that the NOV22a protein contains the domains shown in
the Table
22E.
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Table 22E. Domain Analysis of NOV22a
Identities/
Pfam omain NOV22a Match RegionSimilarities Expect Value
D ~
for the Matched
Region
ig 56..120 11/68 (16%) 0.032
43/68 (63%)
ig 155..215 13/62 (21 %) 0.01
39/62 (63%)
ig 278..335 18/61 (30%) 4.3e-11
44/61 (72%)
ig 368..427 13/63 (21 %) 2.1 e-OS
~ 44163 (70%)
ig 462..520 I 8162 (29%) 1.1 e-07
~ 45/62 (73%)
ig 553..611 19/60 (32%) ~ 5.9e-09
40/60 (67%)
fn3 630..716 28/88 (32%) 6.6e-14
64/88 (73%)
fn3 729..815 27/92 (29%) 5.9e-07--...
62/92 (67%)
fn3 827..922 24/97 (25%) 3.7e-07
66/97 (68%)
fn3 934..1026 20/97 (21 %) 2.1 e-08
66/97 (68%)
fn3 1134..1213 22/85 (26%) 1.5e-08
56/85 (66%)
Example 23.
The NOV23 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 23A.
Table 23A. NOV23 Sequence Analysis
SEQ ID NO: 61 X2497 by
NOV23a, GCCCCGATGGACGCCGCGTTCCTCCTCGTCCTCGGGCTGTTGGCCCAGAGCCTCTGCC
CG105963-O1 TGTCTTTGGGGGTTCCTGGATGGAGGAGGCCCACCACCCTGTACCCCTGGCGCCGGGC
DNA SequeriCeGCCTGCCCTGAGCCGCGTGCGGAGGGCCTGGGTCATCCCCCCGATCAGCGTATCCGAG
AACCACAAGCGTCTCCCCTACCCCCTGGTTCAGGTGAGCAGGTGGAAGCACCAGTTGG
CCAGCGTCATCTCCAGCATCCAGGGCCCCGGCGTGGATGAGGAGCCCCGGGGCGTCTT
CTCTATCGCCCAGTTCACAGGGAAGGTCTTCCTCAATGCCATGCTGGACCGCGAGAAG
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ACTGATCGCTTCAGGCTAAGAGGGTTTGCCCTGGACCTGGGAGGATCCACCCTGGAGG
ACCCCACGGACCTGGAGATTGTAGTTGTGGATCAGAATGACAACCGGCCAGCCTTCCT
GCAGGAGGCGTTCACTGGCCGCGTGCTGGAGGGTGCAGTCCCAGGTACCTATGTGACC
AGGGCAGAGGCCACAGATGCCGACGACCCCGAGACGGACAACGCAGCGCTGCGGTTCT
CCATCCTGCAGCAGGGCAGCCCCGAGCTCTTCAGCATCGACGAGCTCACAGGAGAGAT
CCGCACAGTGCAAGTGGGGCTGGACCGCGAGGTGGTCGCGGTGTACAATCTGACCCTG
CAGGTGGCGGACATGTCTGGAGACGGCCTCACAGCCACTGCTTCAGCCATCATCACCT
TTGATGACATCAATGACAATGCCCCCGAGTTCACCAGGGATGAGTTCTTCATGGAGGC
CATAGAGGCCGTCAGCGGAGTGGATGTGGGACGCCTGGAAGTGGAGGACAGGGACCTG
CCAGGCTCCCCAAACTGGGTGGCCAGGTTCACCATCCTGGAAGGCGACCCCGATGGGC
AGTTCACCATCCGCACGGACCCCAAGACCAACGAGGGTGTTCTGTCCATTGTGAAGGC
CCTGGACTATGAAAGCTGTGAACACTACGAAACTAAAACACACGGGCAGGATAAGACA
GAGAACGCACGGGCAGGGCTGAGGGCTGAGCGGGGCCAGGCCAAGGTCCGCGTGCATG
TGCAGGACACCAACGAGCCCCCCGTGTTCCAGGAGAACCCACTTCGGACCAGCCTAGC
AGAGGGGGCACCCCCAGGCACTCTGGTGGCCACCTTCTCTGCCCGGGACCCTGACACA
GAGCAGCTGCAGAGGCTCAGCTACTCCAAGGACTACGACCCGGAAGACTGGCTGCAAG
TGGACGCAGCCACTGGCCGGATCCAGACCCAGCACGTGCTCAGCCCGGCGTCCCCTTT
CCTCAAGGGCGGCTGGTACAGAGCCATCGTCTTGGCCCAGGATGCCTCCCAGCCCCGC
ACCGCCACCGGCACCCTGTCCATCGAGATCCTGGAGGTGAACGACCATGCACCTGTGC
TGGCCCCGCCGCCGCCGGGCAGCCTGTGCAGCGAGCCACACCAAGGCCCAGGCCTCCT
CCTGGGCGCCACGGATGAGGACCTGCCCCCCCACGGGGCCCCCTTCCACTTCCAGCTG
AGCCCCAGGCTCCCAGAGCTCGGCCGGAACTGGAGCCTCAGCCAGGTCAACCCTCTCT
CCCATCACCGTCTCCACCCAGACCCCCACCTGCCCCATGGCCCCCATTTCATGTCTGT
GGCTCACCAGCTTTTCCCCAGACCCAGCTCCGGAGCCCACAGGCGTGGCCGATGCAGA
AACCTCAGGAAGGTGTGTTGTGAATGTGGGAGGGAGGGTGTGGCGGTCGTGGGCTGTG
CGGGAGTTCTGACTAGGGGAAGTGGGCTCAGCCTGGGCGCACTGGTCATCGTGCTGGC
CAGCGCCCTCCTGCTGCTGGTGCTGGTCCTGCTCGTGGCACTCCGGGCGCGGTTCTGG
AAGCAGTCTCGGGGCAAGGGGCTGCTGCACGGCCCCCAGGACGACCTTCGAGACAATG
~TCCTCAACTACGATGAGCAAGGAGGCGGGGAGGAGGACCAGGACGCCTACGACATCAG
GATGCCCCGCAGGGCCGCCTGCACCCCCAGCCACCCCGAGTGCTGCCCACCAGCCCCC
TGGACATCGCCGACTTCATCAATGATGGCTTGGAGGCTGCAGATAGTGACCCCAGTGT
GCCGCCTTACGACACAGCCCTCATCTATGACTACGAGGGTGACGGCTCGGTGGCGGGG
GAGACTGGGGGCCCCGCTTCGCCCGGCTGGCAGACATGTATGGGCACCCGTGCGGGTT
GGAGTACGGGGCCAGATGGGACCACCAGGCCAGGGAGGGTCTTTCTCCTGGGGCACTG
CTACCCAGACACAGAGGCCGGACAGCCTGACCCTGGGGCGCAACTGGACATGCCACTC
CCC
ORF Start: ATG at 7 ~ ORF Stop: TGA at 2464
SEQ ID NO: 62 819 as MW at 89687.6kD
NOV23a, MDAAFLLVLGLLAQSLCLSLGVPGWRRPTTLYPWRRAPALSRVRRAWVIPPISVSENH
CG105963-Ol KRLPYPLVQVSRWKHQLASVISSIQGPGVDEEPRGVFSIAQFTGKVFLNAMLDREKTD
PTOtelri Se llenCe RFRLRGFALDLGGSTLEDPTDLEIVWDQNDNRPAFLQEAFTGRVLEGAVPGTWTRA
EATDADDPETDNAALRFSILQQGSPELFSIDELTGEIRTVQVGLDREWAVYNLTLQV
ADMSGDGLTATASAIITFDDINDNAPEFTRDEFFMEAIEAVSGVDVGRLEVEDRDLPG
SPNWVARFTILEGDPDGQFTIRTDPKTNEGVLSIVKALDYESCEHYETKTHGQDKTEN
ARAGLRAERGQAKVRVHVQDTNEPPVFQENPLRTSLAEGAPPGTLVATFSARDPDTEQ
LQRLSYSKDYDPEDWLQVDAATGRIQTQHVLSPASPFLKGGWRAIVLAQDASQPRTA
TGTLSIEILEVNDHAPVLAPPPPGSLCSEPHQGPGLLLGATDEDLPPHGAPFHFQLSP
RLPELGRNWSLSQVNPLSHHRLHPDPHLPHGPHFMSVAHQLFPRPSSGAHRRGRCRNL
RKVCCECGREGVAVVGCAGVLTRGSGLSLGALVIVLASALLLLVLVLLVALRARFWKQ
SRGKGLLHGPQDDLRDNVLNYDEQGGGEEDQDAYDISQLRHPTALSLPLGPPPLRRDA
PQGRLHPQPPRVLPTSPLDIADFINDGLEAADSDPSVPPYDTALIYDYEGDGSVAGTL
SSILSSQGDEDQDYDYLRDWGPRFARLADMYGHPCGLEYGARWDHQAREGLSPGALLP
RHRGRTA
Further analysis of the NOV23a protein yielded the following properties shown
in Table
23B.
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Table 23B. Protein Sequence Properties NOV23au '.~.~~
PSort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400
analysis: probability located in plasma membrane; 0.4600 probability located
in Golgi
body; 0.1000 probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 22 and 23
analysis:
A search of the NOV23a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 23C.
Table 23C. Geneseq Results for NOV23a
NOV23a Identities!
Geneseq Protein/Organism/Length Residues!Similarities Expect
[Patent for
Identifier#, Date] Match the Matched Value
ResiduesRegion
ABG30224 Novel human diagnostic 1..819 750/820 (91%)0.0
protein
#30215 - Homo Sapiens, 1..814 766/820 (92%)
814 aa.
[W0200175067-A2, 11-OCT-2001]
ABG30224 Novel human diagnostic 1..819 750/820 (91%)0.0
protein
#30215 - Homo Sapiens, I ..814 766/820 (92%)
814 aa.
[W0200175067-A2, 11-OCT-2001]
AAB24089 Human PRO2198 protein sequence1..819 750/820 (91 0.0
%)
SEQ ID N0:79 - Homo Sapiens,1..814 766/820 (92%)
814
aa. [WO200053755-A2, 14-SEP-
2000]
ABB57233 Mouse ischaemic condition 35..786 313/767 (40%)e-I52
related
protein sequence SEQ ID 149..902436/767 (56%)
NO:606 -
Mus musculus, 906 aa.
[W0200188188-A2, 22-NOV-2001]
AAY70741 Human N-cadherin - Homo 40..786 311/762 (40%)e-151
Sapiens,
906 aa. [W0200021555-A1, 154..902435/762 (56%)
20-
APR-2000]
_ .. . _ _ _.. ~ . _... ._..
. .
In a BLAST search of public sequence databases, the NOV23a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 23D.
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Table 23D. Public BLASTP
Results for NOV23a
P
t NOV23a Identities/
in
ro rrotein/Organism/Length Residues!Similarities Expect
e for
Accession
Number Match the Matched Value
Residues Portion
P55291 Muscle-cadherin precursor1..819 750/820 (91%)0.0
(M-
cadherin) (Cadherin-15) 1..814 766/820 (92%)
(Cadherin-
14) - Homo Sapiens (Human),
814
aa.
P33146 Muscle-cadherin precursor1..787 616/791 (77%)0.0
(M-
cadherin) (Cadherin-15) 1..783 662/791 (82%)
(Cadherin-
14) - Mus musculus (Mouse),
784
aa.
~~~ M-cadherin - mouse, 730 56..787 576/736 (78%)0.0
IJMSCM as
I
(fragment). 1..729 620/736 (83%)
~
Q8UVQ7 N-CADHERIN - Brachydanio 39..786 316/762 (41%)e-157
rerio
(Zebrafish) (Zebra danio),~ 140..8894431762 (57%)
893 aa. ~
Q90275 NEURAL-CADHERIN 39..786 315/763 (41%)e-154
PRECURSOR (N-CADHERIN) 29..779 4421763 (57%)
-
Brachydanio rerio (Zebrafish)
I ..~, _~Zebra danio),..783 aa._..
. .
PFam analysis predicts that the NOV23a protein contains the domains shown in
the Table
23E.
Table 23E. Domain Analysis of NOV23a
Identities/
Pfam Domain NOV23a Match RegionSimilarities Expect Value
for the Matched
Region
cadherin 50..143 23/111 (21%) 0.011
61/I11 (55%)
cadherin 157..251 38/108 (35%) 8.7e-25
74/108 (69%)
cadherin 265..367 34/107 (32%) 6.2e-18
~ 74/107 (69%)
cadherin 380..473 34/109 (31%) 7.7e-20
~ 71/109 (65%)
Cadherin_C_term634..788 83/158 (53%) 5.3e-90
146/158 (92%)
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Example 24.
The NOV24 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 24A.
Table 24A. NOV24 Sequence Analysis
SEQ ID NO: 63 X3617 by
NOV24a, GAGATGGGACTGCAATAGAAATCCGGGCAGCCCGAAGAGGCACCCAGCGCTCCAGCCA
CG105973-O1 CCAGCTGGGCCGCCCGGGAGTCCCTGGCTCTAGACCAGCCGCGAGGAGGCGCCGCGAG
DNA S AGAGCTGGTCCCTGCCCGCGGCCGGAGGAGGGCTAGAGCCCCTGGGCCAGCCCCCCGA
eCluenCe GCCGGCTGGGCGGGCGGGCGGGTGGGAGCAGACGCCGGGCACTGTCACCACGGGTGCG
CCGAGCGCACCGACCCGGGACACGGGCAGCTGGGGACCGCCAGATTCCACCAGCCCCC
CTTGCCCCGCAGGGGTCCTCGGCTCGCGCTCCTGGGTAGCAGCCACCCACCGGGGCGG
AGGGAGATGTCGCCCGGGGCCAGCCGCGGTCCCCGGGGAAGCCAGGCGCCGCTGATCG
i.._......_...~.....~....._____~__~________~____-___._____._.___.___._.
_._.__.
GGCGTTCAACCTGGACGTGGAAAAGCTCACAGTGTACAGCGGCCCCAAGGGCAGCTAC
TTCGGCTACGCCGTGGACTTCCACATACCCGACGCCCGCACAGCGAGTGTCTTGGTGG
GGGCGCCCAAAGCCAACACCAGCCAGCCCGATATCGTGGAAGGGGGAGCCGTCTATTA
CTGTCCTTGGCCCGCGGAGGGGTCTGCGCAGTGCAGGCAGATACCGTTTGACACCACC
AACAACAGAAAGATCAGAGTTAATGGAACCAAAGAACCTATCGAGTTCAAATCCAATC
AGTGGTTTGGAGCAACAGTGAAAGCTCACAAAGGAA.AAGTTGTGGCCTGTGCTCCTTT
ATATCACTGGAGAACTCTTAAACCGACACCAGAA.AAGGACCCAGTTGGCACCTGCTAT
GTAGCAATTCAGAACTTCAGCGCCTATGCCGAGTTCTCTCCTTGCCGGAACAGCAATG
CTGATCCGGAAGGCCAGGGTTACTGCCAAGCAGGATT'TAGTCTGGATTTTTATAAGAA
TGGAGACCTTATTGTGGGAGGACCTGGGAGTTTCTACTGGCAAGGACAAGTGATCACT
GCCAGTGTTGCAGATATCATTGCAAATTACTCATTCAAGGATATCCTCAGGAAACTGG
CAGGAGAAAAGCAGACGGAAGTGGCTCCAGCTTCCTATGATGACAGTTACCTTGGATA
CTCAGTTGCTGCTGGGGAGTTTACTGGGGATTCTCAGCAAGAATTGGTTGCTGGAATT
CCAAGAGGAGCACAGAATTTTGGATATGTTTCCATCATTAACTCTACGGATATGACGT
TTATTCAGAATTTCACGGGAGAACAGATGGCATCTTATTTTGGATATACCGTTGTCGT
ATCAGATGTTAACAGTGATGGACTGGATGATGTCCTGGTTGGGGCACCTCTCTTTATG
GAACGTGAATTTGAGAGCAACCCCAGAGAAGTAGGGCAAATCTACCTGTATTTGCAAG
TGAGCTCTCTCCTCTTCAGAGACCCCCAGATCCTCACTGGCACCGAGACGTTTGGGAG
ATTCGGTAGTGCTATGGCACACTTAGGAGACCTGAACCAAGATGGATACAATGACATT
GCCATCGGAGTGCCTTTTGCAGGCAAGGATCAAAGAGGCAAAGTGCTCATTTATAATG
GGAACAAAGATGGCTTAAACACCAAGCCTTCCCAAGTTCTGCAAGGAGTGTGGGCCTC
ACATGCTGTCCCTTCCGGATTTGGCTTTACTTTAAGAGGAGATTCAGACATAGACAAG
AATGATTACCCAGATTTGATTGT~GGTGCATTTGGAACAGGAAAAGTCGCTGTTTACA
GAGCAAGACCGGTTGTGACTGTAGATGCCCAGCTTCTGCTGCACCCAATGATTATCAA
TCTTGAAAATAAAACTTGCCAGGTTCCAGACTCTATGACATCTGCTGCCTGCTTTTCT
TTAAGAGTATGTGCATCTGTCACAGGCCAGAGCATTGCAAACACAATAGTCTTGATGG
CAGAGGTGCAATTAGATTCCCTGAAACAGAAAGGAGCTATTAAACGGACGCTCTTCCT
TGATAACCATCAGGCTCATCGCGTCTTCCCTCTTGTGATAAAA.AGGCAGAAATCCCAC
CAGTGCCAGGATTTCATCGTTTACCTTCGAGATGAAACTGAATTCCGAGATAAATTAT
CTCCAATCAACATTAGTTTGAATTACAGTTTGGACGAATCCACCTTTAAAGAAGGCCT'
GGAAGTGAAACCAATATTGAACTACTACAGAGAAAACATTGTTAGTGAACAGGCTCAC'
GACCAGATAAGCATCAGGTAATCATTGGAGATGAA.AATCACCTTATGCTCATAATAAA
TGCAAGAAATGAAGGGGAAGGAGCATATGAAGCTGAACTCTTTGTAATGATACCAGAA
GAGGCAGATTATGTTGGAATCGAACGCAACAACAAGGGATTTCGACCACTGAGCTGTG
AGTACAAGATGGAA.AATGTAACCAGGATGGTGGTGTGTGACCTTGGGAACCCTATGGT
GTCTGGAACAAATTATTCCCTGGGCCTCCGATTTGCAGTTCCACGTCTTGAGAAAACA
AACATGAGCATTAACTTCGATCTCCAAATCAGAAGTTCCAACAAGGACAATCCAGACA
GCAATTTTGTGAGCCTGCAAATCAACATCACTGCTGTAGCGCAGGTGGAAATAAGAGG
AGTGTCACACCCTCCGCAGATTGTTCTGCCCATTCATAACTGGGAACCAGAAGAGGAG
CCCCACAAAGAGGAGGAGGTTGGACCATTGGTGGAACATATTTATGAGCTGCACAATA
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TTGGACCAAGTACCATCAGTGACACCATCCTGGAGGTGGGCTGGCCTTTCTCTGCCCFG
GGATGAATTTCTTCTCTATATTTTCCATATTCAAACTCTGGGACCTCTGCAGTGCCAA
CCAAATCCTAATATCAATCCACAGGATATAAAGCCTGCTGCCTCCCCAGAGGACACCC
CTGAGCTCAGCGCCTTTTTGCGAAACTCTACTATTCCTCATCTTGTCAGGAAGAGGGA
TGTACATGTGGTCGAATTCCACAGACAGAGCCCTGCAAAA.ATACTGAATTGTACAAAT
ATCGAGTGTTTACAAATCTCCTGTGCAGTGGGACGACTCGAAGGAGGAGAAAGCGCAG
TCCTGAAAGTCAGGTCACGATTATGGGCCCACACCTTCCTCCAGAGAAAAA.ATGATCC
CTATGCTCTTGCATCCCTGGTGTCCTTTGAAGTTAAGAAGATGCCTTATACAGATCAG
CCAGCAAAACTCCCAGAAGGAAGCATAGTAATTAAGACATCAGTTATTTGGGCAACTC
CGAATGTTTCCTTCTCAATCCCATTATGGGTAATAATACTAGCAATACTTCTTGGATT
GTTGGTTCTCGCCATTTTAACCTTAGCTTTATGGAAGTGTGGATTCTTTGACAGAGCC
AGACCTCCTCAGGAGGACATGACCGACAGGGAACAGCTGACAAATGACAAGACCCCTG
AGGCATGACAAGF,~~AAAAAAAGAAGACCAAAGACCTCAAACACTGGTCCTGTTCAAAG
AAAAAGAAAGAACATGAGGCC
ORF Start: ATG at 355 ORF Stop: TGA at 3544
SEQ ID NO: 64 1063 as MW at 117472.3kD
NOV24a, MSPGASRGPRGSQAPLIAPLCCAAAALGMLLWSPACQAFNLDVEKLTVYSGPKGSYFG
CG105973-O1 YAVDFHIPDARTASVLVGAPKANTSQPDIVEGGAVYYCPWPAEGSAQCRQIPFDTTNN
PPOtelri SeCllleriCeRKIRVNGTKEPIEFKSNQWFGATVKAHKGKVVACAPLYHWRTLKPTPEKDPVGTCYVA
IQNFSAYAEFSPCRNSNADPEGQGYCQAGFSLDFYKNGDLIVGGPGSFYWQGQVITAS
VADIIANYSFKDILRKLAGEKQTEVAPASYDDSYLGYSVAAGEFTGDSQQELVAGIPR
GAQNFGYVSIINSTDMTFIQNFTGEQMASYFGYTVVVSDVNSDGLDDVLVGAPLFMER
EFESNPREVGQIYLYLQVSSLLFRDPQILTGTETFGRFGSAMAHLGDLNQDGYNDIAI
GVPFAGKDQRGKVLIYNGNKDGLNTKPSQVLQGVWASHAVPSGFGFTLRGDSDIDKND
YPDLIVGAFGTGKVAVYRARPVVTVDAQLLLHPMIINLENKTCQVPDSMTSAACFSLR
VCASVTGQSIANTIVLMAEVQLDSLKQKGAIKRTLFLDNHQAHRVFPLVIKRQKSHQC
QDFIVYLRDETEFRDKLSPINISLNYSLDESTFKEGLEVKPILNYYRENIVSEQAHIL
VDCGEDNLCVPDLKLSARPDKHQVIIGDENHLMLIINARNEGEGAYEAELFVMIPEEA
DYVGIERNNKGFRPLSCEYKMENVTRMVVCDLGNPMVSGTNYSLGLRFAVPRLEKTNM
SINFDLQIRSSNKDNPDSNFVSLQINITAVAQVEIRGVSHPPQIVLPIHNWEPEEEPH
KEEEVGPLVEHIYELHNIGPSTISDTILEVGWPFSARDEFLLYIFHIQTLGPLQCQPN
PNINPQDIKPAASPEDTPELSAFLRNSTIPHLVRKRDVHWEFHRQSPAKILNCTNIE
CLQISCAVGRLEGGESAVLKVRSRLWAHTFLQRKNDPYALASLVSFEVKKMPYTDQPA
KLPEGSIVIKTSVIWATPNVSFSIPLWVIILAILLGLLVLAILTLALWKCGFFDRARP
PQEDMTDREQLTNDKTPEA
SEQ ID NO: 65 .3617 by
~
NOV24b, GAGATGGGACTGCAATAGAAATCCGGGCAGCCCGAAGAGGCACCCAGCGCTCCAGCCA
CG105973-02 CCAGCTGGGCCGCCCGGGAGTCCCTGGCTCTAGACCAGCCGCGAGGAGGCGCCGCGAG'
DNA S AGAGCTGGTCCCTGCCCGCGGCCGGAGGAGGGCTAGAGCCCCTGGGCCAGCCCCCCGA'
8Ch1eriC8 GCCG
CT
'
G
GGGCGGGCGGGCGGGTGGGAGCAGACGCCGGGCACTGTCACCACGGGTGCG
CCGAGCGCACCGACCCGGGACACGGGCAGCTGGGGACCGCCAGATTCCACCAGCCCCCi
'
CTTGCCCCGCAGGGGTCCTCGGCTCGCGCTCCTGGGTAGCAGCCACCCACCGGGGCGG
AGGGAGATGTCGCCCGGGGCCAGCCGCGGTCCCCGGGGAAGCCAGGCGCCGCTGATCG
CGCCCCTCTGCTGCGCCGCGGCCGCGCTGGGGATGTTGCTGTGGTCCCCCGCCTGTCA
GGCGTTCAACCTGGACGTGGAAAAGCTCACAGTGTACAGCGGCCCCAAGGGCAGCTAC
TTCGGCTACGCCGTGGACTTCCACATACCCGACGCCCGCACAGCGAGTGTCTTGGTGG
GGGCGCCCAAAGCCAACACCAGCCAGCCCGATATCGTGGAAGGGGGAGCCGTCTATTA
CTGTCCTTGGCCCGCGGAGGGGTCCGCGCAGTGCAGGCAGATACCGTTTGACACCACC
AACAACAGAAAGATCAGAGTTAATGGAACCAAAGAACCTATCGAGTTCAAATCCAATC
AGTGGTTTGGAGCAACAGTGAAAGCTCACAAAGGAAAAGTTGTGGCCTGTGCTCCTTT
ATATCACTGGAGAACTCTTAAACCGACACCAGAAAAGGACCCAGTTGGCACCTGCTAT
GTAGCAATTCAGAACTTCAGCGCCTATGCCGAGTTCTCTCCTTGCCGGAACAGCAATG
CTGATCCGGAAGGCCAGGGTTACTGCCAAGCAGGATTTAGTCTGGATTTTTATAAGAA
TGGAGACCTTATTGTGGGAGGACCTGGGAGTTTCTACTGGCAAGGACAAGTGATCACT
GCCAGTGTTGCAGATATCATTGCAAATTACTCATTCAAGGATATCCTCAGGAAACTGG
CAGGAGAAAAGCAGACGGAAGTGGCTCCAGCTTCCTATGATGACAGTTACCTTGGATA
CTCAGTTGCTGCTGGGGAGTTTACTGGGGATTCTCAGCAAGAATTGGTTGCTGGAATT
CCAAGAGGAGCACAGAATTTTGGATATGTTTCCATCATTAACTCTACGGATATGACGT
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TTATTCAGAATTTCACGGGAGAACAGATGGCATCTTATTTTGGATATACCGTTGTCGT
ATCAGATGTTAACAGTGATGGACTGGATGATGTCCTGGTTGGGGCACCTCTCTTTATG
GAACGTGAATTTGAGAGCAACCCCAGAGAAGTAGGGCAAATCTACCTGTATTTGCAAG
TGAGCTCTCTCCTCTTCAGAGACCCCCAGATCCTCACTGGCACCGAGACGTTTGGGAG
ATTCGGTAGTGCTATGGCACACTTAGGAGACCTGAACCAAGATGGATACAATGACATT
GCCATCGGAGTGCCTTTTGCAGGCAAGGATCAAAGAGGCAAAGTGCTCATTTATAATG
GGAACAAAGATGGCTTAAACACCAAGCCTTCCCAAGTTCTGCAAGGAGTGTGGGCCTC
ACATGCTGTCCCTTCCGGATTTGGCTTTACTTTAAGAGGAGATTCAGACATAGACAAG
AATGATTACCCAGATTTGATTGTGGGTGCATTTGGAACAGGAAAAGTCGCTGTTTACA
GAGCAAGACCGGTTGTGACTGTAGATGCCCAGCTTCTGCTGCACCCAATGATTATCAA
TCTTGAAAATAAAACTTGCCAGGTTCCAGACTCTATGACATCTGCTGCCTGCTTTTCT
TTAAGAGTATGTGCATCTGTCACAGGCCAGAGCATTGCAAACACAATAGTCTTGATGG
CAGAGGTGCAATTAGATTCCCTGAAACAGAAAGGAGCTATTAAACGGACGCTCTTCCT
TGATAACCATCAGGCTCATCGCGTCTTCCCTCTTGTGATAAAAAGGCAGAAATCCCAC
CAGTGCCAGGATTTCATCGTTTACCTTCGAGATGAAACTGAATTCCGAGATAAATTAT
CTCCAATCAACATTAG'T'TTGAATTACAGTTTGGACGAATCCACCTTTAAAGAAGGCCT
GGAAGTGAAACCAATATTGAACTACTACAGAGAAAACATTGTTAGTGAACAGGCTCAC
ATTCTGGTGGACTGTGGAGAAGACAATCTGTGTGTTCCTGACTTGAAGCTGTCGGCTA
GACCAGATAAGCATCAGGTAATCATTGGAGATGAAAATCACCTTATGCTCATAATAAA
TGCAAGAAATGAAGGGGAGGGAGCATATGAAGCTGAACTCTTTGTAATGATACCAGAA
GAGGCAGATTATGTTGGAATCGAACGCAACAACAAGGGATTTCGACCACTGAGCTGTG
AG'T'ACAAGATGGAAAATGTAACCAGGATGGTGGTGTGTGACCTTGGGAACCCTATGGT
GTCTGGAACAAATTATTCCCTGGGCCTCCGATTTGCAGTTCCACGTCTTGAGAAAACA
AACATGAGCATTAACTTCGATCTCCAAATCAGAAGTTCCAACAAGGACAATCCAGACA
GCAATTTTGTGAGCCTGCAAATCAACATCACTGCTGTAGCGCAGGTGGAAATAAGAGG
AGTGTCACACCCTCCGCAGATTGTTCTGCCCATTCATAACTGGGAACCAGAAGAGGAG
CCCCACAAAGAGGAGGAGGTTGGACCATTGGTGGAACATATTTATGAGCTGCACAATA
TTGGACCAAGTACCATCAGTGACACCATCCTGGAGGTGGGCTGGCCTTTCTCTGCCCG'
GGATGAATTTCTTCTCTATATTTTCCATATTCAAACTCTGGGACCTCTGCAGTGCCAA
CCAAATCCTAATATCAATCCACAGGATATAAAGCCTGCTGCCTCCCCAGAGGACACCC'
CTGAGCTCAGCGCCTTTTTGCGAAACTCTACTATTCCTCATCTTGTCAGGAAGAGGGA
TGTACATGTGGTCGAATTCCACAGACAGAGCCCTGCAAAAATACTGAATTGTACAAAT'
TCCTGAAAGTCAGGTCACGATTATGGGCCCACACCTTCCTCCAGAGAAAAAATGATCC
CTATGCTCTTGCATCCCTGGTGTCCTTTGAAGTTAAGAAGATGCCTTATACAGATCAG
CCAGCAAAACTCCCAGAAGGAAGCATAGCAATTAAGACATCAGTTATTTGGGCAACTC
CGAATGTTTCCTTCTCAATCCCATTATGGGTAATAATACTAGCAATACTTCTTGGATT
GTTGGTTCTCGCCATTTTAACCTTAGCTTTATGGAAGTGTGGATTCTTTGACAGAGCC
AGGCATGACAAGF~3AAAAAAAGAAGACCAAAGACCTCAAACACTGGTCCTGTTCAAAG
AAAAAGAAAGAACATGAGGCC
ORF Start: ATG at 355 ORF Sto : TGA at 3544
~EQ ID NO: 66~~~1063 as p yM~W at 117444.2kD
NOV24b, MSPGASRGPRGSQAPLIAPLCCAAAALGMLLWSPACQAFNLDVEKLTVYSGPKGSYFG
CG105973-02 YAVDFHIPDARTASVLVGAPKANTSQPDIVEGGAVYYCPWPAEGSAQCRQIPFDTTNN
PTOtelri SeCllleriCe RKIRVNGTKEPIEFKSNQWFGATVKAHKGKWACAPLYHWRTLKPTPEKDPVGTCYVA
IQNFSAYAEFSPCRNSNADPEGQGYCQAGFSLDFYKNGDLIVGGPGSFYWQGQVITAS
VADIIANYSFKDILRKLAGEKQTEVAPASYDDSYLGYSVAAGEFTGDSQQELVAGIPR
GAQNFGYVSIINSTDMTFIQNFTGEQMASYFGYTVWSDVNSDGLDDVLVGAPLFMER
EFESNPREVGQIYLYLQVSSLLFRDPQILTGTETFGRFGSAMAHLGDLNQDGYNDIAI
GVPFAGKDQRGKVLIYNGNKDGLNTKPSQVLQGVWASHAVPSGFGFTLRGDSDTDKND
YPDLIVGAFGTGKVAVYRARPVVTVDAQLLLHPMIINLENKTCQVPDSMTSAACFSLR
VCASVTGQSIANTTVLMAEVQLDSLKQKGAIKRTLFLDNHQAHRVFPLVIKRQKSHQC
QDFIVYLRDETEFRDKLSPINISLNYSLDESTFKEGLEVKPILNYYRENIVSEQAHIL
VDCGEDNLCVPDLKLSARPDKHQVIIGDENHLMLIINARNEGEGAYEAELFVMIPEEA
DYVGIERNNKGFRPLSCEYKMENVTRMWCDLGNPMVSGTNYSLGLRFAVPRLEKTNM
SINFDLQIRSSNKDNPDSNFVSLQINITAVAQVEIRGVSHPPQIVLPIHNWEPEEEPH
KEEEVGPLVEHIYELHNIGPSTISDTILEVGWPFSARDEFLLYIFHTQTLGPLQCQPN'
PNINPQDIKPAASPEDTPELSAFLRNSTIPHLVRKRDVHWEFHRQSPAKIT.~NCTNIE;
168
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CLQISCAVGRLEGGESAVLKVRSRLWAHTFLQRKNDPYALASLVSFEVKKMPYTDQPA
KLPEGSIAIKTSVIWATPNVSFSIPLWVIILAILLGLLVLAILTLALWKCGFFDRARP
PQEDMTDREQLTNDKTPEA
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 24B.
Table 24B. Comparison of NOV24a against NOV24b.
Protein Sequence NOV24a Residues/ Identities/
Match Residues ~ Similarities for the Matched Region
NOV24b 1..1063 1010/1063 (95%)
1..1063 1010/1063 (95%)
Further analysis of the NOV24a protein yielded the following properties shown
in Table
24C.
Table 24C. Protein Sequence Properties NOV24a
PSort 0.4600 probability located in plasma membrane; 0.1125 probability
located in
analysis: microbody (peroxisome); 0.1000 probability located in endoplasmic
reticulum
(membrane); 0.1000 probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 39 and 40
analysis:
A search of the NOV24a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 24D.
169
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_... ... . ~ - Table 24D. Geneseq . __
Results for NOV24a ._.. .~.
.. . _. ~. ... . .._ .~.
. _ .
NOV24a Identities/
Geneseq Protein/Organism/Length ' Residues/Similarities Expect
for
Identifier(Patent #, Date] Match the Matched Value
Residues Region
AAM39241 Human polypeptide SEQ 29..1063 1031/1035 (99%)0.0
ID NO ,
2386 - Homo Sapiens, 10351..1035 1031/1035 (99%)
aa.
[W0200153312-A1, 26-JUL-
2001 ]
3
AAM41027 Human polypeptide SEQ 24..1063 1024/1046 (97%)0.0
ID NO
5958 - Homo Sapiens, 1044~ 1..10441027/1046 (97%)
aa.
[W0200153312-Al, 26-JUL-
2001]
.xxr 3
ABG18895 Novel human diagnostic 8..1055 0.0
protein 500/1052
(47%)
'
#18886 - Homo sapiens, 25..1049 692/1052 (65%)
1061 aa.
[W0200175067-A2, 11-OCT-
2001 ]
I
ABG18895 Novel human diagnostic 8..1055 500/1052 (47%)0.0
protein
#18886 - Homo sapiens, 25..1049 692/1052 (65%)
1061 aa.
_ [W0200175067-A2, 11-OCT-
2001~
AAB70508 Tissue remodeling protein34..1063 474/1036 (45%)0.0
alpha 5
beta 1 integrin (VLA-5) 37..1049 667/1036 (63%)
protein -
Mammalian, 1049 aa.
[W0200111086-A2, 15-FEB-
2001 ]
In a BLAST search of public sequence databases, the NOV24a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 24E.
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Table 24E. Public BLASTP
Results for NOV24a
NOV24a
Identities/
Protein
AccessionProtein/Organism/Length Residues/Similarities Expect
for
Number Match the Matched Value
ResiduesPortion
P53708 Integrin alpha-8 -Homo 39..10631020/1025 (99%)0.0
Sapiens
(Human), 1025 aa. 1..1025 1020/1025 (99%)
070304 INTEGRIN ALPHA8 - Mus 46..1057910/1012 (89%)0.0
musculus (Mouse), 1012 1..1012 972/1012 (95%)
as
(fragment).
P26009 Integrin alpha-8 precursor27..1063797/1037 (76%)0.0
- Gallus ~
gallus (Chicken), 1044 13..1044907/1037 (86%)
aa.
P26008 Integrin alpha-V precursor35..1055493/1024 (48%)0.0
(Vitronectin receptor 16..1022678/1024 (66%)
alpha
subunit) (CD51) - Gallus
gallus
(Chicken), 1034 aa.
Q9MZD6 INTEGRIN ALPHA V SUBUNIT 8..1055 508/1057 (48%)0.0
PRECURSOR - Bos taurus 12..1035692/1057 (65%)
(Bovine), 1047 aa.
PFam analysis predicts that the NOV24a protein contains the domains shown in
the Table
24F.
Table 24F. Domain Analysis of NOV24a
Identities/
Pfam DomainNOV24a Match RegionSimilarities Expect
. Value
for the Matched
Region
FG-GAP 54..117 22/67 (33%) 1.4e-15
a .
53/67 (79%)
FG-GAP 264..317 19/63 (30%) 2.1e-06
42/63 (67%)
FG-GAP 318..383 23/66 (35%) 1.7e-15
_49/66.(74%) .. . .
FG-GAP 384..443 29/67 (43%) 2e-16
53/67 (79%)
FG-GAP 447..501 22/66 (33%) 7.8e-10
41/66 (62%)
integrin_A 1035..1049 10/15 (67%) 0.00011
14/15 (93%)
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Example 25.
The NOV2S clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 2SA.
Table 25A. NOV2S Sequence Analysis
SEQ ID N067 ~~ , ,1 S24 by
NOV2Sa, TTTGCCATCATGTTGCGGTTGGTGGCAGCTTGCCCTGAGTCATGTGTGGTGTGCACCA
CG106915-O1 ~GATGTAACCCTCTGTCACCAGCTAACCTATATAGTAGCAGCCCCTATGACCACGAG
DNA SequenceGGTTTTAATCATCACCGATGGATATCTCTCCTCTATTGAGAGCACAAACCTGTCTCTC
TTGTTTAATCTTGCCCTGCTCTCCCTAAGCAGAAATGGTATCGAGGATGTTCAGGAAG
ATGCCCTGCATGGGCTTACGATGTTGCGGACCTTGTTGCTGGAGCACAACCAAATATC
CAGCTCTTCGCTCACTGATCACACCTTCAGCAAGCTTCACAGCCTGCAGGTACTGGTG
CTGAGCAATAATGCTCTCCGCACCCTACGAGGGTCTTGGTTCCGAAACACAAGCGGCC
TGACCCGGCTCCAGCTGGATGGGAATCAGATTACTAATCTCACAGACAGT~CTTTCGG
AGGCACGAATCTCCACAGTCTCAGGTATCTGGATTTATCCAACAATTTTATTTCCTAC
ATTGGGAAAGATGCCTTCCGGCCCCTGCCTCAACTACAGGAAGTGGACCTTTCCCGAA
ATAGGTTAGCCCACATGCCGGATGTGTTTACTCCACTGAAGCAGTTAATCCTTCTGAG
CTTAGATAAGAACCAGTGGAGCTGCACTTGTGATCTCCATCCCCTTGCTCGGTTTTTA
AGAAACTACATTAAGTCTTCTGCTCACACGCTCAGGAATGCCAAGGACCTAAATTGCC
AGCCATCTACCGCAGCTGTGGCAGCTGCACAGAGTGTGCTGAGGCTGTCTGAGACCAA
CTGTGATTCCAAAGCTCCCAACTTCACTCTGGTTCTAAAGGACAGAAGTCCCCTCCTC
CCAGGACCAGATGTGGCCCTGCTGACTGTCCTTGGCTTCGCAGGTGCTGTTGGTCTCA
CTTGCCTAGGTTTAGTTGTATTTAACTGGAAACTCCACCAAGGCAAAGCAAATGAACA
CACATCAGAAAACCTTTGTTGCAGAACCTTCGATGAACCCCTGTGTGCTCATGAGGCA
AGAAATTACCACACTAAGGGATACTGCAACTGCCACTTAACTCAGGAAAACGAGATAA
AGGTCATGTCCATTGTGGGGTCCAGAAAAGAAATGCCACTTTTACAGGAAAATAGCCA
TCAAGCAACATCGGCCTCTGAGTCTGCAACCCTTGACAGATCATTTAGAAACCTGAAA
AAGAAAGACCGTGGGGTAGGCAGCACTTTATTTTGCCAGGATGGTAGATTGCTGCATT
CGGAATGTTCAGAGCCTCCTGGAAATATGAGAGCTTTTAATGAAGCAGGCTTACTTAC
AACATATAATCCAAGGAAAGTTCAAAAGCTATGGAATCTTGAGCCTGGAGAAGTCCAG
CCTCAAACTCTGCAACACCATATAATAAGAACAGAAGATATCAGCAGTGACATATTTA
GAAGAAGATATGCAACACCCGCTTCAGCCTTGGCAGGAGAAAGTCTTGAGAAGCGTTT
AACAAATGAATCATGA
ORF Start: ATG at 10 ORF Stop: TGA at 1 S22
SEQ ID NO: 68 S04 as MW at 56079.2kD
NOV2Sa, MLRLVAACPESCVVCTKDVTLCHQLTYIVAAPMTTRVLIITDGYLSSIESTNLSLLFN
CG106915-Ol L~'LSLSRNGIEDVQEDALHGLTMLRTLLLEHNQISSSSLTDHTFSKLHSLQVLVLSN
P1'Oteln N~'RTLRGSWFRNTSGLTRLQLDGNQITNLTDSSFGGTNLHSLRYLDLSNNFISYIGK
Sequence
DAFRPLPQLQEVDLSRNRLAHMPDVFTPLKQLILLSLDKNQG~TSCTCDLHPLARFLRNY
IKSSAHTLRNAKDLNCQPSTAAVAAAQSVLRLSETNCDSKAPNFTLVLKDRSPLLPGP
DVALLTVLGFAGAVGLTCLGLVVFNWKLHQGKANEHTSENLCCRTFDEPLCAHEARNY
HTKGYCNCHLTQENEIKVMSIVGSRKEMPLLQENSHQATSASESATLDRSFRNLKKKD
RGVGSTLFCQDGRLLHSECSEPPGNMRAFNEAGLLTTYNPRKVQKLWNLEPGEVQPQT
LQHHTIRTEDISSDIFRRRYATPASALAGESLEKRLTNES
S Further analysis of the NOV2Sa protein yielded the following properties
shown in Table
2SB.
Table 2513. Protein Sequence Properties NOV25a
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analysis: probability located in plasma membrane; 0.3000 probability located
in
microbody (peroxisome); 0.2622 probability located in mitochondria) matrix
space
SignalP No Known Signal Sequence Predicted
analysis:
A search of the NOV25a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 25C.
Table 25C. Geneseq Results for NOV25a
NOV25a Identities/
~
Geneseq ProteinlOrganismlLength Residues/SimilaritiesExpect
' ~ for
Identifier[Patent #, Date] Match the Matched Value
Residues Region
AAU83655 Human PRO protein, Seq 3..237 79/264 (29%)1 Se-19
ID No
128 - Homo sapiens, 473 22..283 1211264 (44%)
aa.
[W0200208288-A2, 31-JAN-
2002]
3
AAB49891 Human PR0526 protein sequence3..237 79/264 (29%)~ Se-19
-
Homo Sapiens, 473 aa. 22..283 121/264 (44%)
[W0200070050-Al, 23-NOV-
2000]
AAB50908 Human PR0526 protein - 3..237 79/264 (29%)Se-19
Homo
Sapiens, 473 aa. [W0200073452-22..283 121/264 (44%)
A2, 07-DEC-2000]
3
AAU04589 Human Nogo receptor - 3..237 79/264 (29%)Se-19
Homo ~ ~
Sapiens, 473 aa. [W0200151520-22..283 121/264 (44%)
A2, 19-JUL-2001 ]
AAU12362 Human PRO526 polypeptide 3..237 79/264 (29%)~ Se-19
sequence - Homo Sapiens, 22..283 121/264 (44%)
473 aa.
[WO200140466-A2, 07-JUN-
2001 ]
In a BLAST search of public sequence databases, the NOV25a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 25D.
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Table 25D. Public BLASTP Results for NOVZSa
Protein NOV25a Identities/
AccessionProtein/Or anism/Len th Residues/Similarities; Expect
g g for
Match the Matched Value
Number
ResiduesPortion
__ ___ ___ ~
~
Q9BGY6 HYPOTHETICAL 56.5 I~DA 1..504 478/504 (94%)0.0
PROTEIN - Maraca fascicularis1..504 481/504 (94%)
(Crab eating macaque)
(Cynomolgus monkey), 510
aa.
Q961X3 GH01279P - Drosophila ~ 45..233 68/212 (32%)~ 3e-21
melanOgaster (Fruit fly), 313..522103/212 (48%)
615 aa.
Q9NOE3 ~ UNNAMED PROTEIN PRODUCT 3..237 82/264 (31%)~ 1e-19
- Maraca fascicularis (Crab22..283 125/264 (47%)
eating
macaque) (Cynomolgus monkey),
473 aa.
Q9VZ84 CG7509 PROTEIN - Drosophila45..233 69/230 (30%)6e-19
~ melanogaster (Fruit fly),313..540103/230 (44%)
633 aa.
Q9BZR6 NOGO RECEPTOR - Homo 3..237 79/264 (29%)~ 1
e-I
8
sapiens (Human), 473 aa. 22..283 121/264 (44%)f
PFam analysis predicts that the NOV25a protein contains the domains shown in
the Table
25E.
Table 25E. Domain Analysis of NOV25a
Identities/
Pfam Domain NOV25a Match RegionSimilarities Expect Value
for the Matched
Region
LRR 58..81 7/25 (28%) 0.3
19/25 (76%)
LRR 108..131 10/25 (40%) 0.11
19/25 (76%)
LRR 132..155 8/25 (32%) 0.7
18/25 (72%)
LRR 158..181 11/25 (44%) 0.00021
19/25 (76%)
LRR 182..204 10/25 (40%) 0.093
18/25 (72%)
LRRCT 214..270 15/63 (24%) 0.046
42/63 (67%)
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Examule 26.
The NOV26 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 26A.
Table 26A. NOV26 Sequence Analysis
SEQ ID NO: 69 X3757 by
NOV26a, ~CTCTTTGCCATCATGTTGCGGTTGGTGGCAGCTTGCCCTGAGTCATGTGTGGTGTGCA
CG106924-Ol CCAAAGATGTAACCCTCTGTCACCAGCTAACCTATATAGTAGCAGCCCCTATGACCAC
DNA Sequence GAGGGTTTTAATCATCACCGATGGATATCTCTCCTCTATTGAGAGCACAAACCTGTCT
CTCTTGTTTAATCTTGCCCTGCTCTCCCTAAGCAGAAATGGTATCGAGGATGTTCAGG
AAGATGCCCTGCATGGGCTTACGATGTTGCGGACCTTGTTGCTGGAGCACAACCAAAT
ATCCAGCTCTTCGCTCACTGATCACACCTTCAGCAAGCTTCACAGCCTGCAGGTACTG
GTGCTGAGCAATAATGCTCTCCGCACCCTACGAGGGTCTTGGTTCCGAAACACAAGCG
GCCTGACCCGGCTCCAGCTGGATGGGAATCAGATTACTAATCTCACAGACAGTTCTTT
CGGAGGCACGAATCTCCACAGTCTCAGGTATCTGGATTTATCCAACAATTTTATTTCC
TACATTGGGAAAGATGCCTTCCGGCCCCTGCCTCAACTACAGGAAGTGGACCTTTCCC
GAAATAGGTTAGCCCACATGCCGGATGTGTTTACTCCACTGAAGCAGTTAATCCTTCT
GAGCTTAGATAAGAACCAGTGGAGCTGCACTTGTGATCTCCATCCCCTTGCTCGGTTT
TTAAGAAACTACATTAAGTCTTCTGCTCACACGCTCAGGAATGCCAAGGACCTAAATT
GCCAGCCATCTACCGCAGCTGTGGCAGCTGCACAGAGTGTGCTGAGGCTGTCTGAGAC
CAACTGTGATTCCAAAGCTCCCAACTTCACTCTGGTTCTAAAGGACAGAAGTCCCCTC
CTCCCAGGACCAGATGTGGCCCTGCTGACTGTCCTTGGCTTCGCAGGTGCTGTTGGTC
TCACTTGCCTAGGTTTAGTTGTATTTAACTGGAAACTCCACCAAGGCAAAGCAAATGA
T ..T ~r ~.,T..,nr ~r m a T ....~,..,..,~...,..,e-.,.r ~T T
..~..,..,~.,.._..,~_ T ..,.,......,..,...~.,...~...w_ ...~._
GCAAGAAATTACCACACTAAGGGATACTGCAACTGCCACTTAACTCAGGAAAACGAGA
TAAAGGTCATGTCCATTGTGGGGTCCAGAAAAGAAATGCCACTTTTACAGGAAAATAG
CCATCAAGCAACATCGGCCTCTGAGTCTGCAACCCTTGACAGATCATTTAGAAACCTG
AA.AAAGAAAGACCGTGGGGTAGGCAGCACTTTATTTTGCCAGGATGGTAGATTGCTGC
ATTCGGAATGTTCAGAGCCTCCTGGAAATATGAGAGCTTTTAATGAAGCAGGCTTACT
TACAACATATAATCCAAGGAAAGTTCAAAAGCTATGGAATCTTGAGCCTGGAGAAGTC
CAGCCTCAA.ACTCTGCAACACCATATAATAAGAACAGAAGATATCAGCAGTGACATAT
TTAGAAGAAGATATGCAACACCCGCTTCAGCCTTGGCAGGAGAAAGTCTTGAGAAGCG
TTTAACAAATGAATCATGGCAGCCTCCAATAGAAAAAGAAGACAATGGCTTACACCCT
CACAGGCAAAGACATTTTATTACAAGCTCATCATCCAAGCCTTGTGAGCCTGAGGAAC
ACTATGTACAAAATATCGTACAAAAAAATAGATCAAAATATGATGATCCTTGTGGACT
GTTAAAACAGAGCAAACCTAGGTATTTTCAGCCAAACAATTCTCTTATCTGTAAATAT
GTGCCCTGTGAGCAATTTGAAGATTACATGAAAGAAAAGAAGCCAAATCGTAGACAAC
ACTCAAAGCCTGAGAAAGAGCAAATCCAAATTAACAGTGCAATAGAAAAATTTCTTAT
GAGTGAGGACAACATAGATTTATCAGGATTATCAACAAAAACCAAGAAAGCATATTCC
CCAAAGAGGGTTATCTTCCATGATCCTGATTTAGTAGAAATAAATAGGTCGATGATGT
CACCCAAAATATCAACCCCTTGGAAACGACAGAAAAATCAAAGTAACCAACTGACTAA
GTTGGATGTTAAAAAATTTAGCAACACTGGGGAGAGAAACAAAGGAGAA.AAATGGTTT
ACTAATTCATGGGTTCTGAAAAGGAAGAGAACCCCTCAGTCTGACCTCAAAGGGAAAA
TTAAAGGACAAAACTTAAAATTAAATTTACATCCTTTTAGAAAAGTCAGAGTCCATCC
AGAAAAATCCTTGTCAAGTCTCCCAAAGCAATGCAAGCAGGTATTGTTGCCTCCTAAG
AAATTATCCAAAACTTCTGAGACAGAAGCCAA.AATAAATACTGTGTGTTCTGCAGATT
TTCTTCAACAGTCAGAGAGTAGCAACTATGTTAGACTCACTTCAAAGAGGCTGCCTCT'
GAAACATGACTCAAAGCAGACCCCATATTATCAACGAAACACTAAACGTGCCCCCCTG
CTCAGTGCTAACAACTTGCGTGTAGTCAACCAGAGCTCTATAGAAAGCAGCTGTTACT
CAGCTGGCCACATTCCTGATGGAAACACATCAAAATTGCCCCAACCTACACCCACTGA'
TGCTGAGCACAGGCACTCACATTCTCAATTCTCAACTGAGCAAATGGAAGATGCAACT';
CAGCTTGAATCAAAAGTGCTTAGTTATTTAGCAACTACTTGGGAAAT1TACAGGAAGTG~,
ATGTTTTACCATTCCAACATTCCAGGAGGGCTACTGACCAAGGGACAACGGAGTCCACj
TGAGCACATGGGACAGAATGTATCAAAGACCAGTGAGTTAAATCAGTTTTCTTTGTCC'
CCGAGGAATCAAACACAACTTTTAGATGCTCACAAGACTGACAGCTACAACAAGGAAT
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~ACACTTTAGACCAAAATGAAGGCTTACAACACAGAGAGCAAAATTCAAGTCATGCACA
GCTTGAAAATAAAGAAAA.AACATTAATGACAAAACCCCAAATACCACATCAAATTGTG
GAAAATTGTATTATGGATAAGGAAGAAAATGATGTAGAAAAAAAACTTTCAAA.AACAG
AAACTTATGATTCCTCTCTCATTCCCCAAACACAATCCAAGAACAACCTATCATTTAT
GAAGACAAATTCAATTCCATACCAAAATAGAATAGAACTTCCCAAGGATATCAGTACT
TCTCCTGTTAGTAGTCAAGCCGTTTGGCACCTAACCAATAGTAGCGAAAAAGGAATTG
ACAGCACAAATGCATTGCCCAGAAATGACGGCACTGAAGCACTAGAGATAAAAATAGT
AGGGAAAGAAGAGAAAAATATGCTTGATGAAAGCAAGACAGATTCTAGTATGTTAACT
CAGATCTCACAAATGACCTTAAAAGGCATCACAAAAGAAAGGCAGCAAACTTGGGAAA
ATGGAACAAGTGAAAAATATATATTACATGATGCAAGCTCTGCCGAGGAGACCATTAC
AGCTAAAGATTTAAGTATCACAAGTTCCCATGAAACCCAAAATAGAATACTTTGCAGT
GAAGTAGATCCTGAAGTTAACAGTAATGTACATAATTTTAGAGAAGTTCAAAATATTC
AACCAGATAAAGATAGGGCACATAAAGAAGGCGCAATGACAGTGGAGACACATGAAGC
GCTTTCCTTCTTACCAGGGTTAAAAGACAGTTTTGAGGCAGAAAATGAGGTGTTTTTA
GTTCCTAGCAGAATAAATGAAGCTGAAAACTCTGCTCCAAAACCTGTACTGTATCCAC
CATCTGCTGAATATGCTACTACATCACCTTTAGAAACAGAATAAA
ORF Start: ATG at 13 ORF Stop: TAA at 3754
SEQ ID NO: 70 1247 as MW at 140902.2kD
y
NOV26a, MLRLVAACPESCWCTKDVTLCHQLTYIVAAPMTTRVLIITDGYLSSIESTNLSLLFN
CG106924-Ol I'~'LSLSRNGIEDVQEDALHGLTMLRTLLLEHNQISSSSLTDHTFSKLHSLQVLVLSN
Protein SequenceN~'RTLRGSWFRNTSGLTRLQLDGNQITNLTDSSFGGTNLHSLRYLDLSNNFISYIGK
DAFRPLPQLQEVDLSRNRLAHMPDVFTPLKQLILLSLDKNQWSCTCDLHPLARFLRNY
IKSSAHTLRNAKDLNCQPSTAAVAAAQSVLRLSETNCDSKAPNFTLVLKDRSPLLPGP
DVALLTVLGFAGAVGLTCLGLWFNWKLHQGKANEHTSENLCCRTFDEPLCAHEARNY
HTKGYCNCHLTQENEIKVMSIVGSRKEMPLLQENSHQATSASESATLDRSFRNLKKKD
RGVGSTLFCQDGRLLHSECSEPPGNMRAFNEAGLLTTYNPRKVQKLWNLEPGEVQPQT
LQHHIIRTEDISSDIFRRRYATPASALAGESLEKRLTNESWQPPIEKEDNGLHPHRQR
HFITSSSSKPCEPEEHYVQNIVQKNRSKYDI?PCGLLKQSKPRYFQPNNSLICKYVPCE
QFEDYMKEKKPNRRQHSKPEKEQIQINSAIEKFLMSEDNIDLSGLSTKTKKAYSPKRV
IFHDPDLVEINRSMMSPKISTPWKRQKNQSNQLTKLDVKKFSNTGERNKGEKWFTNSW
VLKRKRTPQSDLKGKIKGQNLKLNLHPFRKVRVHPEKSLSSLPKQCKQVLLPPKKLSK
TSETEAKINTVCSADFLQQSESSNYVRLTSKRLPLKHDSKQTPWQRNTKRAPLLSAN
NLRVVNQSSIESSCYSAGHIPDGNTSKLPQPTPTDAEHRHSHSQFSTEQMEDATQLES
KVLSYLATTWENTGSDVLPFQHSRRATDQGTTESTEHMGQNVSKTSELNQFSLSPRNQ
TQLLDAHKTDSYNKEYTLDQNEGLQHREQNSSHAQLENKEKTLMTKPQIPHQIVENCI
MDKEENDVEKKLSKTETYDSSLIPQTQSKNNLSFMKTNSIPYQNRIELPKDISTSPVS
SQAVWHLTNSSEKGIDSTNALPRNDGTEALEIKIVGKEEKNMLDESKTDSSMLTQISQ
MTLKGITKERQQTWENGTSEKYILH17ASSAEETITAKDLSITSSHETQNRILCSEVDP
EVNSNVHNFREVQNIQPDKDRAHKEGAMTVETHEALSFLPGLKDSFEAENEVFLVPSR
INEAENSAPKPVLYPPSAEYATTSPLETE
SEQ ID NO: 71 645 by
NOV26b, GGATCCGCCCTGCTCTCCCTAAGCAGAAATGGTATCGAGGATGTTCAGGAAGATGCCC
2IOO62I44 Z'GCATGGGCTTACGATGTTGCGGACCTTGTTGCTGGAGCACAACCAAATATCCAGCTC
DNA
TTCGCTCACTGATCACACCTTCAGCAAGCTTCACAGCCTGCAGGTACTGGTGCTGAGC
Sequence
p~T~TGCTCTCCGCACCCTACGAGGGTCTTGGTTCCGAAACACAAGCGGCCTGACCC
GGCTCCAGCTGGATGGGAATCAGATTACTAATCTCACAGACAGTTCTTTCGGAGGCAC
GAATCTCCACAGTCTCAGGTATCTGGATTTATCCAACAATTTTATTTCCTACATTGGG
AAAGATGCCTTCCGGCCCCTGCCTCAACTACAGGAAGTGGACCTTTCCCGAAATAGGT
TAGCCCACATGCCGGATGTGTTTACTCCACTGAAGCAGTTAATCCTTCTGAGCTTAGA
TAAGAACCAGTGGAGCTGCACTTGTGATCTCCATCCCCTTGCTCGGTTTTTAAGAAAC
TACATTAAGTCTTCTGCTCACACGCTCAGGAATGCCAAGGACCTAAATTGCCAGCCAT
CTACCGCAGCTGTGGCAGCTGCACAGAGTGTGCTGAGGCTGTCTGAGACCAACTGTGA
~
iTCTCGAG
ORF Start: at I ORF Stop: end o~ sequence
w SEQ ID NO: 72 2IS.aa .~MW at 23982.8kD -..
NOV26b, GSALLSLSRNGIEDVQEDALHGLTMLRTLLLEHNQISSSSLTDHTFSKLHSLQVLVLS
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210062144 ~NI'TALRTLRGSWFRNTSGLTRLQLDGNQITNLTDSSFGGTNLHSLRYLDLSNNFISYIG
PrOteln Sequence ~AFRPLPQLQEVDLSRNRLAHMPDVFTPLKQLILLSLDKNQWSCTCDLHPLARFLRN
((YIKSSAHTLRNAKDLNCQPSTAAVAAAQSVLRLSETNCDLE
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 268.
Table 26B. Comparison of NOV26a against NOV26b.
Protein Sequence ~ NOV26a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOV26b 60..270 199/211 (94%)
3..213 , 199/211 (94%)
Further analysis of the NOV26a protein yielded the following properties shown
in Table
26C.
Table 26C. Protein Sequence Properties NOV26a
PSort 0.8524 probability located in mitochondria) inner membrane; 0.6000
analysis. probability located in endoplasmic reticulum (membrane); 0.3000
probability
located in microbody (peroxisome); 0.2622 probability located in
mitochondria) matrix space
SignalP No Known Signal Sequence Predicted
analysis.. . .~..... ~ . . .. . ... .. . .
S A search of the NOV26a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 26D.
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Table 26D. Geneseq Results
for NOV26a
NOV26a Identities/
Geneseq Protein/Organism/Length Residues/ SimilaritiesExpect
~ for
Identifier[Patent #, Date] Match the MatchedValue
Residues Region
AAU83655 Human PRO protein, Seq 3..237 79/264 (29%)1e-18
ID No
128 - Homo sapiens, 473 22..283 121/264
aa. (44%)
[W0200208288-A2, 31-JAN-
2002J
AAB49891 Human PR0526 protein sequence3..237 79/264 (29%)~1e-18
-
Homo Sapiens, 473 aa. 22..283 I Z 11264
' (44%)
[WO200070050-A1, 23-NOV-
2000) .
AAB50908 Human PR0526 protein - 3..237 79/264 (29%)1e-18
Homo
sapiens, 473 aa. [W0200073452-22..283 121/264
(44%)
A2, 07-DEC-2000]
AAU04589 Human Nogo receptor - 3..237 79/264 (29%)1e-18
Homo ~
Sapiens, 473 aa. [WO200151520-22..283 121/264
~ (44%)
A2, 19-JUL-2001]
AAU12362 Human PR0526 polypeptide 3..237 79/264 (29%)1e-18
sequence - Homo sapiens, 22..283 121/264
473 aa. (44%)
[W0200140466-A2, 07-JUN-
2001 ..
In a BLAST search of public sequence databases, the NOV26a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 26E.
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Table 26E. Public BLASTP Results for NOV26a
.-.~....~. .w.....:,
Protein ' NOV26a Identities/
Accession Protein/Organism/Length Residues/ : Similarities for ~ Expect
Number Match the Matched Value
Residues Portion
Q9BGY6 HYPOTHETICAL 56.5 I~DA 1..504 478/504 (94%) . 0.0
PROTEIN - Maraca fascicularis 1..504 481/504 (94%)
(Crab eating macaque)
(Cynomolgus monkey), 510 aa. .
Q961X3 GH01279P - Drosophila 45..233 68/212 (32%) ~ 9e-21
melanogaster (Fruit fly), 615 aa. 313..522 103/212 (48%)
Q9NOE3 UNNAMED PROTEIN PRODUCT ~ 3..237 82/264 (31%) ~ 3e-19
- Maraca fascicularis (Crab eating 22..283 125/264 (47%)
macaque) (Cynomolgus monkey),
473 aa.
Q9VZ84 CG7509 PROTEIN - Drosophila 45..233 69/230 (30%) ~ l e-18
melanogaster (Fruit fly), 633 aa. 313..540 103/230 (44%)
Q9BZR6 ~ NOGO RECEPTOR - Homo ~ 3..237 79/264 (29%) j 3e-18
sapiens (Human), 473 aa. 22..283 121/264 (44%)
PFam analysis predicts that the NOV26a protein contains the domains shown in
the Table
26F.
Table 26F. Domain Analysis of NOV26a
Identities/
Pfam DomainNOV26a Match RegionSimilarities Expect Value
for the Matched
Region
LRR 58..81 7/25 (28%) 0.3
19/25 (76%)
LRR 108..131 10/25 (40%) 0.11
~ . 19/25 (76%)
LRR 132..155 8/25 (32%) 0.7
18/25 (72%)
LRR 15 8..181 11 /25 (44%) 0.00021
19/25 (76%) ~
_
LRR 182..204 10/25 (40%) ~ 0.093
18/25 (72%)
LRRCT 214..270 15/63 (24%) 0.046
42/63 (67%)
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Example 27.
The NOV27 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 27A.
_Table 27A. NOV27 Sequence Analysis
__.__.__ __. _SEQ_~D NO: 73 ~~ . 358 bp.
NOV27a, ~GACTTCCTGGCTCGCCAGCCCCTTCCTTCCGGAGCCTGACCCGGGCCCGGGCGACCTC
CG106942-Ol CCCGCGCGCTTCCCGGCCGCTGCCCAGGGGGTAGAGCGGGCGCAGCCGATCACTACCT
DNA Sequence G'~CGGCCTTTTTGGCGGCCTGGCCGGGCTGTGCAGGGTGGTAGGGCAAGACGCGCGGC
TCCCAATTCTCCCCGGCGCCTTCGCCGGCCCCGGGCTTCTCGCGCTCCGCTCCGGGCT
GCACCGAGTTGGGCCGGCGCGCCGCGTTGGTGTTGCCGCGCGGCGGCAGCTCAGAGTC
TCCAGGTTGGGGCGGGCCTGGGCCGCACGGCTCCTCCACCCAGGTGACGCTGAGCAGG
CTCAGGGTGAAGCCCAGGGAGATGCCCACGGCCACGGGCCCTGCGGGCCGCAGCACCG
ACAGCAGCAGCGATGCCCGCATGGCGCCCGGACCGCGGGTCCCCGGCCCCGGCGAACC
CCCAGAGCAGCCAGAGGAGTCTCCGAGGGGGCGGGACCGGGGAGGGGGCGGATCCGGA
GGGCTCGGGCCCCGCGGGCGGGCCCGCTCCCTCCCCGCAGAGCAGAGCCAGCGGCCCG
AGCCGAATCCCCGGAGCCGCGCCTCGATTCCCCTCCAGCAGCTGCTCTGGGCTGCGCA
GGGTTCTTGCGCTCGGCACTGGAGCCTCAGCCGCGGCCGCAGCTGTCCGACGTGTCAC
TGCAAGGGCCCCGCCCCCGGGGTGGGGTCTCGGGCTCTCGCTACCGGAGAGGGAGGAG
AAGGGGGAGGTTAAAGGGGAAGGACCCCCGGAAGTGCCCCCTCCTCAGTGCGGGAGAG
GGAGACGCCGGGGGCGGAGTCCCCTGCCTCCCGCGGCGTGGTTGGTGCGTCCCATGTG
ACGTCAGAAGCAGCCCGCCCCTGCCTGGATGGTGCGCCCTGAGTGACGTCAGGAGCAG
AGGCCGGAGCTGTCCATCAGCACCAAAGGCCGCGGGCGGGCTCAGGGCATGGGGCCGC
GGTTCTGGGGCGGCCCGAGCCCCGGCTCCTGCGCCTTCCCCTTCCTCAGGCCCAGCCC
GAGTTCCCGGACGCCGCGGGACTGGAGTGCCAGCCGGTGTTGGACGTGGAGCGGCGCC
GCCACCGCGCCGACACCATTCTCTCCGGCCCAGCAGCCCCCTTCCTCGCACGACGGAC
TTTCCCTGGACCCCAGCACTATGCCGGGGACTGTGGCAACACTGCGGTTCCAGCTGCT
GCCCCCTGAGCCAGATGATGCCTTCTGGGGTGCACCTTGTGAACAGCCCCTGGAGCGC
AGGTACCAGGCACTGCCGGCCCTCGTCTGCATCATGTGCTGTTTGTTTGGAGTCGTCT
ACTGCTTCTTCGGTTACCGCTGCTTCAAGGCAGTGCTCTTTCTCACTGGGTTGCTGTT
TGGCTCGGTGGTCATCTTCCTCCTCTGCTACCGAGAGCGGGTGCTAGAGACACAGCTG
AGTGCTGGGGCGAGCGCGGGCATCGCTCTGGGCATCGGGCTGCTCTGCGGGCTGGTGG
CCATGCTAGTGCGCAGCGTGGGCCTCTTCCTGGTGGGGCTGCTGCTCGGCCTGCTGCT
CGCAGCTGCTGCCCTGCTGGGCTCCGCACCCTACTACCAGCCAGGCTCCGTGTGGGGT
CCACTGGGGCTGTTGCTGGGGGGCGGCCTGCTCTGTGCCCTGCTCACTCTGCGCTGGC
CCCGCCCACTCACCACCCTGGCCACCGCCGTGACTGGTGCTGCGCTGATCGCCACTGC
CGCTGACTACTTCGCCGAGCTGCTACTGCTGGGGCGCTACGTGGTGGAGCGACTCCGG
GCTGCTCCTGTGCCCCCACTCTGCTGGCGAAGCTGGGCCCTGCTGGCACTCTGGCCCC
TGCTCAGCCTGATGGGCGTTCTGGTGCAGTGGAGGGTGACAGCTGAGGGGGACTCCCA
CACGGAAGTGGTCATCAGCCGGCAGCGCCGACGCGTGCAACTGATGCGGATTCGGCAG
CAGGAAGATCGCAAGGAGAAAAGGCGGAAAAAGAGACCTCCTCGGGCTCCCCTCAGAG
GTCCCCGGGCTCCTCCCAGGCCTGGGCCACCAGATCCTGCTTATCGGCGCAGGCCAGT
GCCCATCAAACGCTTCAATGGAGACGTCCTCTCCCCGAGCTATATCCAGAGCTTCCGA
GACCGGCAGACCGGGAGCTCCCTGAGCTCCTTCATGGCCTCACCCACAGATGCGGACT
ATGAGTATGGGTCCCGGGGACCTCTGACAGCCTGCTCAGGCCCCCCAGTGCGGGTATA
GCCATATCTGTCTGTCTAGACTCTGCAGTCACCAGCTCTGACAGCTCGAGGAGGCCGG
TAGGCTGCAATCAGCTTCCGGTTTGGTGGTCCTTCCCA
ORF Start: ATG at 977 ORF Stop: TAG at 2261
SEQ ID NO: 74 428 as MW at 46672.91:D
NOV27a, MGPRFWGGPSPGSCAFPFLRPSPSSRTPRDWSASRCWTWSGAATAPTPFSPAQQPPSS
CG106942-Ol HDGLSLDPSTMPGTVATLRFQLLPPEPDDAFWGAPCEQPLERRYQALPALVCIMCCLF
Protein Sequence G~CFFGYRCFKAVLFLTGLLFGSVVIFLLCYRERVLETQLSAGASAGIALGIGLLC
GLVAMLVRSVGLFLVGLLLGLLLAAAALLGSAPYYQPGSVWGPLGLLLGGGLLCALLT
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LRWPRPLTTLATAVTGAALIA
LWPLLSLMGVLVQWRVTAEGDSHTEWISRQRRRVQLMRIRQQEDRKEKRRKKRPPRA
PLRGPRAPPRPGPPDPAXRRRPVPIKRFNGDVLSPSYIQSFRDRQTGSSLSSFMASPT
DADYEYGSRGPLTACSGPPVRV
Further analysis of the NOV27a protein yielded the following properties shown
in Table
278.
Table 275. Protein Sequence Properties NOV27a
PSort 0.6000 probability located in plasma membrane; 0.4000 probability
located in
analysis: Golgi body; 0.3000 probability located in endoplasmic reticulum
(membrane);
0.2400 probability located in nucleus
SignaIP ': No Known Signal Sequence Predicted
analysis:
A search of the NOV27a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 27C.
Table 27C. Geneseq Results for NOV27a
~ NOV27a Identities/
~
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent ~ ~
i ~ for the
Identifier#, Date) Match Value
~ Matched
Residues
[ Re ion
g
AAM34044 Peptide #8081 encoded by 79..188 60/11 l 2e-25
probe for ~ (54%)
measuring placental gene I9..124 76/111
expression ~ (68%)
~
- Homo sapiens, 124 aa.
[W0200157272-A2, 09-AUG-2001]
AAM20130 Peptide #6564 encoded by 79..188 60/1 I ~ 2e-25
probe for 1 (54%)
measuring cervical gene 19..124 76/I 11
expression - 1 ~ (68%)
Homo Sapiens, 124 aa.
[WO200157278-A2, 09-AUG-2001]
AAM73861 Human bone marrow expressed79..188 60/111 _ 2e-25
~ (54%)
probe encoded protein SEQ 19..124 76/111
ID NO: (68%)
34167 - Homo sapiens, 124
aa.
[W0200157276-A2, 09-AUG-2001]
AAM61147 Human brain expressed single79..188 60/111 2e-25
exon (54%)
probe encoded protein SEQ 19..124 76/111
ID NO: ~ (68%)
33252 - Homo Sapiens, 124
aa.
[W0200157275-A2, 09-AUG-2001]1
. ~ ~
___ _ _ . _ .
ABB24734 . 79..188 60/111 2e-25
Protein #6733 encoded by ~ (54%)
probe for .
measuring heart cell gene 19..124 76/I 11
expression (68%)
- Homo Sapiens, 124 aa.
[W0200157274-A2, 09-AUG-2001]
_ __ _ . _ _ _ __ _.._ _ _ _ .
_. _..
.
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In a BLAST search of public sequence databases, the NOV27a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 27D.
Table 27D. Public BLASTP Results for NOV27a
Protein ~ NOV27a Identities/
AccessionProtein/Organism/Length Residues/Similarities' Expect
for
Number ~ Match the Matched Value
Residues Portion
Q9~WD2 : CG14234 PROTEIN - Drosophila103..392 82/299 (27%)3e-20
melanogaster (Fruit fly),43..329 143/299 (47%)
381 aa.
Q9CRGI 2010003B14RIK PROTEIN I 12..305531208 (2S%)~ 6e-07
- Mus
musculus (Mouse), SS6 286..490 95/208 (4S%)
as
(fragment).
Q9NS93 SEVEN TRANSMEMBRANE . 115..305SO/20S (24%)~ 4e-OS
PROTEIN TM7SF3 - Homo 303..504 88/205 (42%)
sapiens (Human), 570 aa. ~
Q9NUS4 ~ CDNA FLJl 1169 FIS, 115..305 SO/20S (24%)4e-OS
CLONE
PLACE1007282 - Homo sapiens303..504 88/205 (42%)
(Human), 570 aa. j
028838 ~ Hypothetical protein 107..304 S 1/201 (2S%)~ 7e-04
AF1434 -
Archaeoglobus fulgidus, 14..188 821201 (40%)
199 aa.
Example 28.
S The NOV28 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 28A.
Table 28A. NOV28 Sequence
SEQ ID NO: 7S 2177 by
NOV28a, ~ATTCCCCTTGCCGACCCACATACACCATGAAGAGGTGCAGATCGGACGAGCTGCAGCA
CG107S13-Ol ~ACAACAGGGCGAGGAGGATGGAGCTGGGCTGGAAGATGCCGCTTCCCACCTGCCGGGC
DNA Sequence GCGGACCTCCGGCCTGGGGAGACCACGGGTGCTAACTCTGCTGGCGGGCCAACTTCAG
ACGCCGGCGCTGCCGCGGCGCCCAACCCAGGTCCCCGAAGCAAGCCTCCTGATTTAAA
GAAAATCCAGCAGCTGTCAGAGGGCTCCATGTTTGGCCACGGTCTGAAGCACCTGTTC
CACAGCCGCCGTCGGTCTCGGGAAAGGGAGCACCAGACGTCTCAGGATTCCCAGCAGC
ATCAGCAGCAGCAGGGTATGTCCGACCATGACTCCCCAGATGAGAAGGAGCGCTCTCC
GGAGATGCATCGCGTCTCCTACGCCATGTCCCTGCACGACCTGCCCGCCCGGCCCACC
GCCTTCAACCGCGTGCTGCAGCAGATCCGCTCCCGGCCCTCCATCAAGCGGGGCGCCA
GCCTGCACAGCAGCAGTGGGGGCGGCAGCAGCGGGAGCAGCAGCCGGCGCACCAAGAG
TAGCTCCCTGGAGCCCCAGCGTGGCAGCCCTCACCTGCTGCGCAAGGCCCCCCAGGAC
AGCAGCCTGGCCGCCATCCTGCACCAGCACCAGTGCCGTCCCCGCTCTTCCTCCACCA
CCGACACTGCTCTGCTGCTGGCCGACGGCAGCAACGTGTACCTCCTGGCTGAGGAGGC
CGAAGGCATCGGGGACAAGGTGGATAAGGGAGACCTGGTGGCCCTGAGCCTCCCCGCC
GGCCATGGTGACACCGACGGCCCCATCAGCCTGGACGTGCCCGATGGGGCACCGGACC
CCCAGCGGACCAAGGCCGCCATTGACCACCTGCACCAGAAGATCCTGAAGATCACCGA
GCAGATCAAGATTGAGCAGGAGGCTCGCGACGACAATGTGGCAGAGTATCTGAAACTG
GCCAACAACGCGGACAAGCAGCAGGTGTCACGCATCAAGCAAGTGTTCGAGAAGAAGA
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~ACCAGAAGTCAGCCCAGACCATCGCCCAGCTGCACAAGAAGCTGGAGCACTACCGCCG
GCGCCTGAAGGAGATTGAGCAGAACGGGCCCTCGCGGCAGCCCAAGGACGTGCTGCGG
GACATGCAGCAGGGGCTGAAGGACGTGGGCGCCAACGTGCGCGCAGGCATCAGCGGCT
TTGGGGGCGGCGTGGTGGAGGGCGTCAAGGGCAGCCTCTCTGGCCTCTCACAGGCCAC
CCACACCGCCGTGGTGTCCAAGCCCCGGGAGTTTGCCAGCCTCATCCGCAACAAGTTT
GGCAGTGCTGACAACATCGCCCACCTGAAGGACCCCCTGGAAGATGGGCCCCCTGAGG
AGGCAGCCCGGGCACTGAGCGGCAGTGCCACACTCGTCTCCAGCCCCAAGTATGGCAG
CGATGATGAGTGCTCCAGCGCCACGCTCAGCTCAGCCGGGGCAGGCAGCAACTCTGGG
GCTGGGCCTGGTGGGGCGCTGGGGAGCCCTAAGTCCAATGCACTGTATGGTGCTCCTG
GAAACCTGGATGCTCTGCTGGAAGAGCTACGGGAGATCAAGGAGGGACAGTCTCACCT
GGAGGACTCCATGGAAGACCTGAAGACTCAGCTGCAGAGGGACTACACCTACATGACC
CAGTGCCTGCAGGAGGAGCGCTACAGGTACGAGCGGCTGGAGGAGCAGCTCAACGACC
TGACTGAGCTTCATCAGAACGAGATGACGAACCTGAAGCAGGAGCTGGCCAGCATGGA
GGAGAAGGTGGCCTACCAGTCCTATGAGAGGGCACGGGACATCCAGGAGGCCGTGGAG
TCCTGCCTGACCCGGGTCACCAAGCTGGAGCTGCAGCAGCAACAGCAGCAGGTGGTAC
AGCTGGAGGGCGTGGAGAATGCCAACGCGCGGGCGCTGCTGGGCAAGTTCATCAACGT
GATCCTGGCGCTCATGGCCGTGCTGCTGGTGTTCGTGTCCACCATCGCCAACTTCATC
ACGCCCCTCATGAAGACACGCCTGCGCATCACCAGCACCACCCTCCTGGTCCTCGTCC
TGTTCCTCCTCTGGAAGCACTGGGACTCCCTCACCTACCTCCTGGAGCACGTGTTGCT
GCCCAGCTGAGTGGCCAGCCACACCAACCCT
ORF Start: ATG at 27 ORF Stop: TGA at 2154
SEQ ID NO: 76 709 as MW at 77503.9kD
NOV28a, MKRCRSDELQQQQGEEDGAGLEDAASFILPGADLRPGETTGANSAGGPTSDAGAAAAPN
CG107513-OI PGPRSKPPDLKKIQQLSEGSMFGHGLKHLFHSRRRSREREHQTSQDSQQHQQQQGMSD
Protein SeCluenCeHDSPDEKERSPEMHRVSYAMSLHDLPARPTAFNRVLQQIRSRPSIKRGASLHSSSGGG
SSGSSSRRTKSSSLEPQRGSPHLLRKAPQDSSLAAILHQHQCRPRSSSTTDTALLLAD
GSNVYLLAEEAEGIGDKVDKGDLVALSLPAGHGDTDGPISLDVPDGAPDPQRTKAAID
HLHQKILKITEQIKIEQEARDDNVAEYLKLANNADKQQVSRIKQVFEKKNQKSAQTIA
QLHKKLEHYRRRLKEIEQNGPSRQPKDVLRDMQQGLKDVGANVRAGISGFGGGVVEGV
KGSLSGLSQATHTAVVSKPREFASLIRNKFGSADNIAHLKDPLEDGPPEEAARALSGS
ATLVSSPKYGSDDECSSATLSSAGAGSNSGAGPGGALGSPKSNALYGAPGNLDALLEE
LREIKEGQSHLEDSMEDLKTQLQRDYTYMTQCLQEERYRYERLEEQLNDLTELHQNEM
TNLKQELASMEEKVAYQSYERARDIQEAVESCLTRVTKLELQQQQQQVVQLEGVENAN
ARALLGKFINVILALMAVLLVFVSTIANFITPLMKTRLRITSTTLLVLVLFLLWKHWD
SLTYLLEHVLLPS
Further analysis of the NOV28a protein yielded the following properties shown
in Table
28B.
Table 28B. Protein Sequence Properties NOV28a
PSort 0.6000 probability located in plasma membrane; 0.4000 probability
located in
analysis: Golgi body; 0.3000 probability located in endoplasmic reticulum
(membrane);
0.3000 probability located in microbody (peroxisome)
SignalP No Known Signal Sequence Predicted
analysis: ~ .
A search of the NOV28a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 28C.
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Table 28C. Geneseq Results for NOV28a
NOV28a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent for
Identifier#~, Date] Match the Matched Value
Residues Region
AAY94907 Human secreted protein 57..703 359/672 (53%)e-178
clone
ca106_19x protein sequence13..646 438/672 (64%)
SEQ
ID N0:20 - Homo Sapiens,
653 aa.
[W0200009552-Al, 24-FEB-2000]
AAM78708 Human protein SEQ ID NO 249..708 258/466 (55%)e-134
1370 -
Homo Sapiens, 477 aa. 26..476 3261466 (69%)
[W0200157190-A2, 09-AUG-2001]
AAU28090 Novel human secretory protein,258..708 254/457 (55%)e-132
Seq
ID No 259 - Homo Sapiens, 4..445 320/457 (69%)
446 aa.
[W0200166689-A2, 13-SEP-2001]
AAM40705 Human polypeptide SEQ ID 352..703 224/355 (63%)e-117
NO 3
5636 - Homo Sapiens, 369 13..362 267/355 (75%)
aa. '
[W0200153312-Al, 26-JUL-2001]
AAM38919 Human polypeptide SEQ ID 379..703 209/328 (63%)e-108
NO
2064 - Homo Sapiens, 331 2..324 248/328 (74%)
aa.
[W0200153312-Al, 26-JUL-2001]
In a BLAST search of public sequence databases, the NOV28a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 28D.
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_.... . _.-Table 28Dy.._. ub.ic_B _...
_ ... . _ . p ...es _.. .....
_ ..... . _._. ... 2. _
..... P I LAST R ults for NOV
Sa
~ NOV28a ~ Identities!
Protein
AccessionProtein/Organism/Length ~ Residues/Similarities Expect
for
Number Match the Matched Value
Residues Portion
075069 Hypothetical protein I~IAA0481~ 69..709638/641 (99%)0.0
(Cerebral protein-11) 10..650 640!641 (99%)
(hucep-11)
Homo sapiens (Human),
650 as
(fragment).
Q9ULS5 Hypothetical protein KIAAl250..708 258/465 (55%)e-134
145 -
Homo Sapiens (Human), ~ 17..466325/465 (69%)
467 as
(fragment).
AAH26867 . SIMILAR TO KIAA1145 258..708 249/457 (54%)e-129
PROTEIN - Mus musculus 4..445 316/457 (68%)
(Mouse), 446 aa.
~~
094876 Hypothetical protein KIAA0779393..703 202/314 (64%)e-105
- '
Homo Sapiens (Human), 1..313 241/314 (76%)
320 as
(fragment).
Q9VI21 CG1021 PROTEIN - Drosophila88..675 1881409 (45%)~le-84
melan0gaster (Fruit fly),~ 252..638248/409 (59%)
638 aa. ' .
~.__.._ _~....~,'~","~". ~ ~, ......
. ..__ ._... . .
Example 29.
The NOV29 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
axe shown in Table 29A.
Table 29A. NOV29 Sequence Analysis
SEQ ID NO: 77 664 by
NOV29a, ATCGCCCCTCCTGCGCTAGCGGAGGTGATCGCCGCGGCGATGCCGGAGGAGGGTTCGG
CG107533-02 GCTGCTCGGTGCGGCGCAGGCCCTATGGGTGCGTCCTGCGGGCTGCTTTGGTCCCATT
DNA Sequence GGTCGCGGGCTTGGTGATCTGCCTCGTGGTGTGCATCCAGCGCTTCGCACAGGCTCAG
CAGCAGCTGCCGCTCGAGTCACTTGGGGACCTCAGCAGGACCCCAGGCTATACTGGCA
GGGGGGCCCAGCACTGGGCCGCTCCTTCCTGCATGGACCAGAGCTGGACAAGGGGCAG
CTACGTATCCATCGTGATGGCATCTACATGGTACACATCCAGGTGACGCTGGCCATCT
GCTCCTCCACGACGGCCTCCAGGCACCACCCCACCACCCTGGCCGTGGGAATCTGCTC
TCCCGCCTCCCGTAGCATCAGCCTGCTGCGTCTCAGCTTCCACCAAGGTTGTACCATT
GCCTCCCAGCGCCTGACGCCCCTGGCCCGAGGGGACACACTCTGCACCAACCTCACTG
GGACACTTTTGCCTTCCCGAAACACTGATGAGACCTTCTTTGGAGTGCAGTGGGTGCG
CCCCTGACCACTGCTGCTGATTAGGGTTTTTTAAATTTTATTTTATTTTATTTAAGTT
CAAGAGAAAAAGTGTACACACAGGGG
ORF Start: ATG at 40 ORF Stop: TGA at 334
SEQ ID NO: 78 ~98 as MW at 10705.2kD
NOV29a, MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGDLSR
TPGYTGRGAQHWAAPSCMDQSWTRGSYVSIVMASTWYTSR
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CG107533-02
Protein Sequence
Further analysis of the NOV29a protein yielded the following properties shown
in Table
29B.
Table 29B. Protein Sequence Properties NOV29a
PSort 0.7900 probability located in plasma membrane; 0.3000 probability
located in
analysis: Golgi body; 0.2000 probability located in endoplasmic reticulum
(membrane);
0.1000 probability located in rnitochondrial inner membrane
_ . _____ _
SignalP ~ Cleavage site between residues 45 and 46
analysis:
A search of the NOV29a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 29C.
Table 29C. Geneseq Results for NOV29a
NOV29a Identities/
~
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent for
Identifier#, Date] Match the Matched Value
ResiduesRegion
AARSOI21 ~ CD27L - Homo sapiens, 1..54 54/54 (100%)7e-25
193 aa.
[WO9405691-A, 17-MAR-1994) 1..54 54/54 (100%)
AAW41180 CD27 ligand - Homo Sapiens,39..54 16116 (100%)0.16
216 ~
aa. [US5716805-A, 10-FEB-1998]62..77 16/16 (100%)
i
AAR50122 sCD27L-3 - Homo Sapiens, 39..54 16/16 (I00%)0.16
216 aa.
[WO9405691-A, 17-MAR-1994) 62..77 16/16 (100%)
AAR5397I ~ CD27-L type II transmembrane39..54 I6/I~6 (100%)0.16
~
protein - Mammalia, 216 62..77 16/I6 (100%)
aa. -
[W09410308-A, 11-MAY-1994) .
_ ~."_
AAG26041 Zea mays protein fragment 24..72 19/55 (34%) 2.4
SEQ ID
NO: 30347 - Zea mays subsp.6..59 26155 (46%)
mays,
172 aa. [EP1033405-A2, 06-SEP-
2oooJ
In a BLAST search of public sequence databases, the NOV29a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 29D.
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Table 29D. Public BLASTP Results for NOV29a
NOV29a Identities/
Protein Residues!SimilaritiesExpect
for
AccessionProtein/Organism/Length Match the Matched Value
Number
Residues ~ Portion
Q96JS7 ~ TUMOR NECROSIS FACTOR 1..54 ~54/S4 (100%)2e-24
(LIGAND) SUPERFAMILY, 1..54 S4/S4 (100%)
MEMBER 7 - Homo Sapiens
(Human), 193 aa.
P32970 CD27 ligand (CD27-L) (CD701..54 S4/S4 (100%)2e-24
antigen) - Homo Sapiens 1..54 54/S4 (100%)
(Human),
193 aa.
Q9KFY7 HYPOTHETICAL PROTEIN 39..98 18/61 (29%) 7.4
BH0329 - Bacillus halodurans,240..300 28/61 (4S%)
423
aa.
Q9RC64 UNKNOWN - Bacillus halodurans,39..98 18/61 (29%) 7.4
262 aa. 79..139 28161 (4S%)
0342SS PURL PROTEIN - Wolinella 70..93 11/24 (4S%) 9.7
succinogenes, 331 as (fragment).8..31 1S/24 (61%)
Examt~le 30.
The NOV30 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
S are shown in Table 30A.
Table 30A. NOV30 Sequence Analysis
SEQ ID N0: 79 X2840 by
NOV3Oa, CGGCAGTGGCAGGAGCCGCCTTTCCGATTCCCTACGATGCGGGTGCTGAGCTATGGCA
CG107S62-O1~GGGCAGCGAAGTGACGAGCGAGACCCGCGTACGACTGTGAAAGCCACCTGGAGCCA
D CCTTGCCGGGATTGTACCTGCAGGCAGAAAGTCTTCCTACGACCGTCTTTTCCCTTAG
NA Se ltenCe
q
AGGCACCAGAATCCCTGTAACCATTCATCCAGGTGTTGAGAAGATATGTAGCAGCCGA
GCACCCATCTTTTGACACCGTCCTCTGAAATCAGCTTTGGAGATGCTTTCACTCTGTC
CGTCTTCTGCAGCAGCCAGGCAGAGTGCCGACTCCTTCACAGCCGTGAGGAACTCTTC
AGGCTCCAGAAGCTCTTAAACCTGATCTACAATGGAAAAAATTCTTTTTTATCTGTTT
CTCATTGGCATAGCAGTGAAAGCTCAGATCTGTCCAAAGCGTTGTGTCTGTCAGATTT
TGTCTCCTAATCTTGCAACCCTTTGTGCCAAGAAAGGGCTTTTATTTGTTCCACCAAA
CATTGACAGAAGAACTGTGGAACTGCGGTTGGCAGACAATTTTGTTACAAATATTAAA
AGGAAAGATTTTGCCAATATGACCAGCTTGGTGGACCTGACTCTATCCAGGAATACAA
TAAGTTTTATTACACCTCATGCTTTCGCTGACCTACGAAATTTGAGGGCTTTGCATTT
GAATAGCAACAGATTGACTAAAATTACAAATGATATGTTCAGTGGTCTTTCCAATCTT
CATCATTTGATACTGAACAACAATCAGCTGACTTTAATTTCCTCTACAGCGTTTGATG
ATGTCTTCGCCCTTGAGGAGCTGGATCTGTCCTATAATAATCTAGAAACCATTCCTTG
GGATGCTGTTGAGAAGATGGTTAGCTTGCATACCCTTAGTTTGGATCACAATATGATT
GATAACATTCCTAAGGGGACCTTCTCCCATTTGCACAAGATGACTCGGTTAGATGTGA
CATCAAATAAATTGCAGAAGCTACCACCTGACCCTCTCTTTCAGCGAGCTCAGGTACT
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'Su::~ ~npt~ ~sut- u::w. c° A(a-.V... ~~ .
~AGCAACCTCAGGAATCATAAGCCCATCTACTTTTGCATTAAGTTTTGGTGGAAAC~CCC
TTGCATTGCAATTGTGAATTGTTGTGGTTGAGGCGTCTGTCCAGAGAAGATGACTTAG
AGACCTGTGCTTCTCCTCCACTTTTAACTGGCCGCTACTTTTGGTCAATTCCTGAAGA
AGAGTTTTTGTGTGAGCCTCCTCTCATTACTCGTCATACACATGAGATGAGAGTCCTG
GAGGGACAAAGGGCAACACTGAGGTGCAAAGCCAGGGGAGACCCTGAGCCTGCAATTC
ACTGGATTTCTCCTGAAGGGAAGCTTATTTCAAATGCAACAAGATCTCTGGTGTATGA
TAACGGAACACTTGACATTCTTATCACAACTGTAAAGGATACAGGTGCTTTTACCTGC
ATTGCTTCCAATCCTGCTGGGGAAGCAACACAAATAGTGGATCTTCATATAATTAAGC
TCCCTCACTTACTAAATAGTACAAACCATATCCATGAGCCTGATCCTGGTTCTTCAGA
TATCTCAACTTCTACCAAGTCAGGTTCTAATACAAGCAGTAGTAATGGTGATACTAAA
TTGAGTCAAGATAAAATTGTGGTGGCAGAAGCTACATCATCAACGGCACTACTTAAAT
TTAATTTTCAAAGAAATATCCCTGGAATACGTATGTTTCAAATCCAGTACAATGGTAC
TTATGATGACACCCTTGTTTACAGGATGATACCTCCTACGAGCAAAACTTTTCTGGTC
AATAATCTGGCTGCTGGAACTATGTATGACTTGTGTGTCTTGGCCATATATGATGATG
GCATCACTTCCCTCACTGCCACAAGAGTCGTGGGTTGCATCCAGTTTACTACGGAACA
GGATTATGTGCGTTGCCATTTCATGCAGTCCCAGTTTTTGGGAGGCACCATGATTATT
ATTATTGGTGGAATCATTGTAGCATCTGTGCTGGTATTCATCATTATTCTGATGATCC
GGTATAAGGTTTGCAACAATAATGGGCAACACAAGGTCACCAAGGTTAGCAATGTTTA
TTCCCAAACTAACGGGGCTCAAATACAAGGCTGTAGTGTAACGCTGCCCCAGTCCGTG
TCCAAACAAGCTGTGGGACACGAAGAGAATGCCCAGTGTTGTAAAGCTACCAGTGACA
ATGTGATTCAATCTTCAGAAACTTGTTCGAGTCAAGACTCCTCTACCACTACCTCTGC
TTTGCCTCCTTCCTGGACTTCAAGCACTTCTGTGTCCCAAAAGCAGAAAAGAAAGACT
GGCACAAAGCCAAGTACAGAACCACAGAATGAAGCCGTCACAAATGTTGAATCCCAAA
ACACTAACAGGAACAACTCAACTGCCTTGCAGTTAGCTAGCCGTCCTCCCGATTCTGT
CACAGAGGGGCCCACGTCTAAAAGAGCACATATAAAGCCAAGTAAGTTTATCACTTTG
CCTGCTGAGAGATCCGGAGCAAGGCACAAGTACTCCCTCAATGGAGAATTAAAGGAAT
ACTATTGTTATATTAACTCGCCGAACACATGTGGACTGTTTCCTAAAAGAAGCATGTC
TATGAATGTGATGTTTATTCAGTCTGACTGTTCTGATGGTCATAGTGGAAAGGCAACT
CTCAAATTCTGAGGGACTACTGGAAAGCTCTGTGTAATTTATAATTTCTTTTTCATGA
AAAATCATTTTGAGAACTCACATAGAAGATTGGAATTTGCAATTCCAATGCTGTGTAT
AAATCAACCTTCTCAGATGCTTTGCTGACTAATGTTGACCAGATTGTCCAGGAAAC
ORF Start: ATG at 380 ORF Stop: TGA at 2678
SEQ ID NO: 80 766 as MW at 84690.6kD
NOV3Oa, MEKILFYLFLIGIAVKAQICPKRCVCQILSPNLATLCAKKGLLFVPPNIDRRTVELRL
CG107S6Z-Ol ~NFVTNIKRKDFANMTSLVDLTLSRNTISFITPHAFADLRNLRALHLNSNRLTKITN
PrOtelri DMFSGLSNLHHLILNNNQLTLISSTAFDDVFALEELDLSYNNLETIPWDAVEKMVSLH
SeCItlBriCe
TLSLDHNMIDNIPKGTFSHLHKMTRLDVTSNKLQKLPPDPLFQRAQVLATSGIISPST
FALSFGGNPLHCNCELLWLRRLSREDDLETCASPPLLTGRYFWSIPEEEFLCEPPLIT
RFiTHEMRVLEGQRATLRCKARGDPEPAIHWISPEGKLISNATRSLVYDNGTLDILITT
VKDTGAFTCIASNPAGEATQIVDLHIIKLPHLLNSTNHIHEPDPGSSDISTSTKSGSN
TSSSNGDTKLSQDKIWAEATSSTALLKFNFQRNIPGIRMFQIQYNGTYDDTLVYRMI
PPTSKTFLVNNLAAGTMYDLCVLAIYDDGITSLTATRVVGCIQFTTEQDYVRCHFMQS
QFLGGTMIIIIGGIIVASVLVFIIILMIRYKVCNNNGQHKVTKVSNVYSQTNGAQIQG
CSVTLPQSVSKQAVGHEENAQCCKATSDNVIQSSETCSSQDSSTTTSALPPSWTSSTS
VSQKQKRKTGTKPSTEPQNEAVTNVESQNTNRNNSTALQLASRPPDSVTEGPTSKRAH
IKPSKFITLPAERSGARHKYSLNGELKEYYCYINSPNTCGLFPKRSMSMNVMFIQSDC
SDGHSGKATLKF
SEQ ID NO: 81 238_8 by
NOV3Ob, GCTCTTAAACCTGATCTACAATGGAAAAA.ATTCTTTTTTATCTGTTTCTCATTGGCAT
CGIO7S6Z-OZ AGCAGTGAAAGCTCAGATCTGTCCAAAGCGTTGTGTCTGTCAGATTTTGTCTCCTAAT
DNA Se LleriCeCTTGCAACCCTTTGTGCCAAGAAAGGGCTTTTATTTGTTCCACCAAACATTGACAGAA
GAACTGTGGAACTGCGGTTGGCAGACAATTTTGTTACAAATATTAAAAGGAAAGATTT
TGCCAATATGACCAGCTTGGTGGACCTGACTCTATCCAGGAATACAATAAGTTTTATT
ACACCTCATGCTTTCGCTGACCTACGAAATTTGAGGGCTTTGCATTTGAATAGCAACA
GATTGACTAAAATTACAAATGATATGTTCAGTGGTCTTTCCAATCTTCATCATTTGAT
ACTGAACAACAATCAGCTGACTTTAATTTCCTCTACAGCGTTTGATGATGTCTTCGCC
CTTGAGGAGCTGGATCTGTCCTATAATAATCTAGAAACCATTCCTTGGGATGCTGTTG
AGAAGATGGTTAGCTTGCATACCCTTAGTTTGGATCACAATATGATTGATAACATTCC
CA 02447935 2003-11-25
WO 02/099062 PCT/US02/17559
'tie , . f~~ .:nff ~ llux:i-~'-~lia'!
TAAGGGGACCTTCTCCCATTTGCACAAGATGACTCGGTTAGATGTGACATCAAATAAA
TTGCAGAAGCTACCACCTGACCCTCTCTTTCAGCGAGCTCAGGTACTAGCAACCTCAG
GAATCATAAGCCCATCTACTTTTGCATTAAGTTTTGGTGGAAACCCCTTGCATTGCAA
TTGTGAATTGTTGTGGTTGAGGCGTCTGTCCAGAGAAGATGACTTAGAGACCTGTGCT
TCTCCTCCACTTTTAACTGGCCGCTACTTTTGGTCAATTCCTGAAGAAGAGTTTTTGT
GTGAGCCTCCTCTCATTACTCGTCATACACATGAGATGAGAGTCCTGGAGGGACAAAG
GGCAACACTGAGGTGCAAAGCCAGGGGAGACCCTGAGCCTGCAATTCACTGGATTTCT
CCTGAAGGGAAGCTTATTTCAAATGCAACAAGATCTCTGGTGTATGATAACGGAACAC
TTGACATTCTTATCACAACTGTAAAGGATACAGGTGCTTTTACCTGCATTGCTTCCAA
TCCTGCTGGGGAAGCAACACAAATAGTGGATCTTCATATAATTAAGCTCCCTCACTTA
CTAAATAGTACAAACCATATCCATGAGCCTGATCCTGGTTCTTCAGATATCTCAACTT
CTACCAAGTCAGGTTCTAATACAAGCAGTAGTAATGGTGATACTAAATTGAGTCAAGA
TAAAATTGTGGTGGCAGAAGCTACATCATCAACGGCACTACTTAACTTTACTTTTCAA
AGAACTATCCCTGGAATACGTATGTTTCAAATCCAGTACAATGGTACTTATGATGACA
CCCTTGTTTACAGGATGATACCTCCTACGAGCAAAACTTTTCTGGTCAATAATCTGGC
TGCTGGAACTATGTATGACTTGTGTGTCTTGGCCATATATGATGATGGCATCACTTCC
CTCACTGCCACAAGAGTCGTGGGTTGCATCCAGTTTACTACGGAACAGGATTATGTGC
GTTGCCATTTCATGCAGTCCCAGTTTTTGGGAGGCACCATGATTATTATTATTGGTGG
AATCATTGTAGCATCTGTGCTGGTATTCATCATTATTCTGATGATCCGGTATAAGGTT
TGCAACAATAATGGGCAACACAAGGTCACCAAGGTTAGCAATGTTTATTCCCAAACTA
ACGGGGCTCAAATACAAGGCTGTAGTGTAACGCTGCCCCAGTCCGTGTCCAAACAAGC
TGTGGGACACGAAGAGATTGCCCAGTGTTGTAAAGCTACCAGTGACAATGTGATTCAA
TCTTCAGAAACTTGTTCGAGTCAGGACTCCTCTACCACTACCTCTGCTTTGCCTCCTT
CCTGGACTTCAAGCACTTCTGTGTCCCAAAAGCAGAAAAGAAAGACTGGCACAAAGCC
AAGTACAGAACCACAGAATGAAGCCGTCACAAATGTTGAATCCCAAAACACTAACAGG
AACAACTCAACTGCCTTGCAGTTAGCTAGCCGTCCTCCCGATTCTGTCACAGAGGGGC
CCACGTCTAAAAGAGCACATATAAAGCCAAATGCTTTGCTGACTAATGTTGACCAGAT
TGTCCAGGAAACACAGAGGCTGGAGTTAATCTGAAGAGCACCACTTCTCCTCTCTCTC
CTGAAAAAATTTGCCACTGATATTTTTACTGGATAAAATTCAAAAATGTTTCAATTCA
CAAAGGCTAA'T'TGTTGAACTGGTGTCGTAGAAGAAATTGTCTACAGGAGCCAAGGTGA
AAGTCTCTGATGACGGCGGAACTGGCTCCATTAGACCATGGTTCATCCTCTTTTAAAA
ACAAATTTTT
ORF Start: ATG at 21 ORF Stop: TGA at 2178
SEQ ID NO: 82 719 as MW at 79402.7kD
NOV3Ob, MEKILFYLFLIGIAVKAQICPKRCVCQILSPNLATLCAKKGLLFVPPNIDRRTVELRL
CG107562-02 ~N~TNIKRKDFANMTSLVDLTLSRNTISFITPHAFADLRNLRALHLNSNRLTKITN
Protein S8C1l1eriCeDMFSGLSNLHHLILNNNQLTLISSTAFDDVFALEELDLSYNNLETIPWDAVEKMVSLH
TLSLDHNMIDNIPKGTFSHLHKMTRLDVTSNKLQKLPPDPLFQRAQVLATSGIISPST
FALSFGGNPLHCNCELLWLRRLSREDDLETCASPPLLTGRYFWSIPEEEFLCEPPLIT
RHTHEMRVLEGQRATLRCKARGDPEPAIHWISPEGKLISNATRSLVYDNGTLDILITT
VKDTGAFTCIASNPAGEATQIVDLHIIKLPHLLNSTNHIHEPDPGSSDISTSTKSGSN
TSSSNGDTKLSQDKIWAEATSSTALLNFTFQRTIPGIRMFQIQYNGTYDDTLVYRMI
PPTSKTFLVNNLAAGTMYDLCVLAIYDDGITSLTATRVVGCIQFTTEQDYVRCHFMQS
QFLGGTMIIIIGGIIVASVLVFIIILMIRYKVCNNNGQHKVTKVSNVYSQTNGAQIQG
CSVTLPQSVSKQAVGHEEIAQCCKATSDNVIQSSETCSSQDSSTTTSALPPSWTSSTS
VSQKQKRKTGTKPSTEPQNEAVTNVESQNTNRNNSTALQLASRPPDSVTEGPTSKRAH
IKPNALLTNVDQIVQETQRLELI
SEQ ID NO: 83 1545 by
NOV3OC, GGATCCCAGATCTGTCCAAAGCGTTGTGTCTGTCAGATTTTGTCTCCTAATCTTGCAA
210086373 CCCTTTGTGCCAAGAAAGGGCTTTTATTTGTTCCACCAAACATTGACAGAAGAACTGT
DNA
GGAACTGCGGTTGGCAGACAATTTTGTTACAAATATTAAAAGGAAAGATTTTGCCAAT
S2qtleriCe
ATGACCAGCTTGGTGGACCTGACTCTATCCAGGAATACAATAAGTTTTATTACACCTC
ATGCTTTCGCTGACCTACGAAATTTGAGGGCTTTGCATTTGAATAGCAACAGATTGAC
TAA.AATTACAAATGATATGTTCAGTGGTCTTTCCAATCTTCATCATTTGATACTGAAC
AACAATCAGCTGACTTTAATTTCCTCTACAGCGTTTGATGATGTCTTCGCCCTTGAGG
AGCTGGATCTGTCCTATAATAATCTAGAAACCATTCCTTGGGATGCTGTTGAGAAGAT
GGTTAGCTTGCATACCCTTAGTTTGGATCACAATATGATTGATAACATTCCTAAGGGG
ACCTTCTCCCATTTGCACAAGATGACTCGGTTAGATGTGACATCAAATAAATTGCAGA
189
CA 02447935 2003-11-25
WO 02/099062 PCT/US02/17559
AGCTACCACCTGACCCTCTCTTTCAGCGAGCTCAGGTACTAGCAACCTCAGGAATCAT
AAGCCCATCTACTTTTGCATTAAGTTTTGGTGGAAACCCCTTGCATTGCAATTGTGAA
TTGTTGTGGTTGAGGCGTCTGTCCAGAGAAGATGACTTAGAGACCTGTGCTTCTCCTC
CACTTTTAACTGGCCGCTACTTTTGGTCAATTCCTGAAGAAGAGTTTTTGTGTGAGCC
TCCTCTCATTACTCGTCATACACATGAGATGAGAGTCCTGGAGGGACAAAGGGCAACA
CTGAGGTGCAAAGCCAGGGGAGACCCTGAGCCTGCAATTCACTGGATTTCTCCTGAAG
GGAAGCTTATTTCAAATGCAACAAGATCTCTGGTGTATGATAACGGAACACTTGACAT
TCTTATCACAACTGTAAAGGATACAGGTGCTTTTACCTGCATTGCTTCCAATCCTGCT
GGGGAAGCAACACAAATAGTGGATCTTCATATAATTAAGCTCCCTCACTTACTAAATA
GTACAAACCATATCCATGAGCCTGATCCTGGTTCTTCAGATATCTCAACTTCTACCAA
GTCAGGTTCTAATACAAGCAGTAGTAATGGTGATACTAAATTGAGTCAAGATAAAATT
GTGGTGGCAGAAGCTACATCATCAACGGCACTACTTAAATTTAATTTTCGAAGAAATA
TCCCTGGAATACGTATGTTTCAAATCCAGTACAATGGTACTTATGATGACACCCTTGT
TTACAGAATGATACCTCCTACGAGCAAAACTTTTCTGGTCAATAATCTGGCTGCTGGA
ACTATGTATGACTTGTGTGTCTTGGCCATATATGATGATGGCATCACTTCCCTCACTG
CCACAAGAGTCGTGGGTTGCATCCAGTTTACTACGGAACAGGATTATGTGCGTTGCCA
TTTCATGCAGTCCCAGTTTTTGGGAGGCACCCTCGAG
ORF Start: at 1 ORF Stop: end of sequence
SEQ ID NO: 84 515 as MW at 57372.8kD
NOV3OC, GSQICPKRCVCQILSPNLATLCAKKGLLFVPPNIDRRTVELRLADNFVTNIKRKDFAN
210086373 MTSLVDLTLSRNTISFITPHAFADLRNLRALHLNSNRLTKITNDMFSGLSNLHHLILN
Protein Sequence~QZ'TLISSTAFDDVFALEELDLSYNNLETIPWDAVEKWSLHTLSLDHNMIDNIPKG
TFSHLHKMTRLDVTSNKLQKLPPDPLFQRAQVLATSGIISPSTFALSFGGNPLHCNCE
LLWLRRLSREDDLETCASPPLLTGRYFWSIPEEEFLCEPPLITRHTHEMRVLEGQRAT
LRCKARGDPEPAIHWISPEGKLISNATRSLVYDNGTLDILITTVKDTGAFTCIASNPA
GEATQIVDLHIIKLPHLLNSTNHTHEPDPGSSDISTSTKSGSNTSSSNGDTKLSQDKI
WAEATSSTALLKFNFRRNIPGIRMFQIQYNGTYDDTLVYRMIPPTSKTFLVNNLAAG
TMYDLCVLAIYDDGITSLTATRWGCIQFTTEQDYVRCHFMQSQFLGGTLE
SEQ ID NO: 85 1545 by
NOV3Od, GGATCCCAGATCTGTCCAAAGCGTTGTGTCTGTCAGATTTTGTCTCCTAATCTTGCAA
210086403 CCCTTTGTGCCAAGAAAGGGCTTTTATTTGTTCCACCAAACATTGACAGAAGAACTGT
DNA
GGAACTGCGGTTGGCAGACAATTTTGTTACAAATATTAAAAGGAAAGATTTTGCCAAT
SeqLlenCe
ATGACCAGCTTGGTGGACCTGACTCTATCCAGGAATACAATAAGTTTTATTACACCTC
ATGCTTTCGCTGACCTACGAAATTTGAGGGCTTTGCATTTGAATAGCAACAGATTGAC
TAAAATTACAAATGATATGTTCAGTGGTCTTTCCAATCTTCATCATTTGATACTGAAC
AACAATCAGCTGACTTTAATTTCCTCTACAGCGTTTGATGATGTCTTCACCCTTGAGG
AGCTGGATCTGTCCTATAATAATCTAGAAACCATTCCTTGGGATGCTGTTGAGAAGAT
GGTTAGCTTGCATACCCTTAGTTTGGATCACAATATGATTGATAACA'I'TCCTAAGGGG
ACCTTCTCCCATTTGCACAAGATGACTCGGTTAGATGTGACATCAAATAAATTGCAGA
AGCTACCACCTGACCCTCTCTTTCAGCGAGCTCAGGTACTAGCAACCTCAGGAATCAT
AAGCCCATCTACTTTTGCATTAAGTTTTGGTGGAAACCCCTTGCATTGCAATTGTGAA
TTGTTGTGGTTGAGGCGTCTGTCCAGAGAAGATGACTTAGAGACCTGTGCTTCTCCTC
CACTTTTAACTGGCCGCTACTTTTGGTCAATTCCTGAAGAAGAGTTTTTGTGTGAGCC
TCCTCTCATTACTCGTCATACACATGAGATGAGAGTCCTGGAGGGACAAAGGGCAACA
CTGAGGTGCAAAGCCAGGGGAGACCCTGAGCCTGCAATTCACTGGATTTCTCCTGAAG
GGAAGCTTATTTCAAATGCAACAAGATCTCTGGTGTATGATAACGGAACACTTGACAT
TCTTATCACAACTGTAAAGGATACAGGTGCTTTTACCTGCATTGCTTCCAATCCTGCT
GGGGAAGCAACACAAATAGTGGATCTTCATATAATTAAGCTCCCTCACTTACTAAATA
GTACAAACCATATCCATGAGCCTGATCCTGGTTCTTCAGATATCTCAACTTCTACCAA
GTCAGGTTCTAATACAAGCAGTAGTAATGGTGATACTAAATTGAGTCAAGATAAAATT
GTGGTGGCAGAAGCTACATCATCAACGGCACTACTTAA.ATTTAATTTTCAAAGAAATA
TCCCTGGAATACGTATGTTTCAAATCCAGTACAATGGTACTTATGATGACACCCTTGT
TTACAGAATGATACCTCCTACGAGCAAAACTTTTCTGGTCAATAATCTGGCTGCTGGA
ACTATGTATGACTTGTGTGTCTTGGCCATATATGATGATGGCATCACTTCCCTCACTG
CCACAAGAGTCGTGGGTTGCATCCAGTTTACTACGGAACAGGATTATGTGCGTTGCCA
TTTCATGCAGTCCCAGTTTTTGGGAGGCACCCTCGAG
ORF Start: at I ~ ORF Stop: end of sequence
190
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
CONTENANT LES PAGES 1 A 190
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
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CONTAINING PAGES 1 TO 190
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