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CA 02446437 2003-11-07
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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.
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,
CA 02446437 2003-11-07
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such signaling pathways involve extracellular 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 patlr6lagies 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.
Antibodies are multichain proteins that bind specifically to a given antigen,
arid
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
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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.
S 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 further
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 palypeptide comprisi.»b a
:::~t~,:.re
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
15% of the amino acid residues in the sequence are so changed. The 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
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' 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 determining 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 of the 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
prophylaetically-
effective amount of this pharmaceutical composition.
In still another aspect, the invention provides the use of a therapeutic in
the
ar!anufacture of a medicament for treating a syndrome associated :vi~h a
h~amar. dise~ ~~:
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
antibody that binds the NOVX polypeptide in an amount sufficient to modulate
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
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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 1 and 46, 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 polypPptide. The NOVX antibody may be monoclonal, humanized, or a _ful_ly
human antibody. Preferably, the antibody has a dissociatdor: crustant fo: ~~:e
~u.ding 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.
In a further aspect, the invention provides for the use of a therapeutic 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.
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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.
TABLE 1. NOVX Polynucleotide and Polypeptide Sequences and
Corresponding SEQ ID Numbers
SEQ
ID
Internal Identificationn ep Homology
O e
Assignment (n (po p
c tid
leic
acid
1 1 2 HYALURONIC ACID
CG100051-02 RECEPTOR like, homo
sa iens
2a 3 4 fibronectin-malate
dehydrogenase like,
homo
CG100104-01 sa iens
2b 5 6 fibronectin-malate
. dehydrogenase like,
homo
198362674 sa iens
2c 7 8 fibronectin-malate
dehydrogenase like,
homo
198362686 sa iens
3 CG100114-01 9 10 JUNCTION ADHESION
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MOLECULE
4a 11 12 Leucine Rich Repeat
Membrane
CG100619-01 ' Protein like, homo
sa lens
4b 13 14 Leucine Rich Repeat
Membrane
210168777 Protein like, homo
sa lens
15 16 GTP:AMP phosphotransferase
CG56785-01 mitochondrial like,
homo sa lens
6a 17 18 Thrombospondin like,
homo
CG56914-01 sa lens
6b CG56914-02 19 20 Fibuiin tike, homo
sa lens
7 CG57242-01 21 22 KIAA0900 like, homo
sa lens
8a 23 24 Complement Decay-
Accelerating Factor
like, homo
CG57279-02 sa lens
8b 25 26 Complement Decay-
Accelerating Factor
like, homo
CG57279-04 sa lens
8c 27 28 . Complement Decay-
Accelerating Factor
like, homo
CG57279-05 sa lens
8d 29 30 Complement Decay-
Accelerating Factor
like, homo
175070639 sa lens
9 31 32 MHC CLASS I ANTIGEN
like,
CG94630-01 homo sa lens
10a CG94831-01 33 34 Tetras an-2 like, homo
sa lens
IOb CG94831-02 35 36 Tetras an-2 like, homo
sa lens
11 37 38 CUB domain containing
membrane protein like,
homo
CG94892-01 sa lens
12a 39 40 Collagen alpha 2 (VIII)
chain
CG95227-01 like, homo sa lens
12b 41 42 Collagen alpha 2 (VIN)
chain
CG95227-02 tike, homo sa lens
13a CG96384-01 43 44 Plasma Membrane Protein
13b CG96384-02 45 46 Plasma Membrane Protein
13c 209749131 47 48 Plasma Membrane Protein
13d 209749030 49 50 Plasma Membrane Protein
14 ( 51 52 ~ SodiumlHydrogen Exchanger
CG96432-01 like, ( Fomo sa i2i
~s
15a CG96545-02 53 54 EPHRIN-A5 PRECURSOR
ISb CG96545-03 55 56 EPHRiN-A5 PRECURSOR
16 57 58 BENZODIAZEPINE RECEPTOR
CG97101-01 RELATED like, homo
sa lens
17 59 60 ATP-Binding Cassette
CG97168-01 trans orter A like,
homo sa lens
18a 61 62 MAGE-domain Containing
like,
CG97420-01 homo sa lens
18b 63 64 MADE-domain Containing
like,
CG97420-02 homo sa lens
19a 65 66 collagen and scavenger
receptor
CG97430-01 domain like, homo sa
lens
19b 67 68 collagen and scavenger
receptor
CG97430-02 domain like, homo sa
lens
20a 69 70 CUB domain-containing
like,
CG97440-01 homo sa lens
20b 71 72 CUB domain-containing
tike,
199652779 homo sa lens
21 CG97451-01 73 74 GI cine-rich membrane
rotein
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like, homo sa iens
22a CG97852-01 75 76 atectin 9 tike, homo
sa iens
22b CG97852-03 77 78 alectin 9 like, homo
sa iens
23a 79 80 T Cetl Surface Glycoprotein
CG99575-01 CD1
like, homo sa iens
23b 81 82 T Cell Surface Glycoprotein
CG99575-02 CD1
like, homo sa iens
24 83 84 1110002C08RtK PROTEIN
CG99608-01 tike,
homo sa iens
25 85 86 EPITHELIAL V LIKE,
CG99674-01 ANTIGEN
PRECURSOR
26a CG99732-02 87 88 MACROPHAGE LECTIN 2
26b CG99732-03 89 90 MACROPHAGE LECTIN 2
27 CG99767-01 9~ 92 . a I membrane rotein
Table 1 indicates the homology of NOVX polypeptides to 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 1, 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
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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
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.
The NOVX genes and their corresponding encoded proteins are useful for
preventing, treating or ameliorating medical conditions, e.g., by protein or
gene therapy.
I 5 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 1 and 46; (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
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between 1 and 46, wherein any amino acid in the mature form is changed to a
different
amino acid, provided that no more than 1 S% 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: 2n, wherein n is an integer between 1 and 46; (d) a
variant of the
S amino acid sequence selected from the group consisting of SEQ ID N0:2n,
wherein n is an
integer between 1 and 46 wherein any amino acid specified in the chosen
sequence is
changed to a different amino acid, provided that no more than 1 S% 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
acid sequence selected from the group consisting of (a) a mature form of the
amino acid
sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 46; (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 46 wherein any amino acid in the
mature
1 S form of the chosen sequence is changed to a different amino acid, provided
that no more
than 1 S% 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 l and 46; (d) a variant of the amino acid sequence
selected from the
group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 46,
in which
any amino acid specified in the chosen sequence is changed to a different
amino acid,
provided that no more than 1 S% of the amino acid residues in the sequence are
so changed;
(e) a nucleic acid fragment encoding at least a portion of a nolypeptide
comprising the
amino acid sequence selected from the group consisting of S>~:Q _Tn NC)' Vin,
Wherein n ;~ an
integer between 1 and 46 or any variant of said polypeptide wherein any amino
acid of the
2S 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-1, wherein n is an integer between 1 and.46; (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
1 and 46
is changed from that selected from the group consisting of the chosen sequence
to a
CA 02446437 2003-11-07
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different nucleotide provided that no more than I S% 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 46; 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 1 and 46 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.
NOVX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that
encode
NOVX polypeptides or biologically active portions thereof. Also included in
the invention
are nucleic acid fragments sufficient for use as hybridization probes to
identify NOVX-
encoding nucleic acids (e.g., NOVX 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
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 occurnng polypeptide or precursor form or proprotein. The
naturally
occurring polypeptide, precursor or proprotein ineludc-s~, 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 I 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
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having residues I 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.
The term "probes", as utilized herein, refers to nucleic acid sequences of
variable
length, preferably between at least about 10 nucleotides (nt), 100 nt, or as
many as
approximately, e.g., 6,000 nt, depending upon the specific use. Probes are
used in the
detection of identical, similar, or complementary nucleic acid sequences.
Longer length
probes are generally obtained from a natural or recombinant source, are highly
specific, and
much slower to hybridize than shorter-length oligomer probes. Probes may be
single- or
double-stranded and designed to have specificity in PCR, membrane-based
hybridization
technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one, which
is
separated from other nucleic acid molecules which are present in the natural
source of the
nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences
which naturally
flank the nucleic acid (i. e., sequences located at the 5'- and 3'-termini of
the nucleic acid) in
the genomic DNA of the organism from which the nucleic acid is derived. For
example, in
various embodiments, the isolated NOVX nucleic acid molecules can contain less
than
about 5 kb, 4 kb, 3 kb, 2 kb, I kb9 0.5 kb or 0.1 kb of nucleotide sequences
which naturally
fl_ auk the nucleic acid molecule in genomic DNA of the cPl1/tiss»e from which
the nT~ele~e
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 1-46,
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 N0:2n-1, wherein n is an
integer between
1-46, as a hybridization probe, NOVX molecules can be isolated using standard
hybridization and cloning techniques (e.g., as described in Sambrook, et al.,
(eds.),
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MOLECULAR CLONING: A LABORATORY MANUAL 2°d Ed., Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY', John Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers
according to standard PCR amplification techniques. The nucleic acid so
amplified can be
cloned into an appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can
be
prepared by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a
PCR reaction. A short oligonucleotide sequence may be based on, or designed
from, a
genomie or eDNA 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 nt in length. In one embodiment
of the
invention, an oligonucleotide comprising a nucleic acid molecule less than 100
nt in length
would further comprise at least 6 contiguous nucleotides of SEQ ID N0:2n-1,
wherein n is
an integer between 1-46, or a complement thereof. Oligonucleotides may be
chemically
synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a nucleic acid molecule that is a complement of the nucleotide
sequence SEQ ID
N0:2n-l, wherein n is an integer between 1-46, or a portion nftl~is
_n_ncleot~d~Pq"PnCe
(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-l, wherein n is an
integer
between 1-46, is one that is su~ciently complementary to the nucleotide
sequence of SEQ
ID NO:2n-l, wherein n is an integer between 1-46, that it can hydrogen bond
with little or
no mismatches to the nucleotide sequence of SEQ ID N0:2n-l, wherein n is an
integer
between 1-46, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base pairing between nucleotides units of a nucleic acid molecule, and the
term "binding"
means the physical or chemical interaction between two polypeptides or
compounds or
associated polypeptides or compounds or combinations thereof. Binding includes
ionic,
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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
S without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous)
nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to
allow for specific
hybridization in the case of nucleic acids or for specific recognition of an
epitope in the
case of amino acids, respectively, and are at most some portion less than a
full length
sequence. Fragments may be derived from any contiguous portion of a nucleic
acid or
amino acid sequence of choice. Derivatives are nucleic acid sequences or amino
acid
sequences formed from the native compounds either directly or by modification
or partial.
substitution. Analogs are nucleic acid sequences or amino acid sequences that
have a
structure similar to, but not identical to, the native compound but differs
from it in respect
1 S 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
codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence
lacking an
ATG start codon therefore encodes a truncated C-terminal fragment of the
respective
NOVX polypeptide, and requires that the corresponding full-length cDNA extend
in the S'
direction of the disclosed sequence. Any disclosed NOVA nucleotide sequence
lacking an
in-frame stop codon similarly encodes a truncated N-terminal fragment of the
respective
2S 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 9S%
identity (with a preferred identity of 80-9S%) 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
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WO 02/090504 PCT/US02/14342
of hybridizing to the complement of a sequence encoding the aforementioned
proteins
under stringent, moderately stringent, or low stringent conditions. See e.g.
Ausubel, et al.,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993,
and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed.above. Homologous nucleotide sequences encode
those
sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed
in
different tissues of the same organism as a result of, for example,
alternative splicing of
RNA. Alternatively, isoforms can be encoded by different genes. In the
invention,
homologous nucleotide sequences include nucleotide sequences encoding for 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
N0:2n-I, wherein n is an integer between 1-46, as well as a polypeptide
possessing NOVX
biological activity. Various biological activities of the NOVX proteins are
described
below.
A NOVX polvpeptide 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 OR,F
is
uninterrupted by a stop colon. An ORF that represents the coding sequence for
a full
protein begins with an ATG "start" colon and terminates with one of the three
"stop"
colons, namely, TAA, TAG, or TGA. For the purposes of this invention, an OR.F
may be
any part of a coding sequence, with or without a start colon, a stop colon, 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 o$en 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 andlor
cloning NOVX homologues in other cell types, e.g. from other tissues, as well
as NOVX
CA 02446437 2003-11-07
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homologues from other vertebrates. The p~obe/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 N0:2n-
l, wherein n is an integer between 1-46; or an anti-sense strand nucleotide
sequence of
SEQ >D N0:2n-1, wherein n is an integer between 1-46; or of a naturally
occurring mutant
of SEQ ID N0:2n-1, wherein n is an integer between 1-46.
Probes based on the human NOVX nucleotide sequences can be used to detect
transcripts or genomic sequences encoding the same or homologous proteins. In
various
embodiments, the probe further comprises a label group attached thereto, e.g.
the label
group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor.
Such probes can be used as a part of a diagnostic test kit for identifying
cells or tissues
which mis-express 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 1-46, that encodes a polypeptide having a NOVX
biological activity (the biological activities of the NOVX proteins are
described below),
ea~pressing the encoded portion of NOVX protein (e.g., by recombinant
expression ~ 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
nucleotide sequences of SEQ TD N0:2n-1, wherein n is an integer between 1-46,
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 n is an integer between
1-46. 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 N0:2n,
wherein n
is an integer between 1-46.
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In addition to the human NOVX nucleotide sequences of SEQ ID N0:2n-1, wherein
n is an integer between 1-46, 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
S 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
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
NO:2n-1, wherein n is an integer between 1-46, 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 sennPncP of SEQ _TD NO:2n-1,
wherein n
is an integer between 1-46. 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.
Fiomologs (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.
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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
I 0 excess, at Tm, 50% of the probes are occupied at equilibrium. Typically,
stringent
conditions will be those in which the salt concentration is less than about
1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion (or other salts) at 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 B10LOGY, john Wiley &
Sons,
N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences
at least about
65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically
remain
hybridized to each other. A non-limiting example of stringent hybridization
conditions are
hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, (1.52% WSA, and 500 mg/ml denal,~rerl salmon
sne,_m
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-l, wherein n is an integer between 1-
46,
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-1, wherein n
is an
integer between I-46, or fragments, analogs or derivatives thereof, under
conditions of
moderate stringency is provided. A non-limiting example of moderate stringency
hybridization conditions are hybridization in 6X SSC, SX Reinhardt's solution,
0.5% SDS
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and 100 mg/ml denatured salmon sperm DNA at SS °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, 1990; GENE TRANSFER
S 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 N0:2n-l, wherein n is
an integer
between I-46, or fragments, analogs or derivatives thereof, under conditions
of low ,
stringency, is provided: A non-limiting example of low stringency
hybridization conditions
are hybridization in 3S% formamide, SX SSC, SO mM Tris-HCI (pH 7.S), S mM
EDTA,
0.02% PVP, 0.02% Ficoll, 0.2°/ BSA, I00 mglml denatured salmon sperm
DNA, 10%
(wt/volt) dextran sulfate at 40 °C, followed by one or more washes in
2X SSC, 2S mM
Tris-HCl (pH 7.4), S mM EDTA, and 0.1% SDS at SO °C. Other conditions
of low
stringency that may be used are well known in the art (e.g., as employed for
cross-species
1 S hybridizations). See, e.g., Ausubel, et al. (eds.),1993, CURRENT PROTOCOLS
IN
MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER
AND
ElIPRESSIQN, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg,
1981.
Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may
exist in the population, the skilled artisan will further appreciate that
changes can be
introduced by mutation into the nucleotide sequence~~~of SEQ ID N0:2r_-J ,
WhPrP;" ~ is ~
integer between 1-46, thereby leading to changes in the amino acid sequences
of the
2S 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 NO:2n, wherein n is
an integer
between 1-46. 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.
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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:2n-I,
wherein
n is an integer between 1-46, yet retain biological activity. In one
embodiment, the isolated
S 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 N0:2n, wherein n is an integer between 1-46.
Preferably, the
protein encoded by the nucleic acid molecule is at least about 60% homologous
to SEQ ID
N0:2n, wherein n is an integer between 1-46; more preferably at least about
70%
homologous to SEQ ID N0:2n, wherein n is an integer between 1-46; still more
preferably
at least about 80% homologous to SEQ ID N0:2n, wherein n is an integer between
1-46;
even more preferably at least about 90% homologous to SEQ ID NO:Zn, wherein n
is an
integer between 1-46; and most preferably at least about 95% homologous to SEQ
ID
N0:2n, wherein n is an integer between 1-46.
An isolated nucleic acid molecule encoding a NOVX protein homologous to the
protein of SEQ ID N0:2n, wherein n is an integer between 1-46, 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 1-46, 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 NO:2n-1, wherein n is an
integer
between 1-46, 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 a=mino acid
substitution" i~
one in which the amino acid residue is replaced with an amino acid residue
having a similar
side chain. Families of amino acid residues having similar side chains have
been defined
within the art. These families include amino acids with basic side chains
(e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid),
uncharged polar
side chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a
predicted non-essential amino acid residue in the NOVX .protein is replaced
with another
amino acid residue from the same side chain family. Alternatively, in another
embodiment,
mutations can be introduced randomly along all or part of a NOVX coding
sequence, such
CA 02446437 2003-11-07
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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 N0:2n-I, wherein n is an integer between 1-46, 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
may be substituted for each other. Likewise, the "weak" group of conserved
residues may
be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK,
NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the
single
letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to
form protein:protein interactions with other NOVX proteins, other cell-surface
proteins, or
biologically-active portions thereof, (ii) complex formation between a mutant
NOVX
protein and 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 N0:2n-1, wherein n is an integer between 1-46,
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,
S0, 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 n is an integer between 1-46, or
antisense
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nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID N0:2n-1,
wherein n is an integer between 1-46, 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).
Given the coding strand sequences encoding the NOVX protein disclosed herein,
antisense nucleic acids of the invention can be designed according to the
rules of Watson
and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can
be
complementary to the entire coding region of NOVX mRNA, but more preferably is
an
oligonucleotide that is antisense to only a portion of the coding or noncoding
region of
NOVX mRNA. For example, the antisense oligonucleotide can be complementary to
the
region surrounding the translation start site of NOVX mRNA. An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45
or 50
nucleotides in length. An antisense nucleic acid of the invention can be
constructed using
chemical synthesis or enzymatic ligation reactions using procedures known in
the art. For
example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically
synthesized using naturally-occurring nucleotides or variously modif ed
nucleotides
designed to increase the biological stability of the molecules or to increase
the physicaa
stability of the duplex formed between the antisense and sense nucleic acids
(e.g.,
phosphorothioate derivatives and acridine substituted nucleotides can be
used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: S-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil,
dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, l-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine,
S-methylaminomethyluracil, S-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
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WO 02/090504 PCT/US02/14342
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-
thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid
(v),
5-methyl-2-thiouracil, 3-(3-amino-3 N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced
biologically
using an expression vector into which a nucleic acid has been subcloned in an
antisense
orientation (i. e., RNA transcribed from the inserted nucleic acid will be of
an antisense
orientation to a target nucleic acid of interest, described further in the
following
subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated in situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding a 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 de1_ivered to cells using the vectors described
herein. To ach;PVe
sufficient nucleic acid molecules, vector constructs in which the antisense
nucleic acid
molecule is placed under the control of a strong pot II or pol III promoter
are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is
an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
(3-units,
the strands run parallel to each other. See, e.g., Gaultier, et al., 1987.
Nucl. Acids Res. 15:
6625-6641. The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (See, e.g., moue, et al. 1987. Nuel. Acids Res. 15:
6131-6148) or
a chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987. FEBSLett. 215: 327-
330.
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Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified
bases, and nucleic acids whose sugar phosphate backbones are modified or
derivatized.
These modifications are carned out at least in part to enhance the chemical
stability of the
modified nucleic acid, such that they may be used, for example, as antisense
binding
nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activiTy that are
capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described
in
Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically
cleave
NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme
having specificity for 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
NO:Zn-l,
wherein n is an integer between 1-46). For example, a derivative of a
Tetrahymena L-19
IVS RNA can be constructed in which the nucleotide sequence of the active site
is
complementary to the nucleotide sequence to be cleaved in a 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 rev?gin 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.
Anticaneer Drug
Des. 6: 569-84; Helene, et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher,
1992.
Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization,
or solubility of the molecule. For example, the deoxyribose phosphate backbone
of the
nucleic acids can be modified to generate peptide nucleic acids. See, e.g.,
Hyrup, et al.,
1996. Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic
acids" or
"PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in which the
deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only the four
natural
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WO 02/090504 PCT/US02/14342
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'Keefe, et al.,
1996. Proc. Natl.
S Acad. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For
example, PNAs can be used as antisense or antigens agents for sequence-
specific
modulation of gene expression by, e.g., inducing transcription or translation
arrest or
inhibiting replication. PNAs of NOVX can also be used, for example, in the
analysis of
single base pair mutations in a gene (e.g., PNA directed PCR clamping; as
artificial
restriction enzymes when used in combination with other enzymes, e.g., Sl
nucleases (See,
Hyrup, et al., I996.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
1 S stability or cellular uptake, by attaching lipophilic or other helper
groups to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of NOVX can be
generated
that may combine the advantageous properties of PNA and DNA. Such chimeras
allow
DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with
the DNA
portion while the PNA portion would provide high binding affinity and
specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths selected
in terms of
base stacking, number of bonds between the nucleotide teases, and orientation
(see, Hyrup,
et al., 1996. supra). The syntfi-esis of PNA-DNA ch?t?-!Pras ran be performed
as describPri
in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-
3363. For
2S example, a DNA chain can be synthesized on a solid support using standard
phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g.,
S'-(4-methoxytrityl)amino-S'-deoxy-thymidine phosphoramidite, cari be used
between the
PNA and the S' 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 S' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra.
Alternatively, chimeric molecules can be synthesized with a S' DNA segment and
a 3' PNA
segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lets. S: 1119-
11124.
In other embodiments, the oligonucleotide may include other appended groups
such
as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport
2S
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WO 02/090504 PCT/US02/14342
across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acad. Sci. U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In
addition, oligonucleotides can be modified with hybridization triggered
cleavage agents
S (see, e.g., Krol, et a1.,1988. BioTechniques 6:958-976) or intercalating
agents (see, e.g.,
Zon, 1988. Pharm. Res. S: 539-549). To this end, the oligonucleotide may be
conjugated to
another molecule, e.g., a peptide, a hybridization triggered cross-linking
agent, a transport
agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino acid sequence of NOVX polypeptides whose sequences are provided in any
one of
SEQ ID N0:2n, wherein n is an integer between 1-46. 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-
46, while
1 S still encoding a protein that maintains its NOVX activities and
physiological functions, or a
functional fragment 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 fi.wther include the possibility of inserting an additional
residue or
residues between taro 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-
2S active portions thereof, or derivatives, fragments, analogs or homologs
thereof. Also
provided are polypeptide fragments suitable for use as immunogens to raise
anti-NOVX
antibodies. In one embodiment, native NOVX proteins can be isolated from cells
or tissue
sources by an appropriate purification scheme using standard protein
purification
techniques. In another embodiment, NOVX proteins are produced by recombinant
DNA
techniques. Alternative to recombinant expression, 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
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WO 02/090504 PCT/US02/14342
cell or tissue source from which the NOVX protein is derived, or substantially
free from
chemical precursors or other chemicals when chemically synthesized. The
language
"substantially free of cellular material" includes preparations of NOVX
proteins in which
the protein is separated from cellular components of the cells from which it
is isolated or
recombinantly-produced. In one embodiment, the language "substantially free of
cellular
material" includes preparations of NOVX proteins having less than about 30%
(by dry
weight) of non-NOVX proteins (also referred to herein as a "contaminating
protein"), more
preferably less than about 20% of non-NOVX proteins, still more preferably
less than about
10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX
proteins. When the NOVX protein or biologically-active portion thereof is
recombinantly-
produced, it is also preferably substantially free of culture medium, i.e.,
culture medium
represents less than about 20%, more preferably less than about 10%, and most
preferably
less than about 5% of the volume of the NOVX protein preparation.
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 e~mYr_cing
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 1-46) that include fewer amino acids than the full-length NOVX
proteins, and
exhibit at least one activity of a NOVX protein. Typically, biologically-
active port' ions
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.
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WO 02/090504 PCT/US02/14342
In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID
N0:2n, wherein n is an integer between 1-46. In other embodiments, the NOVX
protein is
substantially homologous to SEQ ID NO:2n, wherein n is an integer between 1-
46, and
retains the functional activity of the protein of SEQ ID N0:2n, wherein n is
an integer
between 1-46, 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
1-46, and retains~the functional activity of the NOVX proteins of SEQ ID
N0:2n, wherein
n is an integer between 1-46.
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
]der~t,_ty
between _ __.two sequences. The homology may be determined using computer
r_rog_ramc
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 n is an integer between 1-46.
The team "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
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WO 02/090504 PCT/US02/14342
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I,
in the case of
nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing
the number of matched positions by the total number of positions in the region
of
comparison (i. e., the window size), and multiplying the result by 100 to
yield the
percentage of sequence identity. The term "substantial identity" as used
herein denotes a
characteristic of a polynucleotide sequence, wherein the polynucleotide
comprises a
sequence that has at least g0 percent sequence identity, preferably at least
85 percent
identity and often 90 to 95 percent sequence identity, more usually at least
99 percent
sequence identity as compared to a reference sequence over a comparison
region.
Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein,
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 1-46, 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 iwo biologically-active portions of a NOVX prote~_ pn~yet
~nnther
embodiment, a NOVX fusion protein comprises at least 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 are fused to the C-terminus of the GST (glutathione S-
transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
NOVX
polypeptides.
In another embodiment, the fusion protein is a NOVX protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host
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WO 02/090504 PCT/US02/14342
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 are fused to sequences derived from a
member of
the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of
the
invention can be incorporated into pharmaceutical compositions and
administered to a
subject to inhibit an interaction between a NOVX ligand and a NOVX protein on
the
surface of a cell, to thereby suppress NOVX-mediated signal transduction in
vivo. The
NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability
of 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-immunoglobuIin 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
liga+.lon. In another embodiment, the fusion gene car+ be synthesized by
conventional
techniques including automated DNA synthesizers. Alternatively, FC..R
_amplification ~f
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.
NOVX 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
CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
can be generated by mutagenesis (e.g., discrete point mutation or truncation
of the NOVX
protein). An agonist of the NOVX protein can retain substantially the same, or
a subset of,
the biological activities of the naturally occurring form of the NOVX protein.
An
antagonist of the NOVX protein can inhibit one or more of the activities of
the naturally
occurring form of the NOVX protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which includes
the NOVX
protein. Thus, specific biological effects can be elicited by treatment with a
variant of
limited function. In one embodiment, treatment of a subject with a variant
having a subset
of the biological activities of the naturally occurring form of the protein
has fewer side
effects in a subject relative to treatment with the naturally occurnng 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 t:r .produce libraries of potential NOVX ~~a.~iants from a
degenerate
oligonucleotide sequer_ce_ ~'hP~nica~ synthesis of a degenerate gene sequence
can bP
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. A~nu. Rev.
Bioehem. 53: 323;
Itakura, et al., 1984. Science 198: 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
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WO 02/090504 PCT/US02/14342
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,
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 F(a6~2 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 IgG~, IgG2, and others.
Furthermore, in
humans, the light chain may be a kappa chain or a lambda chain. Reference
herein to
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WO 02/090504 PCT/US02/14342
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
immunospecificalIy bind the antigen, using standard techniques for polyclonal
and
monoclonal antibody preparation. The full-length protein can be used or,
alternatively, the
invention provides antigenic peptide fragments of the antigen for use as
immunogens. An
antigenic peptide fragment comprises at least 6 amino acid residues of the
amino acid
sequence of the full length protein, such as an amino acid sequence of SEQ ID
N0:2n,
wherein n is an integer between 1-46, and encompasses an epitope thereof such
that an
antibody raised against the peptide forms a specific immune complex with the
i~~1_i_ length
protein or with any fragment that contains the epitope. Preferably, the
antigenic peptide
comprises at least 10 amino acid residues, or at least 15 amino acid residues,
or at least 20
amino acid residues, or at least 30 amino acid residues. Preferred epitopes
encompassed by
the antigenic peptide are regions of the protein that are located on its
surface; commonly
these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of NOVX 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 hydrophiiicity and
hydrophobicity may be generated by any method well known in the art,
including, for
example, the Kyte Doolittle or the Hopp Woods methods, either with or without
Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc: Nat. Acad. Sci. USA 78:
3824-
3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated
herein by
reference in their entirety. Antibodies that are specific for one or more
domains within an
antigenic protein, or derivatives, fragments, analogs or homologs thereof, are
also provided
herein.
The term "epitope" includes any protein determinant capable of specific
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
33
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WO 02/090504 PCT/US02/14342
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 <_1 pM,
preferably <_
100 nM, more preferably <_ 10 nM, and most preferably <_ 100 pM to about 1 pM,
as
S measured by assays such as radioligand binding assays or similar assays
known to those
skilled in the art.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of
polyclonal or monoclonal antibodies directed against a protein of the
invention, or against
derivatives, fragments, analogs homologs or orthologs thereof (see, for
example,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring
Harbor .
Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).
Some of
1 S these antibodies are discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals
(e.g.,
rabbit, goat, mouse or other mammal) may be immunized by one or more
injections with
the native protein, a synthetic variant thereof, or a derivative of the
foregoing. An
appropriate immunogenic preparation can contain, for example, the naturally
occurring
immunogenic protein, a chemically synthesized polypeptide representing the
immunogenic
protein, or a recombinantly expressed imununogenic prote3:~. Furt~hernore, ~e
protei:~ may
be conjugated to a second protein known to be immunogenic in the mammal being
immunized. Examples of such immunogenic proteins include but are not limited
to
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin
inhibitor. The preparation can further include an adjuvant. Various adjuvants
used to
increase the immunological response include, but are not limited to, Freund's
(complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface active
substances (e.g.,
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium
parvum,
or similar immunostimulatory agents. Additional examples of adjuvants which
can be
employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
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The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the immunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for example, by
D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA,
Vol. 14, No. 8
(April 17, 2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs)
of the monoclonal antibody are identical in all the molecules of the
population. MAbs thus
contain an antigen binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding 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 i~: m~arized
with an
immunizing agent to elicit lymphocytes that produce or are capable of
rrouuci~~g 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 are desired. The lymphocytes are then fused with an
immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-
103). Immortalized cell lines are usually transformed mammalian cells,
particularly
myeloma cells of rodent, bovine and human origin. Usually, rat or mouse
myeloma cell
CA 02446437 2003-11-07
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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-def cient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell lines are marine
myeloma lines, which can be obtained, for instance, from the Salk Institute
Cell
Distribution Center, San Diego, California and the American Type Culture
Collection,
Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines
also
have been described for the production of human monoclonal antibodies
(I~ozbor, 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 PoIIard,
Anal, Biochem.,107:220 (1980). It is an objective, especiahy ~inportant r"
therape"t;c
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.
36
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The monoclonal antibodies can also be made by recombinant DNA methods, such
as those' described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies
of the invention can be readily isolated and sequenced using conventional
procedures (e.g.,
by using oligonucleotide probes that are capable of binding specifically to
genes encoding
the heavy and light chains of marine antibodies). The hybridoma cells of the
invention
serve as a preferred source of such DNA. Once isolated, the DNA can be placed
into
expression vectors, which are then transfected into host cells such as simian
COS cells,
Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce
irnmunoglobulin protein, to obtain the synthesis of monoclonal antibodies in
the
recombinant host cells. The DNA also can be modified, for example, by
substituting the
coding sequence for human heavy and light chain constant domains in place of
the
homologous marine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368,
812-13
(1994)) or by covalently joining to the immunoglobulin coding sequence all or
part of the
coding sequence for a non-immunoglobulin polypeptide. Such a non-
immunoglobulin
polypeptide can be substituted for the constant domains of an antibody of the
invention, or
can be substituted for the variable domains of one antigen-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 im_mt~~e response by the
h»xnan aga~net
the a~m?l~~StPred immunoglobulin. Humanized forms of antbodies arP crdmeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
F(ab'~ 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
37
CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
comprise substantially all of at least one, and typically two, variable
domains, in which all
or substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the framework regions are those
of a human
immunoglobulin consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
human immunoglobulin (Jones et al., 1986; Riechmann et a1.,1988; and Presta,
Curr. Op.
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 triorna technique;
the human
B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and
the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
Human
monoclonal antibodies may be utilized in the practice of the present invention
and may be
produced by using human hybridomas (see Cote, et a1.,1983. Proc Natl Acad Sci
USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro
(see Cole, et
2O al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. L1SS, Inc.,
pp:
77-96).
In addition, human antibodies can also be produced using additional
technictues,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in all respects, including gene rearrangement, assembly, and antibody
repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technology 10, 779-
783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature
368, 812-13
(1994)); Fishwild et al,( Nature Biotechnology 14, 845-SI (1996)); Neuberger
(Nature
38
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WO 02/090504 PCT/US02/14342
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
S animal's endogenous antibodies in response to challenge by an antigen. (See
PCT
publication W094/02602). The endogenous genes encoding. the heavy and light
immunoglobulin chains in the nonhuman host have been incapacitated, and active
loci
encoding human heavy and light chain immunoglobulins are inserted into the
host's
genome. The human genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal which
provides
all the desired modifications is then obtained as progeny by crossbreeding
intermediate
transgenic animals containing fewer than the full complement of the
modifications. The
preferred embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse~ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This
animal produces B cells which secrete fully human immunoglobulins. The
antibodies can
be obtained directly from the animal after immunization with an immunogen of
interest, as,
for example, a preparation of a polyclonal antibody, or alternatively from
immortalized B
cells derived from the animal, such as hybridomas producing monoclonal
antibodies.
Additionally, the genes encoding the immunoglobulins with human variable
regions can be
recovered and expressed to obtain the antibodies directly, or can be further
modified to
obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobuli_n heavy chain is disclosed
in T T.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
39
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WO 02/090504 PCT/US02/14342
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.
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 F~, fragments with the desired
specificity for a
protein or derivatives, fragments, analogs or homologs thereof. Antibody
fragments that
contain the idiotypes to a protein antigen may be produced by techniques known
in the art
including, but not limited to: (i) an F~ab~2 fragment produced by pepsin
digestion of an
antibody molecule; (ii) an Fab fragment generated by reducing the disulfide
bridges of an
F(ab72 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 spP~ificities 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
CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
93/08829, 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 immunoglobulin constant domain sequences. The
fusion ,
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least
part of the hinge, CH2, and CH3 regions. It is preferred to have the first
heavy-chain
constant region (CH1) containing the site necessary for light-chain binding
present in at
least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if
desired, the immunoglobulin light chain, are inserted into separate expression
vectors, and
are co-transfected into a suitable host organism. For further details of
generating bispecific
antibodies see, for example, Suresh et al., Methods. in Enzymology, 121:210
(1986).
According to another approach described in WO 96/27011, the interface between
a
pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
which are recovered from recombinant cell culture. The preferred interface
comprises at
least a part of the CH3 region of an antibody constant domain. In this method,
one or more
small amino acid side chains from the interface of the first antibody molecule
are replaced
with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical
or similar size to the large side chains) are created on the interface of the
second antibody
molecule by replacing Iarge 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'')a bispecific antibodies). Techniq~ae~ for generating
bispecific
antibodies from antibody fragments have been described in the literature. For
example,
bispecific antibodies can be prepared using chemical linkage. Brennan et al.,
Science
229:81 (1985) describe a procedure wherein intact antibodies are
proteolytically cleaved to
generate Flab'}a fragments. These fragments are reduced in the presence of the
dithiol
complexing agent sodium arsenite to stabilize vicinal dithiols and prevent
intermolecular
disulfide formation. The Fab' fragments generated are then converted to
thionitrobenzoate
(TNB). derivatives. One of the Fab'-TNB derivatives is then reconverted to the
Fab'-thiol
by reduction with mercaptoethylamine and is mixed with an equimolar amount of
the other
Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies
produced
can be used as agents for the selective immobilization of enzymes.
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Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')a
molecule. Each
Fab' fragment was separately secreted from E. coli and subjected to directed
chemical
coupling in vitro to form the bispecific antibody. The bispecific antibody
thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well
as trigger the lytic activity of human cytotoxic lymphocytes against human
breast tumor
targets.
Various techniques for making and isolating bispecific antibody fragments
directly
from recombinant cell culture have also been described. For example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553
(1992). The leucine zipper peptides from the Fos and Jun proteins were linked
to the Fab'
portions of two different antibodies by gene fusion. The antibody homodimers
were
reduced at the hinge region to, form monomers and then re-oxidized to form the
antibody
heterodimers. This method can also be utilized for the production of antibody
homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
USA
90:6444-6448 (1993) has provided an alternative mechanism for making
bispecific
antibody fragments. The fragments comprise a heavy-chain variable domain (VH)
connected to a light-chain variable domain (VL) by a linker which is too short
to allow
pairing between the two domains on the same chain. Accordingly, the VH and VL
domains
of one fragment are forced to pair with the complementary VL and VH domains of
another
fragment, thereby forming two antigen-binding sites. Another strategy for
making
bispecific antibody fragments by'the use of single-chain Ft. \yF,~) d;mPrS 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 (Fc~yR), such as FcyRI (CD64), FcyRII
(CD32) and
FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell
expressing the
particular antigen. Bispecific antibodies can also be used to direct cytotoxic
agents to cells
which express a particular antigen. These antibodies possess an antigen-
binding arm and
42
CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
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 are also within the scope of the present invention:
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such
antibodies have, for example, been proposed to target immune system cells to
unwanted
cells (tT.S. Patent No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared
in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking
agents. For example, 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 a1. 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
43
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WO 02/090504 PCT/US02/14342 .-
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. Enzymatically active toxins and fragments thereof that
can be used
include diphtheria A chain, nonbinding active fragments of diphtheria toxin,
exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A
chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca
americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,
enomycin, and the
tricothecenes. A variety of radionuclides are available for the production of
radioconjugated antibodies. Examples include zl2Bi, i3ih l3iln, 9oY, and
lasRe.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling agents such as N-succinimidyl-3-(2-
pyridyldithiol)
propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as
dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate),
aldehydes
(such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-
ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-
active fluorine
compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin
can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
Carbon-14-
labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-
DTPA)
is an exemplary chelating agent for conjugation of radionucleotide to the
antibody. See
W094111026.
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.,
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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
S derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded
through filters
of defined pore size to yield liposomes with the desired diameter. Fab'
fragments of the
antibody of the present invention can be conjugated to the liposomes as
described in Martin
et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within
the liposome.
See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
Diagnostic Applications of Antibodies Directed Against the Proteins of the
Invention
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, fox 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 fox a protein of the invention can be used to isolate the
protein
by standard techniques, such as immunoaffmity chromatography or
immunoprecipitation.
Such an antibody can facilitate the purification of the natural protein
antigen from cells and
of recombinantly produced antigen expressed in host cells. Moreover, such an
antibody
can be used to detect the antigenic protein (e.g., in a cellular lysate or
cell supernatant) in
order to evaluate the abundance and pattern of expression of the antigenic
protein.
Antibodies directed against the protein can be used diagnostically to monitor
protein levels
in tissue as part of a clinical testing procedure, e.g., to, for example,
determine the efficacy
of a given treatment regimen. Detection can be facilitated by coupling (i. e.,
physically
linking) the antibody to a detectable substance. Examples of detectable
substances include
various enzymes, prosthetic groups, fluorescent materials, luminescent
materials,
bioluminescent materials, and radioactive materials. Examples of suitable
enzymes include
horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or
acetylcholinesterase;
CA 02446437 2003-11-07
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examples of suitable prosthetic group complexes include streptavidin/biotin
and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride
or phycoerythrin; an example of a luminescent material includes luminol;
examples of
bioluminescent materials include luciferase, Iuciferin, and aequorin, and
examples of
suitable radioactive material include lash 1311, 3sS or 3H.
Antibody Therapeutics
Antibodies of the invention, including polyclonal, monoclonal, humanized and
fully
human antibodies, may used as therapeutic agents. Such agents will generally
be employed
to treat or prevent a disease or pathology in a subj ect. An antibody
preparation, preferably
one having high specificity and high affinity for its target antigen, is
administered to the
subj ect 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. Tn the first
instance,
administration of the antibody may abrogate or inhibit the binding of the
target with an
endogenous ligand to which it naturally binds. In this case, the antibody
binds to the target
and masks a binding site of the naturally occurnng ligand, wherein the ligand
serves as an
effector molecule. Thus the receptor mediates a signal transduction pathway
for which
Iigand 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. Tn 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 obj ective. As noted above, this
may be a
binding interaction between the antibody and its target antigen that, in
certain cases,
interferes with the functioning of the target, and in other cases, promotes a
physiological
response. The amount required to be administered will furthermore depend on
the binding
affinity of the antibody for its specific antigen, and will also depend on the
rate at which an
administered antibody is depleted from the free volume other subject to which
it is
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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
of components are provided, for example, in Remington : The Science And
Pxactice Of
Pharmacy 19th ed. (Alfonso R. Gennaxo, 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. USA, 90: 7889-
7893
(1993). The formulation herein can also contain more than one active compound
as
necessary for the particular indication being treated, preferably those with
complementary
activities that do not adversely affect each other. Alternatively, or in
addition, the
composition can comprise an agent that enhances its function, such as, for
example, a
cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
Such
molecules are suitably present in combination in amounts that are effective
for the purpose
intended.
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,
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WO 02/090504 PCT/US02/14342
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 _
microcapsuIes. Examples of sustained-release matrices include polyesters,
hydrogels (for
example, poly(2-hydroxyethyl-methacrylate),, or poly(vinylalcohol)),
polylactides (U.S.
I O Pat. No. 3,773,919), copolymers of L-glutamic acid and 'y ethyl-L-
glutamate, non-
degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid
copolymers such as
the LUPRON DEPOT ~ (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
I S 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
20 polyclonal, or more preferably, monoclonal. An intact antibody, or a
fragment thereof
(e.g., Fab or Flab>a) can be used. The term "labeled", with regard to the
probe or antibody, is
intended to encompass direct labeling of the probe or antibody by coupling (i.
e. , physically
linking) a detectable substance to the probe or antibody, ~s'w~ll as indirect
labeling of the
probe or antibody by reactivity with another reagent that is directly labeled.
Examples of
25 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
30 sample", therefore, is blood and a fraction or component of blood including
blood serum,
blood plasma, or lymph. That is, the detection method of the invention can be
used to
detect an analyte mRNA, protein, or genomic DNA in a biological sample in
vitro as well
as in vivo. For example, in vitro techniques for detection of an analyte mRNA
include
Northern hybridizations and in situ hybridizations. In vitro techniques for
detection of an
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analyte protein include enzyme linked immunosorbent assays (ELISAs), Western
blots,
irnmunoprecipitations, and immunofluorescence. In vitro techniques for
detection of an
analyte genomic DNA include Southern hybridizations. Procedures for conducting
immunoassays are described, for example in "ELISA: Theory and Practice:
Methods in
Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, 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.
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.
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 episomai manunali~u-~ vectois).
~th er ~~ctns
(e.g., non-episomal mammalian vectors) are integrated mto 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.
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The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means
that the recombinant expression vectors include one or more regulatory
sequences, selected
on the basis of the host cells to be used for expression, that is operatively-
linked to the
nucleic acid sequence to be expressed. Within a recombinant expression vector,
"operably-
linked" is intended to mean that the nucleotide sequence of interest is linked
to the
regulatory sequences) in a manner that allows for expression of the nucleotide
sequence
(e.g., in an in vitro transcription/translation system or in a host cell when
the vector is
introduced into the host cell).
I O The term "regulatory sequence" is intended to includes promoters,
enhancers and
other expression control elements (e.g., polyadenylation signals). Such
regulatory
sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory
sequences include those that direct constitutive expression of a nucleotide
sequence in
many types of host cell and those that direct expression of the nucleotide
sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It will be
appreciated by
those skilled in the art that the design of the expression vector can depend
on such factors
as the choice of the host cell to be transformed, the level of expression of
protein desired,
etc. The expression vectors of the invention can be introduced into host cells
to thereby
produce proteins or peptides, including fusion proteins or peptides, encoded
by nucleic
acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins,
fusion
nrmtPj~S, Pty':).
The recombinant expression vectors of the inver_tion ran bP designed fox
expression
of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX
proteins can be
expressed in bacterial cells such as Escherichia coli, insect cells (using
baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host cells are
discussed further
in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic
Press, San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be
transcribed and translated in vitro, for example using T7 promoter regulatory
sequences
and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia
eoli
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
CA 02446437 2003-11-07
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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
pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E
binding protein, or protein A, respectively, to the target recombinant
protein.
Examples of suitable inducible non-fusion E. eoli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET 11 d (Studier et al., GENE
E3~PRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CaIif.
(1990)
I S 60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the
recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another
strategy is
to alter the nucleic acid sequence of the nucleic acid to be inserted into an
expression vector
so that the individual codons for each amino acid are those preferentially
utilized in E. coli
(see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such
alteration of nucleic
acid-sequences of the invention can be carried out by standard DN A s; nthesis
techniques.
In another embodiment, the NOVX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include
pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,1982.
Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (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
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WO 02/090504 PCT/US02/14342
expression vectors include pCDM8 (Seed,1987. Nature 329: 840) and pMT2PC
(Kaufman,
et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the
expression vector's
control functions are often provided by viral regulatory elements. For
example, commonly
used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and
simian virus
40. For other suitable expression systems for both prokaryotic and eukaryotic
cells see,
e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY
MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable
of directing expression of the nucleic acid preferentially in a particular
cell type (e.g.,
tissue-specific regulatory elements are used to express the nucleic acid).
Tissue-specific
regulatory elements are known in the art. Non-limiting examples of suitable
tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.
Genes Dev. 1:
268-277), lymphoid-specific promoters (Calame and Eaton,1988. Adv. Immunol.
43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore,
1989. EMBO
J. 8: 729-733) and immunoglobulins (Banerji, et a1.,1983. Cell 33: 729-740;
Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the
neurofilament
promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477),
pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and
mammary
gland-speci$c promoters (e.g., milk whey promoter; IT.S. Pat. No. 4,873,316
and European
Application Publication No. 264,166). Developmentally-regulated promoters are
also
encompassed, e.~ , the marine hox promoters (I~essel and Grass, 1990. Science
249:
374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. P'enes nev
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
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WO 02/090504 PCT/US02/14342
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, VoI. I (I ) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms
refer not only to the particular subject cell but also to the progeny or
potential progeny of
such a cell. Because certain modifications may occur in succeeding generations
due to
either mutation or environmental influences, such progeny may not, in fact, be
identical to
the parent cell, but are still included within the scope of the term as used
herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVx
protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells
(such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host
cells are
known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or trar~fecting host cells
can be found
in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
N.Y.~ 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Various selectable
markers include those that confer resistance to drugs, such as 6418,
hygromycin and
methotrexate. Nucleic acid encoding a selectable marker'can be introduced into
a host cell
on the same vector as that encoding NOVX or can be introduced on a separate
vector.
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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 fiu-ther
provides methods for producing NOVX protein using the host cells of the
invention. In one
embodiment, the method comprises culturing the host cell of invention (into
which a
recombinant expression vector encoding NOVX protein has been introduced) in a
suitable
medium such that NOVX protein is produced. In another embodiment, the method
further
comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte
or an embryonic stem cell into which NOVX protein-coding sequences have been
I S introduced. Such host cells can then be used to create non-human
transgenic animals in
which exogenous NOVX sequences have been introduced into their genome or
homologous recombinant animals in which endogenous NOVX sequences have been
altered. Such animals are useful for studying the function and/or activity of
NOVX protein
and for identifying and/or evaluating modulators of NOVX protein activity. As
used
herein, a "transgenic animal" is a non-human animal, preferably a mammal, more
preferably a rodent such as a rat or mouse, in which one or more of the cells
of the animal
includes a transgene. Other exarryics 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
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WO 02/090504 PCT/US02/14342
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-1, wherein n is an integer between 1-46, can be introduced as a
transgene into the
genome of a non-human animal. Alternatively, a non-human homologue of the
human
NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization
to the
human NOVX cDNA (described further supra) and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the transgene to
increase the
efficiency of expression of the transgene. A tissue-specific regulatory
sequences) can be
operably-linked to the NOVX transgene to direct expression of NOVX protein to
particular
cells. Methods for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become conventional in
the art and
are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and
4,873,191; and
Hogan, 1986. In: MANIPULATING 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, 0.g., ~:nctioraily di~i-apt, the NOVX gene. The
NOVX gene
can be a human gene (e.g., the cDNA of any one of SEQ ID N0:2n-I, wherein n is
an
integer between 1-46), 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 NO:2n-1,
wherein n is an integer between I-46, 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
CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
alter the expression of the endogenous NOVX protein). In the homologous
recombination
vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-
termini by
additional nucleic acid of the NOVX gene to allow for homologous recombination
to occur
between the exogenous NOVX gene carried by the vector and an endogenous NOVX
gene
in an embryonic stem cell. The additional flanking NOVX nucleic acid is of
sufficient
length for successful homologous recombination with the endogenous gene.
Typically,
several kilobases of flanking DNA (both at the S'- and 3'-termini) are
included in the
vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of
homologous
recombination vectors. The vector is ten introduced into an embryonic stem
cell line (e.g.,
. by electroporation) and cells in which the introduced NOVX gene has
homologously-
recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al.,
1992. Cell
69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to
form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152. A chimeric embryo can then be implanted into a suitable
pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the
homologously-
recombined DNA in their germ cells can be used to breed animals in which all
cells of the
animal contain the homologously-recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination vectors and
homologous
recombinant animals are described further in Bradley, 1991. Curr. O~ain.
Biotechnol. 2:
823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO
92/0968;
and WO 93/04169. w --
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 a1.,1992. Proc. Natl.
Acad. Sci.
USA 89: 6232-6236. Another example of a recombinase system is the FLP
recombinase
system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science
251:1351-1355.
If a cre/loxP recombinase system is used to regulate expression of the
transgene, animals
containing transgenes encoding both the Cre recombinase and a selected protein
are
required. Such animals can be provided through the construction of "double"
transgenic
animals, e.g., by mating two transgenic animals, orie containing a transgene
encoding a
selected protein and the other containing a transgene encoding a recombinase.
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WO 02/090504 PCT/US02/14342
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilmut, et dl., 1997. Nature 385: 810-
813. In brief,
a cell (e.g., a somatic cell) from the transgenic animal can be isolated and
induced to exit
the growth cycle and enter Go phase. The quiescent cell can then be fused,
e.g., through the
use of electrical pulses, to an enucleated oocyte from an animal of the same
species from
which the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it
develops to morula or blastocyte and then transferred to pseudopregnant female
foster
animal. The offspring borne of this female foster animal will be a clone of
the animal from
which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies
(also referred to herein as "active compounds") of the invention, and
derivatives, fragments,
analogs and homologs thereof, can be incorporated into pharmaceutical
compositions
suitable for administration. Such compositions typically comprise the nucleic
acid
molecule, protein, or antibody and a pharmaceutically acceptable carrier. As
used herein,
"pharmaceutically acceptable Garner" is intended to include any and all
solvents, dispersion
media, coatings, antibacterial and antifungal~agents, isotonic and absorption
delaying
agents, and the like, compatible with pharmaceutical administration. Suitable
carriers are
described in the most recent edition of Remington's Pharmaceutical Sciences, a
standard
reference text in the field, which is incorporated herein by reference.
Preferred examples of
such carriers or diluents include, but are not limited to, water, saline,
finger's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and non- aqueous
vehicles
such as fixed oils may also be used. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active compound, use
thereof in the
compositions is contemplated. Supplementary active compounds can also be
incorporated
into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with
its intended route of administration. Examples of routes of administration
include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation), transdermaI
(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,
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WO 02/090504 PCT/US02/14342
glycerine, propylene glycol or other synthetic solvents; antibacterial agents
such as benzyl
alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate;
chelating agents such as ethylenediaminetetraacetie acid (EDTA); buffers such
as acetates,
citrates or phosphates, and agents for the adjustment of tonicity such as
sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampoules, disposable
syringes or
multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration,
suitable carriers include physiological saline, bacteriostatic water,
Cremophor EL'~ (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable
under the conditions of manufacture and storage and must be preserved against
the
contaminating action of microorganisms such as bacteria and fungi. The carrier
can be a
solvent or dispersion medium containing, for example, water, ethanol, polyol
(for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and
suitable
mixtures thereof. The proper fluidity can be maintained, for example, by the
use of a
coating such as lecithin, by the maintenance of the required particle size in
the case of
dispersion and by the use of surfactants. Prevention of the action of
microorganisms can be
achieved by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases,
it will be
- ~ preferable to include isotonic agents, for example, sugars,
pcly,~.lecl~ols auc:: ~a :nanit3l,
sorbitol, sodium chloride in the composition. Prolonged absorption of the
injectable
compositions can be brought about by including in the composition an agent
which delays
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound
(e.g., a NOVX protein or anti-NOVX antibody) in the required amount in an
appropriate
solvent with one or a combination of ingredients enumerated above, as
required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active
compound into a sterile vehicle that contains a basic dispersion medium and
the required
other ingredients from those enumerated above. In the case of sterile powders
for the
preparation of sterile injectable solutions, methods of preparation are vacuum
drying and
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WO 02/090504 PCT/US02/14342
freeze-drying that yields a powder of the active ingredient plus any
additional desired
ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible earner. 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 earner is
applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible
binding agents, and/or adjuvant materials can be included as part of the
composition. The
tablets, pills, capsules, troches and the like can contain any of the
following ingredients, or
compounds of a similar nature: a binder such as microcrystalline cellulose,
gum tragacanth
or gelatin; an excipient such as starch or lactose, a disintegrating agent
such as alginic acid,
Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such
as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring
agent such as peppermint, methyl salicylate, or orange flavoring.
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 thrnn~ the
base ef
nasal sprays or suppositories. For transdermaI 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
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CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
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
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 subj ect 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
pl:a..rrnaceutical preparation can include one or more Jells that produce the
oenP dP1_1vP_r~,' ~- --
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
insu~cient or
CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
excessive production of NOVX protein or production of NOVX protein forms that
have
decreased or aberrant activity compared to NOVX wild-type protein (e.g.;
diabetes
(regulates insulin release); obesity (binds and transport lipids); metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting
disorders associated with chronic diseases and various cancers, and infectious
disease(possesses anti-microbial activity) and the various dyslipidemias. In
addition, the
anti-NOVX antibodies of the invention can be used to detect and isolate NOVX
proteins
and modulate NOVX activity. In yet a further aspect, the invention can be used
in methods
to influence appetite, absorption of nutrients and the disposition of
metabolic substrates in
both a positive and negative fashion.
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 or NOVX
protein
activity. The invention also includes compounds identified in the screening
assays
described herein.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which bind to or modulate the activity of the membrane-bound form of
a
NOVX protein or polypeptide or biologically-active portion thereoL The test
compounds
of the invention can be obtained using any of the numerous approaches in
combinatorial
library methods known in the art, including: biological libraries; spatially
addressable
parallel solid phase or solution phase libraries; synthetic library methods
requiring
deconvolution; the "one-bead one-compound" library method; and synthetic
library
methods using affinity chromatography selection. The biological library
approach is
limited to peptide libraries, while the other four approaches are applicable
to peptide,
non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam,
1997.
Anticancer Drug Design 12: 145.
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,
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CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
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
S art, for example in: DeWitt, et a1.,1993. Proc. Natl. Acad. Sci. U.S.A. 90:
6909; Erb, et al.,
1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J.
Med. Chem. 37:
2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Ange~rv.
Chem. Int. Ed.
Engl. 33: 2059; Carell, et al., 1994. Angew Chem. Int. Ed. Engl. 33: 2061; and
Gallop, et
al., 1994. J. Med. Chem. 37: 1233.
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: SSS-SS6), bacteria (Ladner, U.S. Patent No. 5,223,409),
spores (Ladner,
U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin,
1990.
1S 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
enzyniafic label Sorb +.hat binding of the test compound to the NOVX protein
or
biologically-active portion thereof can be determined by detecting the labeled
compound in
2S a complex. For example, test compounds can be labeled with Iash 3sS, ~4C,
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
62
CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342 .. ..,..-
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
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
surface 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., Iuciferase), or detecting a cellular
response, for
example, cell survival, cellular differentiation, or cell proliferation.
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CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
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
with a NOVX protein, wherein determining the ability of the test compound to
interact with
I O 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 erribodiment, 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 cari 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 retl_-free assay comprises contacting tie 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 are amenable to use of both the soluble
form or
the membrane-bound form of NOVX protein. In the case of cell-free assays
comprising the
membrane-bound form of NOVX protein, it may be desirable to utilize a
solubilizing agent
such that the membrane-bound form of NOVX protein is maintained in solution.
Examples
of such solubilizing agents include non-ionic detergents such as n-
octylglucoside,
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CA 02446437 2003-11-07
WO 02/090504 PCT/US02/14342
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
to accommodate automation of the assay. Binding of a test compound to NOVX
protein, or
interaction of NOVX protein with a target molecule in the presence and absence
of a
candidate compound, can be accomplished in any vessel suitable for containing
the
reactants. Examples of such vessels include microtiter plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be provided
that adds a
domain that allows one or both of the proteins to be bound to a matrix. For
example, GST-
NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto
glutathione
sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized
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'ue uissociated from the matrix, and the levee of NC1VX r~rr~tei_rl
h,'_nding 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
streptavidm-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
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WO 02/090504 PCT/US02/14342
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.
S In another embodiment, modulators of NOVX protein expression are identified
in a
method wherein a cell is contacted with a candidate compound and the
expression of
NOVX mRNA or protein in the cell is determined. The level of expression of
NOVX
mRNA or protein in the presence of the candidate compound is compared to the
level of
expression of NOVX mRNA or protein in the absence of the candidate compound.
The
candidate compound can then be identified as a modulator of NOVX mRNA or
protein
expression based upon this comparison. For example, when expression of NOVX
mRIVA
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. T'he 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. 5,283,317;
Zervos, et a1.,1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et
al., 1993.
Oncogene 8: 1693-1696; and Brent WO 94/10300), to :dPr'ta~ -tier 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
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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.
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 SEA 1 D N0:2n-l, wherein n is an integer between 1-46, 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.
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Somatic cell hybrids are prepared by fusing somatic cells from different
mammals
(e.g., human and mouse cells). As hybrids of human and mouse cells grow and
divide, they
gradually lose human chromosomes in random order, but retain the mouse
chromosomes.
By using media in which mouse cells cannot grow, because they lack a
particular enzyme,
but in which human cells can, the one human chromosome that contains the gene
encoding
the needed enzyme will be retained. By using various media, panels of hybrid
cell lines
can be established. Each cell line in a panel contains either a single human
chromosome or
a small number of human chromosomes, and a full set of mouse chromosomes,
allowing
easy mapping of individual genes to specific human chromosomes. See, e.g.,
D'Eustachio,
et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only
fragments of
human chromosomes can also be produced by using human chromosomes with
translocations and deletions.
PCR .mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
sequence to a particular chromosome. Three or more sequences can be assigned
per day
using 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?~e
idPnt;fPd
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
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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 Fiopkins University Welch Medical Library). The relationship
between
genes and disease, mapped to the same chromosomal region, can then be
identified through
linkage analysis (co-inheritance of physically adjacent genes), described in,
e.g., Egeland,
et al., 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the NOVX gene, can be determined. If
a
mutation is observed in some or all of the affected individuals but not in any
unaffected
individuals, then the mutation is likely to be the causative agent of the
particular disease.
Comparison of affected and unaffected individuals generally involves first
looking for
structural alterations in the chromosomes, such as deletions or translocations
that are
visible from chromosome spreads or detectable using PCR based on that DNA
sequence.
Ultimately, complete 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 qne or more restriction enzymes, and probed on a Southern blot to yield
unique band.
for identification. The sequences of the invention are useful as additional
DNA markers for
RFLP ("restriction fragment length polyrnorphisms," 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
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to obtain such identif cation sequences from individuals and from tissue. The
NOVX
sequences of the invention uniquely represent portions of the human genome.
Allelic
variation occurs to some degree in the coding regions of these sequences, and
to a greater
degree in the noncoding regions. It is estimated that allelic variation
between individual
humans occurs with a frequency of about once per each 500 bases. Much of the
allelic
variation is due to single nucleotide polymorphisms (SNPs), which include
restriction
fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes.
I 0 Because greater numbers of polymorphisms occur in the noncoding regions,
fewer
sequences are necessary to differentiate individuals. The noncoding sequences
can
comfortably provide positive individual identification with a panel ofperhaps
10 to 1,000
primers that each yield a noncoding amplified sequence of 100 bases. If coding
sequences,
such as those of SEQ ID N0:2n-1, wherein ~ is an integer between 1-46, 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
prophylactically.
Accordingly, one aspect of the invention relates to diagnostic assays for
determining
NOVX protein and/or nucleic acid expression as well as NOVX activity, in the
context of a
biol~gi~~1 sample (~.~ , blood, serum, cells, tissue) to thereby determine
whether a~n
individual is afflicted with a disease or disorder, or is at risk of
developing a disorder,
associated with aberrant NOVX expression or activity. The disorders include
metabolic
disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated
cachexia,
cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's
Disorder, immune
disorders, and hematopoietic disorders, and the various dyslipidemias,
metabolic
disturbances associated with obesity, the metabolic syndrome X and wasting
disorders
associated with chronic diseases and various cancers. The invention also
provides for
prognostic (or predictive) assays for determining whether an individual is at
risk of
developing a disorder associated with NOVX protein, nucleic acid expression or
activity.
For example, mutations in 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
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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").
Pharnacogenomics allows for the selection of agents (e.g., drugs) for
therapeutic or
prophylactic treatment of an individual based on the genotype of the
individual (e.g., the
genotype of the individual examined to determine the ability of the individual
to respond to
a particular agent.) .
Yet another aspect of the invention pertains to monitoring the influence of
agents
(e.g., drugs, compounds) on the expression or activity of NOVX in clinical
trials.
These and other agents are described in further detail in the following
sections.
Diagnostic 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., mIZNA, 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-1, wherein n is an
integer between
1-46, ox a r onion thei:eof; 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
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antibody and end-labeling of a DNA probe with biotin such that it can be
detected with
fluorescently-labeled streptavidin. The term "biological sample" is intended
to include
tissues, cells and biological fluids isolated from a subject, as well as
tissues, cells and fluids
present within a subject. That is, the detection method of the invention can
be used to
detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as
well as
in vivo. For example, in vitro techniques for detection of NOVX mRNA include
Northern
hybridizations and i~ situ hybridizations. In vitro techniques for detection
of NOVX
protein include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence. In vitro techniques for
detection of
NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques
for detection of NOVX protein include introducing into a subject a labeled
anti-NOVX
antibody. For example, the antibody can be labeled with a radioactive marker
whose
presence and location in a subject can be detected by standard imaging
techniques.
In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test
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 ~f 1':CtVX 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
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NOVX expression or activity. For example, the assays described herein, such as
the
preceding diagnostic assays or the following assays, can be utilized to
identify a subject
having or at risk of developing a disorder associated with NOVX protein,
nucleic acid
expression or activity. Alternatively, the prognostic assays can be utilized
to identify a
subject having or at risk for developing a disease or disorder. Thus, the
invention provides
a method for identifying a disease or disorder associated with aberrant NOVX
expression
or activity in which a test sample is obtained from a subject and NOVX protein
or nucleic
acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX
protein or
nucleic acid is diagnostic for a subject having or at risk of developing a
disease or disorder
associated with aberrant NOVX expression or activity. As used herein, a "test
sample"
refers to a biological sample obtained from a subject of interest. For
example, a test sample
can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug
candidate) to
treat a disease or disorder associated with aberrant NOVX expression or
activity. For
example, such methods can be used to determine whether a subject can be
effectively
treated with an agent for a disorder. Thus, the invention provides methods for
determining
whether a subject can be effectively treated with an agent for a disorder
associated with
aberrant NOVX expression or activity in which a test sample is obtained and
NOVX
protein or nucleic acid is detected (e.g., wherein the presence of NOVX
protein or nucleic
acid is diagnostic for a subject that can be administered the agent to treat a
disorder
associated with aberrant NOVX expression or activityl.
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
Ievel of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification
of a
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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,
(viiz~ 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.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in
a polyrnerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
4,683,202),
such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain
reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et
al., 1994.
Proc. Natl. Acad. Sci. USA 91: 360-364), the latter of which can be
particularly useful for
detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl.
Acids Res.
23: 675-682). This method can include the steps of collecting a sample of
cells from a
patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells
of the sample,
contacting the nucleic acid sample with one or more primers that specifically
hybridize to 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
ampli~catiowstep z~ co~?junction with any of the techniques used for
detectir_g mutations
described herein.
Alternative amplification methods include: self sustained sequence replication
(see,
Guatelli, et al., 1990. PYac. Natl. Acad. Sci. USA 87: 1874-1878),
transcriptional
amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTechnolo~ 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.
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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
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. Biotechniques 19: 448), including sequencing by mass spectrometry (see,
e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In
general,
CA 02446437 2003-11-07
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the art technique of "mismatch cleavage" starts by providing heteroduplexes of
formed by
hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded
duplexes are treated with an agent that cleaves single-stranded regions of the
duplex such
as which will exist due to basepair mismatches between the control and sample
strands.
For instance, RNAfDNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with Sl nuclease to enzyrnatically digesting the mismatched regions.
In other
embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with .
hydroxylamine or osmium tetroxide and with piperidine in order to digest
mismatched
regions. After digestion of the mismatched regions, the resulting material is
then separated
by size on denaturing polyacrylamide gels to determine the site of mutation.
See, e.g.,
Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 8S: 4397; Saleeba, et al.,
1992. Methods
Enzymol. 217: 286-295. In an embodiment; the control DNA or RNA can be labeled
for
detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point
mutations
in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of
E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa
cells
cleaves T at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 1S:
1657-1662. '
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). 'lie duplex is treated ~,~~ah a DNA mismatch repair enzyme, and uhe
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 denatwed 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
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fragments may be labeled or detected with labeled probes. The sensitivity of
the assay may
. be enhanced by using RNA (rather than DNA), in which the secondary structure
is more
sensitive to a change in sequence. In one embodiment, the subject method
utilizes
heteroduplex analysis to separate double stranded heteroduplex molecules on
the basis of
changes in electrophoretic mobility. See, e.g., Keen, et al., 1991. Trends
Genet. 7: S.
In yet another embodiment, the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing
gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature
313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure
that it does
not completely denature, for example by adding a GC clamp of approximately 40
by of
high-melting GC-rich DNA by PCR. In a further embodiment, a temperature
gradient is
used in place of a denaturing gradient to identify differences in the mobility
of control and
sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265:
12753.
Examples of other techniques for detecting point mutations include, but are
not
limited to, selective oligonucleotide hybridization, selective amplification,
or selective
primer extension. For example, oligonucleotide primers may be prepared in
which the
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. Acid. 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, ailtie specif a a,npli~cation-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
polymerise
extension (see, e.g., Prossner, 1993. Tibtech. 11: 238). In addition it may be
desirable to
introduce a novel restriction site in the region of the mutation to create
cleavage-based
detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is
anticipated that in
certain embodiments amplification may also be performed using Taq ligase for
amplification. See, e.g., Barany, 1991. Proc. Natl. Acid. Sei. USA 88: 189. In
such cases,
ligation will occur only if there is a perfect match at the 3'-terminus of the
5' sequence,
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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.
However, any biological sample containing nucleated cells may be used,
including, for
example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity
(e.g., NOVX gene expression), as identified by a screening assay described
herein can be
administered to individuals to treat (prophylactically or therapeutically)
disorders (The
disorders include metabolic disorders, diabetes, obesity, infectious disease,
anorexia,
cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's
Disease,
Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the
various
dyslipidemias, metabolic disturbances associated with obesity, the metabolic
syndrome X
and wasting disorders associated with chronic diseases and various cancers.)
In
conjunction with such treatment, the pharmacogenomics (i. e., the study of the
relationship
between an individual's genotype and that.individual's.response to a foreign
compn~.~nd 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
pharmacogenornics of the
individual perrriits the selection of effective agents (e.g., drugs) for
prophylactic or
therapeutic treatments based on a consideration of the individual's genotype.
Such
pharmacogenomics can further be used to determine appropriate dosages and
therapeutic
regimens. Accordingly, the activity of NOVX protein, expression of NOVX
nucleic acid,
or mutation content of NOVX genes in an individual can be determined to
thereby select
appropriate 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.
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See e.g., Eichelbaum,1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985;
Linder, 1997.
Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions
can be
differentiated. Genetic conditions transmitted as a single factor altering the
way drugs act
on the body (altered drug action) or genetic conditions transmitted as single
factors altering
the way the body acts on drugs (altered drug metabolism). These
pharmacogenetic
conditions can occur either as rare defects or as polymozphisms. For example
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited
enzymopathy in which the main clinical complication is hemolysis after
ingestion of
oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of
fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
determinant of both the intensity and duxation of drug action. The discovery
of genetic
polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and
cytochrome pregnancy zone protein precursor enzymes CYP2D6 and CYP2C19) has
provided an explanation as to why some patients do not obtain the expected
drug effects or
show exaggerated drug response and serious toxicity after taking the standard
and safe dose
of a drug. These polymorphisms are expressed in two phenotypes in the
population, the
extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is
different
among different populations. For example, the gene coding for CYP2D6 is highly
polymorphic and several mutations have been identified in PM, which all lead
to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite
frequently experience exaggerated drug response and side effects when they
receive
standard doses. If a mctawlite is the active _~ __ r_ tthPra,~utac mrripty~ pM
show ne ther%~pe~at_ic
response, as demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. At the other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
Thus, the activity of 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
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when treating a subj ect 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 of NOVX (e.g., the ability to modulate aberrant cell proliferation
and/or
differentiation) can be applied not only in basic drug screening, but also in
clinical trials.
For example, the effectiveness of an agent determined by a screening assay as
described
herein to increase NOVX gene expression, protein levels, or upregulate NOVX
activity,
can be monitored in clinical trails of subjects exhibiting decreased NOVX gene
expression,
protein levels, or downregulated NOVX activity. Alternatively, the
effectiveness of an
agent determined by a screening assay to decrease NOVX gene expression,
protein levels,
or downregulate NOVX activity, can be monitored in clinical trails of subjects
exhibiting
increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such
clinical trials, the expression or activity of NOVX and, preferably, other
genes that have
been implicated in, for example, a cellular proliferation or immune disorder
can be used as
a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule)
that modulates NOVX activity (e.g., identified in a screening assay as
described herein) can
be identified. Thus, to study the effect of agents on cellular proliferation
disorders, fox
example, in a clinical trial, cells can be isolated and RNA prepared and
analyzed for the
levels of expression of NOVX and other genes i-ml I»ated ~ 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
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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
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; adenocarcinoma, lymphoma, uterus cancer, fertility,
hemophilia,
hypercoaguladon, idiopathic thrombocytopenic purpura, immunodeficiencies,
graft versus
host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis,
treatment of
Albright Hereditary Ostoeodystrophy, and other diseases, disorders and
conditions of the
like.
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
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may be utilized include, but are not limited to: (i) an aforementioned
peptide, or analogs,
derivatives, fragments or homologs thereof; (iz~ antibodies to an
aforementioned peptide;
(iii) nucleic acids encoding an aforementioned peptide; (iv) administration of
antisense
nucleic acid and nucleic acids that are "dysfunctional" (i. e., due to a
heterologous insertion
within the coding sequences of coding sequences to an aforementioned peptide)
that are
utilized to "knockout" endogenous function of an aforementioned peptide by
homologous
recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v)
modulators ( i.e.,
inhibitors, agonists and antagonists, including additional peptide mimetic of
the invention
or antibodies specific to a peptide of the invention) that alter the
interaction between an
aforementioned peptide and its binding partner.
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
m>ZNAs
(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
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upon the type of NOVX aberrancy, 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
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 i~ 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-regi.~lates 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 and/or 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 subject has a
gestational
disease (e.g., preclampsia).
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Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays
are
performed to determine the effect of a specific Therapeutic and whether its
administration
is indicated for treatment of the affected tissue.
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,
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 compositioii~ 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,
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which immunospecifically-bind to the novel substances of the invention for use
in
therapeutic or diagnostic methods.
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 1 A.
Table 1A.
NOVl Sequence
Analysis
SEQ ID NO: I I 127 by
NOVla, CATATCACCAGTGGCCATCTGAGGTGTTTCCCTGGCTCTGAAGGGGTAGGCACGATGG
CGI00051-02 CCAGGTGCTTCAGCCTGGTGTTGCTTCTCACTTCCATCTGGACCACGAGGCTCCTGGT
DNA Sequence CCAAGGCTCTTTGCGTGCAGAAGAGCTTTCCATCCAGGTGTCATGCAGAATTATGGGA
CTAAGTTTGGCCGGCAAGGACCAAGTTGAAACAGCCTTGAAAGCTAGCTTTGAAACTT
GCAGCTATGGCTGGGTTGGAGATGGATTCGTGGTCATCTCTAGGATTAGCCCAAACCC
CAAGTGTGGGAAAAATGGGGTGGGTGTCCTGATTTGGAAGGTTCCAGTGAGCCGACAG
TTTGCAGCCTATTGTTACAACTCATCTGATACTTGGACTAACTCGTGCATTCCAGAAA
TTATCACCACCAAAGATCCCATATTCAACACTCAAACTGCAACACAAACAACAGAATT
TATTGTCAGTGACAGTACCTACTCGGTGGCATCCCCTTACTCTACAATACCTGCCCCT
ACTACTACTCCTCCTGCTCCAGCTTCCACTTCTATTCCACGGAGAAAAAAATTGATTT
GTGTCACAGAAGTTTTTATGGAAACTAGCACCATGTCTACAGAAACTGAACCATTTGT
TGAAAATAAAGCAGCATTCAAGAATGAAGCTGCTGGGTTTGGAGGTGTCCCCACGGCT
CTGCTAGTGCTTGCTCTCCTCTTCTTTGGTGCTGCAGCTGGTCTTGGATTTTGCTATG
TCAAAAGGTATGTGAAGGCCTTCCCTTTTACAAACAAGAATCAGCAGAAGGAAATGAT
CGAAACCAAAGTAGTAAAGGAGGAGAAGGCCAATGATAGCAACCCTAATGAGGAATCA
AAGAAAACTGATAAAAACCCAGAAGAGTCCAAGAGTCCAAGCAAAACTACCGTGCGAT
GCCTGGAAGCTGAAGTTTAGATGAGACAGAAATGAGGAGACACACCTGAGGCTGGTTT
CTTTCATGCTCCTTACCCTGCCCCAGCTGGGGAAATTCAAAAGGGCCAAAGAACCAAA
GAAGGAAAGTCCACCCTTGGTTCCTAACTGGGATTCAGCTCAGGGACTGCCATTTGGA
CTATTGGGAGTTGCACCAAAGGAGA
ORF Start: ATG at ORF Stop: TAG at 946
55
SEQ ID NO: 2 297 as MW at.32454.8kD
NOVla, MARCFSLVLLLTSIWTTRLLVQGSLRAEELSIQVSCRIMGLSLAGKDQVETALKASFE
CGI00051-02 TCSYGWVGDGFWISRISPNPKCGKNGVGVLIWKVPVSRQFAAYCYNSSDTWTNSCIP
Protein SequenceEIITTI~PIFNTQTATQTTEFIVSDSTYSVASPYSTIPAPTTTPPAPASTSIPRRKKL
ICVTEVFMETSTMSTETEPFVENKAAFKNEAAGFGGVPTALLVLALLFFGAAAGLGFC
YVKRYVKAFPFTNKNQQKEMIETKVVKEEKANDSNPNEESKKTDKNPEESKSPSKTTV
RCLEAEV
Further analysis of the NOV 1 a protein yielded the following properties shown
in Table 1 B.
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Table 1B. Protein Sequence Properties NOVla
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
SignalP Cleavage site between residues 27 and 28
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.
Table 1C. Geneseq Results
for NOVla
NOVla Identities/
Geneseq Protein/Organism/Length (PatentResidues/SimilaritiesExpect
#, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAB80247Human PR0263 protein - Homo I ..297 297/322 (92%)e-167
Sapiens,
322 aa. [WO200104311-Al, 1..322 297/322 (92%)
18-JAN-
2001
AAB88391Human membrane or secretory 1..297 297/322 (92%)e-167
protein
clone PSEC0135 - Homo sapiens,1..322 297/322 (92%)
322 aa.
[EP1067182-A2, 10-JAN-2001]
AAB87528Human PRO263 - Homo Sapiens,1..297 297/322 (92%)e-167
322 aa.
[WO200116318-A2, 08-MAR-2001]1..322 297/322 (92%)
AAY87287Human signal peptide containing1..297 297/322 (92%)( e-167
protein
HSPP-64 SEQ ID NO:64 - Homo 1..322 297/322 (92%)
Sapiens,
322 aa. [wO200000610-A2,
06-JAN-
2000]
AAY13379Amino acid sequence of protein1..297 297/322 (92%)e-167
PR0263
- Homo Sapiens, 322 aa. [W09914328-1..322 297/322 (92%)
A2, 25-MAR-1999]
In a BLAST search of public sequence datbases, the NOV 1 a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 1 D.
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Table 1D. Public BLASTP Results for NOVla
Identities/
Protein Similarities
NOVla Residues/ Expect
AccessionProtein/Organism/Length Match Residuesfor the Value
Number Matched
Portion
Q9UNF4 HYALURONIC ACID RECEPTOR 1..297 297/322 ~ e-167
- (92%)
Homo Sapiens (Human), 1..322 297/322
322 aa. (92%)
Q9YSY7 LYMPHATIC ENDOTHELIUM- 1..297 294/322 e-165
(91%)
SPECIFIC HYALURONAN 1..322 295/322
(91%)
RECEPTOR LYVE-1 - Homo
Sapiens
(Human), 322 aa.
Q99NE4 HYALURONAN RECEPTOR 6..297 202/316 e-106
(63%)
PRECURSOR - Mus musculus 6..318 230/316
(71%)
(Mouse), 318 aa.
Q98SR5 T CELL ANTIGEN CD44 ~ISOFORM36..116 30/81 (37%)Se-09
B - Anas platyrhynchos 53..132 48/81 (59%)
(Domestic
duck), 265 aa.
Q90ZL8 T CELL ANTIGEN CD44 ISOFORM36..116 30/81 (37%)Se-09
A - Anas platyrhynchos 53..132 48181 (59%)~
(Domestic
duck), 398 aa.
PFam analysis predicts that the NOV 1 a protein contains the domain shown in
the Table 1 E.
Table 1E.
Domain
Analysis
of NOVla
Identities! ~
Pfam Doma~isNOVIa Match Region Similarities ~ ~ Expect
:'aIue ~
for the Matched Region
Xlink 43..104 19/74 (26%) 8.7e-12
43/74 (58%)
Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 2A.
Table 2A. NOV2 Sequence Analysis
SEQ ID NO: 3 2289 by
NOV2a, GTATTCTGGTAGAGGAGGCATCAAGAGTCCTGGGAGGCCGGTGGTAATCATGTAGGCA
CG1OO104- .O1 _CCATGGAAACTGCTATGTGCGTTTGCTGTCCATGTTGTACATGGCAGAGATGTTGTCC
DNA Sequence T~GTTATGCTCCTGTCTGTGCTGCAAGTTCATCTTCACCTCAGAGCGGAACTGCACC
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TGCTTCCCCTGCCCTTACAAAGATGAGCGGAACTGCCAGTTCTGCCACTGCACCTGTT
GTGCTGCTGCACAGCCAGCAGCAATCTCAACTGCTACTACTATGAGAGCCGCTGCTGC
CGCAATACCATCATCACTTTCCACAAGGGCCGCCTCAGGAGCATCCATACCTCCTCCA
AGACTGCCCTGCGCACTGGGAGCAGCGATACCCAGGTGGATGAAGTAAAGTCAATACC
AGCCAACAGTCACCTGGTGAACCACCTCAATTGCCCCATGTGCAGCCGGCTGCGCCTG
CACTCATTCATGCTGCCCTGCAACCACAGCCTGTGCGAGAAGTGCCTGCGGCAGCTGC
AGAAGCACGCCGAGGTCACCGAGAACTTCTTCATCCTCATCTGCCCAGTGTGCGACCG
CTCGCACTGCATGCCCTACAGCAACAAGATGCAGCTGCCCGAGAACTACCTGCACGGG
CGTCTCACCAAGCGCTACATGCAGGAGCACGGCTACCTCAAGTGGCGCTTTGACCGCT
CCTCCGGGCCCATCCTCTGCCAGGTCTGCCGCAACAGGCGCATCGCTTACAAGCGCTG
CTGCACCTTCTGCAAGTTCTCTTTCCACAATGGCCACGACACCATTAGCCTCATCGAC
GATATGAAATTGATAATGACCTAATGGAATTCAACATCTTAAAAAACAGCTTTAAAGC
TGACAAGGAGGCAAAGCGAAAAGAGATCAGAAATGGCTTTCTCAAGTTGCGCAGCATT
CTTCAGGAGAAAGAGAAGATCATCATGGAGCAGATAGAGAATCTAGAAGTGTCCAGGC
AGAAGGAAATTGAAAAATATGTGTATGTTAC__AACCATG.AAAGTGAACGAGATGGATGG
TCTGATCGCCTACTCCAAGGAAGCCCTGAAGGAGACTGGCCAGGTGGCATTCCTGCAG
TCAGCCAAGATCCTGGTGGACCAGATCGAGGACGGCATCCAGACCACCTACAGGCCTG
ACCCACAGCTCCGGCTGCACTCAATAAACTACGTGCCCTTGGACTTTGTTGAGCTTTC
CAGTGCCATCCATGAGCTCTTCCCCACAGGGCCCAAGAAGGTACGCTCCTCAGGGGAC
TCCCTGCCCTCCCCCTACCCCGTGCACTCAGAAACAATGATTGCCAGGAAGGTCACTT
TCAGCACCCACAGCCTCGGCAACCAGCACATATACCAGCGAAGCTCCTCCATGTTGTC
CTTCAGCAACACTGACAAGAAGGCCAAGGTGGGTCTGGAGGCCTGTGGGAGAGCCCAG
TCAGCCACCCCCGCCAAACCCACAGACGGCCTCTACACCTACTGGAGTGCTGGAGCAG
CTCTGTGAAGACCCCAGGCCCAATTGTTATCTACCAGACTCTGGTGTACCCAAGAGCT
GCCAAGGTTTACTGGACATGTCCAGCAGAAGACGTGGACTCTTTTGAGATGGAATTCT
TTTAAAGTTAGAGCCATCAATGATAATGGTCCTGGGCAATGGAGTGATATCTGCAAGG
TGGTAACACCAGATGGACATGGGAAGAACCGAGCTAAGTGGGGCCTGCTGAAGAATAT
CCAGTCTGCCCTCCAGAAGCACTTCTGAGCCCCTTCAGAGCAGGAAACAACCTCAGAC
TCATCACAAAGTAGACATATACACACA
ORF Start: ATG at 61 ORF Stop: TGA at 2230
SEQ ID NO: 4 ~ X23 as ~~J at 82771.BkD
NOV2a, METAMCVCCPCCTWQRCCPQLCSCLCCKFIFTSr:~vc:~rc:r~rc:YYII~ERNCQr~CHCTCS
CG1001O4-O1 ESPNCHWCCCSWANDPNCKCCCTASSNLNCYYYESRCCRNTIITFHKGRLRSIHTSSK
Protein Sequence T~RTGSSDTQVDEVKSIPANSHLVNHLNCPMCSRLRLHSFMLPCNHSLCEKCLRQLQ
KHAEVTENFFILICPVCDRSHCMPYSNKMQLPENYLHGRLTKRYMQEHGYLKW12FDRS
SGPILCQVCRNRRIAYKRCITCRLNLCNDCLKAFHSDVAMQDHVFVDTSAEEQDEKIC
IHHPSSRIIEYCRNDNKLLCTFCKFSFHNGHDTISLIDACSERAASLFSAIAKFKAVR
YEIDNDLMEFNILKNSFKADKEAKRKEIRNGFLKLRSILQEKEKIIMEQIENLEVSRQ
PQLRLHSINYVPLDFVELSSAIHELFPTGPKKVRSSGDSLPSPYPVHSETMIARKVTF
STHSLGNQHTYQRSSSMLSFSN'TDKKAKVGLEACGRAQSATPAKPTDGLYTYWSAGAD
SQSVQNSSSF'HNWYSFNDGSVKTPGPIVIYQTLVYPRAAICVYWTCPAEDVDSFEMEFY
SEQ ID NO: 5 X579 by
198362674 DNA
Sequence
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AATGGAATTCAACATCTTAAAAAACAGCTTTAAAGCTGACAAGGAGGCAAAGCGAAAA
GAGATCAGAAATGGCTTTCTCAAGTTGCGCAGCATTCTTCAGGAGAAAGAGAAGATCA
TCATGGAGCAGATAGAGAATCTAGAAGTGTCCAGGCAGAAGGAAATTGAAAAATATGT
GTATGTTACAACCATGAAAGTGAACGAGATGGATGGTCTGATCGCCTACTCCAAGGAA
GCCCTGAAGGAGACTGGCCAGGTGGCATTCCTGCAGTCAGCCAAGATCCTGCTCGAG
OItF Start: at 1
~ OIZF Stop: end
of sequence
SEQ ID NO: 6 193 MW at 22238.2kD
as
NOV2b, GSDCLKAFHSDVAMQDHVFVDTSAEEQDEKICIHHPSSRIIEYCRNDNKLLCTFCKFS
198362674 Protein~G~TISLIDACSERAASLFSAIAKFKAVRYEIDNDLMEFNILKNSFKADKEAKRK
Sequence EIRNGFLKLRS~ILQEKEKIIMEQIENLEVSRQKEIEKYVYVTTMFCVNEMDGLIAYSKE
ALKETGQVAFLQSAKILLE
SEQ ID NO: 7 579 by
NOV2C, GGATCCGACTGCCTCAAGGCCTTCCACTCGGATGTGGCCATGCAAGACCACGTCTTTG
198362686 DNA TGGACACCAGCGCCGAGGAACAGGACGAGAAGATCTGCATCCACCACCCATCCAGCCG
Sequence CATCATCGAGTACTGCCGCAATGACAACAAATTGCTCTGCACCTTCTGCAAGTTCTCT
TTCCACAATGGCCACGACACCATTAGCCTCATCGACGCCTGCTCCGAGAGGGCCGCCT
CACTCTTCAGCGCCGTCGCCAAGTTCAAAGCAGTCCGATATGAAATTGATAATGACCT
AATGGAATTCAACATCTTAAAAAACAGCTTTAAAGCTGACAAGGAGGCAAAGCGAAAA
GAGATCAGAAATGGCTTTCTCAAGTTGCGCAGCATTCTTCAGGAGAAAGAGAAGATCA
TCATGGAGCAGATAGAGAATCTAGAAGTGTCCAGGCAGAAGGAAATTGAAAAATATGT
GTATGTTACAACCATGAAAGTGAACGAGATGGATGGTCTGATCGCCTACTCCAAGGAA
GCCCTGAAGGAGACTGGCCAGGTGGCATTCCTGCAGTCAGCCAAGATCCTGCTCGAG
ORF Start: at 1 ORF Stop:
end
of sequence
SEQ ID NO: 8 193 as
MW at
22224.21eD
NOV2C, GSDCLKAFIiSDVAMQDHVFVDTSAEEQDEKICIHHPSSRIIEYCRNDNKLLCTFCKFS
198362686 PIOteiri~G~TISI'IDACSERAASLFSAVAKFKAVRYEIDNDLMEFNILKNSFKADKEAKRK
Sequence EIRNGFLKLRSILQEKEKIIMEQIENLEVSRQKEIEKYVYVTTMKVNEMDGLIAYSKE
ALKETGQVAFLQSAKILLE
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 2B.
Table 2B. Comparison of
NOV2a against NOV2b and
NOV2c.
NOV2a Residues/ Identities/
Protein Sequence Similarities for the
Match Residues Matched Region
NOV2b 260..451 175/192 (91%)
2..193 178/192 (92%)
NOV2c 260..451 174/192 (90%)
2..193 178/192 (92%)
Further analysis of the NOV2a protein yielded the following properties shown
in Table 2C.
Table 2C. Protein Sequence Properties NOV2a
PSort 0.4600 probability located in mitochondria) matrix space; 0.3000
probability located in
analysis: microbody (peroxisome); 0.1562 probability located in mitochondria)
inner
membrane; 0.1562 probability located in mitochondria) intermembrane space
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SignalP Cleavage site between residues 25 and 26
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 2D.
Table 2D. Geneseq Results
for NOV2a
NOV2a Identities/
Geneseq Protein/Organism/Length Residues/. SimilaritiesExpect
[Patent #, for
IdentifierDatej Match the Matched Value
ResiduesRegion
AAM34792Peptide #8829 encoded by 14..113 100/100 (100%)2e-66
probe for
measuring placental gene i ..100 l "vG/100
expression - ( l 00 lo)
Homo Sapiens, 100 aa. [W0200157272-
A2, 09-AUG-2001 ]
ABB41017Peptide #8523 encoded by 14..113 100/100 (140%)2e-66
human foetal
liver single exon probe.- 1..100 100/100 (100%)
Homo sapiens,
100 aa. [W0200I57277-A2,
09-AUG-
2001]
AAM35060Peptide #9097 encoded by 515..620106/106 (100%)2e-56
probe for
measuring placental gene 1..106 106/106 (100%)
expression -
Homo Sapiens, 106 aa. [WO200157272-
A2, 09-AUG-2001 ]
AAM74944Human bone marrow expressed515..6201061106 (100%)2e-56
probe
encoded protein SEQ ID NO: 1..106 106/106 (100%)
35250 -
Homo Sapiens, 106 aa. [W0200157276-
A2, 09-AUG-2001
AAM62140Human brain expressed singleSI5..620~ 106!106 2e-56
exon (100%)
proba encoded protein SEQ 1..106 ~ ? Q5/1
ID NO: ~5 (.1.00%)
34245 - Homo sapiens, 106
aa.
[W0200I57275-A2, 09-AUG-2001]
In a BLAST search of public sequence datbases, the NOV2a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 2E.
Table 2E. Public BLASTP Results for NOV2a
Protein NOV2a Identities/
AccessionProtein/Organism/Length Residues/ SimilaritiesExpect
for
Number Match the Matched Value
Residues Portion
Q9D2H5 4930486B16RIK PROTEIN -Mus 1..723 629/723 (86%)0.0
musculus (Mouse), 723 aa. 1..723 679/723 (92%)
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Q90WDl MIDLINE-1 - Gallus gallus 140..52594/411 (22%) Ie-21
(Chicken),
667 aa. 4..402 169/411 (40%)
Q9QUS6 MIDLINE 2 PROTEIN - Mus musculus140..52594/411 (22%) 2e-21
(Mouse), 685 aa. 4..402 168/411 (40%)
P82458 MIDLINE 1 PROTEIN (RING FINGER140..52594/411 (22%) 3e-21
PROTEIN) - Rattus norvegicus4..402 170/411 (40%)
(Rat),
667 aa.
Q9UJV3 RING FINGER PROTEIN 140..52594/411 (22%) 4e-21
(HYPOTHETICAL 77.9 KDA 4..402 167/41 I (39%)
PROTEIN) - Homo sapiens (Human),
685 aa.
PFam analysis predicts that the NOV2a protein contains the domains shown in
the Table
2F.
Table 2F. Domain Analysis of NOV2a
Identities/
Pfam DomainNOV2a Match Similarities Expect
Region Value
for the Matched
Region
zf C3HC4 146..191 16/54 (30%) ~ 0.0023
28/54 (52%)
zf B box 233..280 10/50 (20%) 0.46
3 1/50 (62%)
zf B_box 285..326 14/49 (29%) 0.0033
24/49 (49%)
fn3 601..691 17/94 (18%) 0.00093
62/94 (66%)
Example 3.
The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 3A.
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TGGGGGTCATCGTGGCAGCCGTCCTTGTAACCCTGATTCTCCTGGGAATCTTGGTTTT
TGGCATCTGGTTTGCCTATAGCCGAGGCCACTTTGACAGAACAAAGAAAGGGACTTCG
AGTAAGAAGGTGATTTACAGCCAGCCTAGTGCCCGAAGTGAAGGAGAATTCAAACAGA
CCTCGTCATTCCTGGTGTGAGCCTGGTCGGCTCACCGCCTATCATCTGCATTTG
ORF Start: ATG at 39 ORF Stop:
TGA
at 714
SEQ ID NO: 10 225 as MW at 24525.6kD
NOV3a, MGTKAQVERKLLCLFILAILLCSLALGSVTVHSSEPEVRIPENNPVKLSCAYSGFSSP
CG100114-O1 R~~~QGDTTIGNRAVLTCSEQDGSPPSEYTWFKDGIVMPTNPKSTRAFSNSSYVL
Protein Sequence NPTTGELVFDPLSASDTGEYSCEARNGYGTPMTSNAVRMEAVERNVGVIVAAVLVTLI
LLGILVFGIWFAYSRGIiFDRTKKGTSSKKVIYSQPSARSEGEFKQTSSFLV
Further analysis of the NOV3a protein yielded the following properties showwin
Table 3B.
Table 3B. Protein Sequence Properties NOV3a
PSort 0.4600 probability located in plasma membrane; 0.1000 probability
located in
analysis: endoplasmic reticulum (membrane); 0.1000 probability located in
endoplasmic
r_eticulum (lumen); 0.1000 probability located in outside .
SignalP Cleavage site between residues 28 and 29
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 Identifies/
Geneseq Protein/Organism~/Lengtli Residues/SipnilaritiesExpect
[Patent #, fo:
IdentifierDate]' Matc'a the l~atslDedVal;:c
"
ResiduesRegion
ABB72215Human protein isolated from70..225 156/156 (100%)4e-86
skin cells
SEQ ID NO: 331 - Homo sapiens,144..299156/156 (100%)
299
aa. [W0200190357-A1, 29-NOV-2001]
ABB72150Human protein isolated from70..225 156/156 (100%)4e-86
skin cells ~
SEQ ID NO: 189 - Homo sapiens,144..299156/156 (100%)
299
aa. [W0200190357-A1, 29
NOV-2001]
AAB53086Human angiogenesis-associated70..225 156/156 (100%)4e-86
protein
PR030I, SEQ ID N0:119 - 144..299156/156 (100%)
Homo
sapiens, 299 aa. [W0200053753-A2,
14-
SEP-2000]
AAB56015Skin cell protein, SEQ ID 70..225 156/156 (100%)4e-86
NO: 331-
Homo Sapiens, 299 aa. [W0200069884-144..299156/156 (100%)
~ A2, .
23-NOV-2000]
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AAB55950 Skin cell protein, SEQ ID NO: 189 - 70..225 156/156 (100%) 4e-86
Homo sapiens, 299 aa. [W0200069884- 144..299 156/156 (100%)
A2, 23 NOV-2000]
In a BLAST search of public sequence datbases, the NOV3a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 3D.
Table 3D. Public BLASTP Results
for NOV3a
NOV3a Identities/
Protein Residues/Similarities Expect
for
AccessionProtein/Organism/Length Match the Matched Value
Number ResiduesPortion
Q9YSB2 JUNCTION ADHESION MOLECULE I ..225 198/279 (70%)3e-98
-
Homo sapiens (Human), 259 1..259 202/279 (71%)
aa.
Q9Y624 functional adhesion molecule70..225 156/156 (100%)9e-86
1 precursor
(JAM) (Platelet adhesion 144..299156/156 (100%)
molecule 1)
(PAM-1 ) (Platelet F 11 receptor)
- Homo
sapiens (Human), 299 aa.
Q9XT56 functional adhesion molecule70..225 1191156 (76%)1e-66
l precursor
(JAM) - Bos taurus (Bovine),143..298138/156 (88%)
298 aa.
Q9JHY1 JUNCTIONAL ADHESION 70..225 125/158 (79%)2e-65
MOLECULE JAM - Rattus norvegicus143..300140/158 (88%)
(Rat), 300 aa.
Q9JKD5 JUNCTIONAL ADHESION 70..225 125/158 (79%)2e-65
MOLECULE - Rattus norvegicus16..173 140/158 (88%)
(Rat),
173 as (fragment).
PFam analysis predicts that the NOV3a protein contains the domains shown in
the Table
~3E.
Table 3E. Domain Analysis of NOV3a
Identities/
Pfam Domain NOV3a Match Region Similarities Expect Value
for the Matched Region
Ig 43..67 8/27 (30%) 0.1 S
21/27 (78%)
1g 72..140 15/71 (21%) 1.2e-08
52/71 (73%) .
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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: 11 1545
by
NOV4a, _CATGAGTGTGGTGCTGGTGCTACTTCCTACACTGCTGCTTGTTATGCTCACGGGTGCT
CG1O0619-O1 CAGAGAGCTTGCCCAAAGAACTGCAGATGTGATGGCAAAATTGTGTACTGTGAGTCTC
DNA SequenceATGCTTTCGCAGATATCCCTGAGAACATTTCTGGAGGGTCACAAGGCTTATCATTAAG
GTTCAACAGCATTCAGAAGCTCAAATCCAATCAGTTTGCCGGCCTTAACCAGCTTATA
TGGCTTTATCTTGACCATAATTACATTAGCTCAGTGGATGAAGATGCATTTCAAGGGA
TCCGTAGACTGAAAGAATTAATTCTAAGCTCCAACAAAATTACTTATCTGCACAATAA
AACATTTCACCCAGTTCCC"~-~AT~mCCGCAAmCTGC',p~CCTCTCCTACAATAAGCTTCAG
ACATTGCAATCTGAACAATTTAAAGGCCTTCGGAAACTCATCATTTTGCACTTGAGAT
CTAACTCACTAAAGACTGTGCCCATAAGAGTTTTTCAAGACTGTCGGAATCTTGATTT
TTTGGATTTGGGTTACAATCGTCTTCGAAGCTTGTCCCGAAATGCATTTGCTGGCCTC
TTGAAGTTAAAGGAGCTCCACCTGGAGCACAACCAGTTTTCCAAGATCAACTTTGCTC
ATTTTCCACGTCTCTTCAACCTCCGCTCAATTTACTTACAATGGAACAGGATTCGCTC
CATTAGCCAAGGTTTGACATGGACTTGGAGTTCCTTACACAACTTGGATTTATCAGGG
AATGACATCCAAGGAATTGAGCCGGGCACATTTAAATGCCTCCCCAATTTACP~AAAAT
TGAATTTGGATTCCAACAAGCTCACCAATATCTCACAGGAAACTGTCAATGCGTGGAT
ATCATTAATATCCATCACATTGTCTGGAAATATGTGGGAATGCAGTCGGAGCATTTGT
CCTTTATTTTATTGGCTTAAGAATTTCAAAGGAAATAAGGAAAGCACCATGATATGTG
CGGGACCTAAGCACATCCAGGGTGAAAAGGTTAGTGATGCAGTGGAAACATATAATAT
CTGTTCTGAAGTCCAGGTGGTCAACACAGAAAGATCACACCTGGTGCCCCAAACTCCC
CAGAAACCTCTGATTATCCCTAGACCTACCATCTTCAAACCTGACGTCACCCAATCCA
CCTTTGAAACACCAAGCCCTTCCCCAGGGTTTCAGATTCCTGGCGCAGAGCAAGAGTA
TGAGCATGTTTCATTTCACAAAATTATTGCCGGGAGTGTGGCTCTCTTTCTCTCAGTG
GCCATGATCCTCTTGGTGATCTATGTGTCTTGGAAACGCTACCCAGCCAGCATGAAAC
AACTCCAGCAACACTCTCTTATGAAGAGGCGGCGGAAAAAGGCCAGAGAGTCTGAAAG
ACAAATGAATTCCCCTTTACAGGAGTATTATGTGGACTACAAGCCTACAAACTCTGAG
ACCATGGATATATCGGTTAATGGATCTGGGCCCTGCACATATACCATCTCTGGCTCCA
GGGAATGTGAGGTATGAACCATGATCCTCCTAAAAGC
ORF Start: ATG at
2 ORF Stop: TGA
at 1523
SEQ ID NO: 12 507 MW at 57899.I1cD
as
NOV4a, MSVVLVLLPTLLLVMLTGAQRACPKNCRCDGKIVYCESHAFADIPENISGGSQGLSLR
CG100619-O1 ~SIQ~KSNQFAGLNQLIWLYLDHNYISSVDEDAFQGIRRLKELILSSNKITYLHNK
Protein SequenceT~PVPNLRNLDLSYNKLQTLQSEQFKGLRKLIILHLRSNSLKTVPIRVFQDCRNLDF
LDLGYNRLRSLSRNAFAGLLKLKELHLEHNQFSKINFAHFPRLFNLRSIYLQWNRIRS
ISQGLTWTWSSLHNLDLSGNDIQGIEPGTFKCLPNLQKLNLDSNKLTNISQETVNAWI
SLISITLSGNMWECSRSICPLFYWLKNFKGNKESTMICAGPKHIQGEKVSDAVETYNI
CSEVQVVNTERSHLVPQTPQKPLIIPRPTIFKPDVTQSTFETPSPSPGFQIPGAEQEY
EHVSFHKIIAGSVALFLSVAMILLVIYVSWKRYPASMKQLQQHSLMKRRRKKARESER
QMNSPLQEYYVDYKPTNSETMDISVNGSGPCTYTISGSRECEV
SEQ ID NO: 13 1194
by
NOV4b, GGTACCCAGAGAGCTTGCCCAAAGAACTGCAGATGTGATGGCAAAATTGTGTACTGTG
210168777 AGTCTCATGCTTTCGGAGATATCCCTGAGAACATTTCTGGAGGGTCACAAGGCTTATC
DNA
Sequence ATTAAGGTTCAACAGCATTCAGAAGCTCAAATCCAATCAGTTTGCCGGCCTTAACCAG
CTTATATGGCTTTATCTTGACCATAATTACATTAGCTCAGTGGATGAAGATGCATTTC
AAGGGATCCGTAGACTGAAAGAATTAATTCTAAGCTCCAACAAAATTACTTATCTGCA
CAATAAAACATTTCACCCAGTTCCCAATCTCCGCAATCTGGACCTCTCCTACAATAAG
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Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 4B.
Table 4B. Comparison of NOV4a against NOV4b.
Protein Sequence NOV4a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOV4b 18..414 384/397 (96%)
1..397 385/397 (96io)
Further analysis of the NOV4a protein yielded the following properties shown
in Table 4C.
Table 4C. Protein Sequence Properties NOV4a
Psort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400
probability
analysis: located in plasma membrane; 0.4600 probability located in Golgi
body; 0.1000
probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 20 and 21
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 4D.
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Table 4D. Geneseq Results
for NOV4a
NOV4a Identities/
Geneseq Protein/Organism/Length [Patent Expect
#,
IdentifierDate] Residues/SimilaritiesValue
for
Match the Matched
Residues Region
AAB66201Protein of the invention 2..507 327/512 0.0
#113 - (63%)
Unidentified, 513 aa. [W0200078961-A1,13..513 403/512
(77%)
28-DEC-2000]
AAB87587Human PR01693 - Homo Sapiens,2..507 327/512 0.0
513 aa. (63%)
[W0200116318-A2, 08-MAR-2001]13..513 403/512
(77%)
AAU12439Human PRO1693 polypeptide 2..507 327/512 0.0
sequence - (63%)
Homo Sapiens, S 13 aa. [W0200140466-13..513 403/512
(77%)
A2, 07-JUN-2001 ]
AAY99452Human PRO1693 (UNQ803) amino~ 2..507 327/512 0.0
acid (63%)
sequence SEQ ID N0:385 - ~ 13..513403/512
Homo (77%)
Sapiens, 513 aa. [W0200012708-A2,
09-
MAR-2000]
AAB65236Human PR01309 (UNQ675) protein~ 3..507 244/514 e-135
(47%)
sequence SEQ ID N0:278 - 20..522 339/514
Homo (65%)
sapiens, 522 aa. [W0200073454-Al,
07-
DEC-2000]
In a BLAST search of public sequence datbases, the NOV4a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 4E.
Table 4E. Public BLASTP Results for Nv~ V :a
Protein ' NOV4a Identities/
AccessionProtein/Organism/Length Residues/SimilaritiesExpect
for
Number Match the Matched Value
ResiduesPortion
Q9BGP6 HYPOTHETICAL 65.9 IRDA PROTEIN2..507 326/512 (63%)0.0
- Macaca fascicularis (Crab13..513 403/512 (78%)
eating
macaque) (Cynomolgus monkey),
581
aa.
Q95KI8 HYPOTHETICAL 65.9 KDA PROTEIN2..507 324/512 (63%)0.0
- Macaca fascicularis (Crab13..513 402/512 (78%)
eating
macaque) (Cynomolgus monkey),
581
aa.
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Q96DN1 CDNA FLJ32082 FIS, CLONE OCBBF2000231,3..507 244/514 e-
(47%)
WEAKLY SIMILAR TO PHOSPHOLIPASE A2 20..522340/514 135
(65%)
INHIBITOR SUBUNIT B PRECURSOR - Homo
sapiens (Human), 522 aa.
Q9H9T0 CDNA FLJ12568 FIS, CLONE NT2RM4000857,301..507207/207 e-
(100%)
WEAKLY SIMILAR TO LEUCINE-RICH ALPHA- 1..207 207/207 120
(100%)
2-GLYCOPROTEIN (HYPOTHETICAL 23.6 KDA
PROTEIN) - Homo Sapiens (Human), 207 aa.
043300 KIAA0416 PROTEIN - Homo Sapiens 1..507 227/5/ 1 e-
(Human), 516 (44%)
aa. 20..516327/511 118
(63%)
PFam analysis predicts that the NOV4a protein contains the domains shown in
the Table
4F.
Table 4F. Domain
Analysis of
NOV4a
Identities/
Pfam DomainNOV4a Match Similarities Expect
Region Value
for the Matched
Region
LRRNT 22..49 9131 (29%) 0.043
18/31 (58%)
LRR 75..98 7125 (28%) 0.068
20/25 (80%}
LRR 99..122 9/25 (36%) 0.33
18/25 (72%)
LRR 123..146 12/25 (48%) 0.0015
21/25 (84%)
' LRI~ ~ 147..170 7/25 (28%) 0.48
20/25 (80%)
LRR 171..194 11/25 (44%) 0.014
20/25 (80%)
LRR 243..266 10/25 (40%) 0.0004
20125 (80%)
LRRCT 300..350 11/55 (20%) 0.25
34/55 (62%)
Example 5.
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table SA.
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Table SA. NOVS Sequence
Analysis
SEQ ID NO: I 5 743 by
NOVSa, GTGCCAGCGCGGCGTGGGCCTCGGTCTGCGGCCATGGGGGTGTCCTCGCGGCTGCTGC
CG56785-O1 GTGTGGTGATCATGGGGGCCCCCGGCTCGGGCAAGGGCACTGTGTCGTCGCGTATCAC
DNA
Sequence TCAATACTTCGAGCTAAAGCACCTTTCCAGCGGGGACCTGCTCCGGGACAACATGCTG
CGGGGCGCAGAAATTGGCCTGTTAGCCAAGGCTTTCATTGACCAAGGGAAACTCATCC
CAAATGATGTCATCTTGGGCGTGGCCCTTCAGGAACTGCAAAATCTCACCCAGTCTAG
GCTGTTGGATAGTTTTCCAAGGACACTTCCACAGGCAGAAGCCCTAGATAAAGCTGAT
CAGACCGACACAGTGATTAACCTGAATATGTCCTTTGAGGTCATTAAACAACGCCTTA
CTGCTCACTGGATTCATCTCACCAATGGCCAAGTCTACAACATTGGATTCAACCCTCC
CACAACTGTGGGCATTGATGCTCTGACAGGGGAGCCGCTCATTCAGCGTGAGGATGAT
AAACCAGAGATGGTTATCAAGAGACTAAAGGCTTATGAAGCCAAACAAAGCCAGTCCT
GGACTATTACCAGAP.AAAAAGCGGTGTTGGAAACATTCTCCAGAACAGAAACCAACAA
GATTTGGCCCTGTGGATATGCTTTCCTCCAAACTGACGTTCCTCAAACAAGCCAGGAA
GCTTCAGTTACTCTATAAGGAGAAATGTGTGGAACTATTAGTAGTAA
ORF Start: ATG at
34 ORF Stop: TAA
at 712
SEQ ID NO: 16 226 MW at 25110.6kD
as
NOVSa, MGVSSRLLRWIMGAPGSGKGTVSSRITQYFELKHLSSGDLLRDNMLRGAEIGLLAKA
CG56785-O1 FIDQGKLIPNDVILGVALQELQNLTQSRLLDSFPRTLPQAEALDKADQTDTVINLNMS
Protein SequenceFEVIKQRLTAHWIFiLTNGQVYNIGFNPPTTVGIDALTGEPLIQREDDKPEMVIKRLKA
YEAKQSQSWTITRKKAVLETFSRTETNKIWPCGYAFLQTDVPQTSQEASVTL
Further analysis of the NOVSa protein yielded the following properties shown
in Table SB.
Table SB. Protein Sequence Properties NOVSa
PSort 0.3600 probability located in mitochondria) matrix space; 0.3000
probability located
analysis: in microbody (peroxisome); 0.2224 probability located in lysosome
(lumen); 0.0000
probability located in endoplasmic reticulum (membrane)
SignalP No Known Signal Sequence Predicted
analysis:
A search o...f .t>ae NOVSa protein against the Geneseq database, 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/ Similarities for the Expect Value
Identifier [Patent #, Date) Match Matched Region
Residues
AAW81101 Human mitochondria) adenylate kinase 1..225 177/226 (78%) 1e-92
protein - Homo Sapiens, 227 ad. 1..226 191/226 (84%)
[W09844124-A1, 08-OCT-1998]
98
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AAB85885 Human adenylate kinase 3 1..225 177/226 (78%)
(AK3)-like ~ 1 e-92
protein - Homo Sapiens, 227 aa. 1..226 191/226 (84%)
['1V0200109346-A1, 08-FEB-2001)
AAB93487 Human protein sequence SEQ 1..225 177/226 (78%)
ID 1 e-92
N0:12786 - Homo Sapiens, 227 aa. 1..226 191/226 (84%)
[EP1074617-A2, 07-FEB-2001)
AAB93066 Human protein sequence SEQ 1..225 177/226 (78%)
ID 1 e-92
N0:11883 - Homo Sapiens, 227 aa. 1..226 191/226 (84%)
[EP 1074617-A2, 07-FEB-2001 )
AAB92887 Human protein sequence SEQ 1..225 177/226 (78%)
ID 1 e-92
N0:11492 - Homo Sapiens, 227 aa. 1..226 191/226 (84%)
[EP1074617-A2, 07-FEB-2001)
In a BLAST search of public sequence datbases, the NOVSa protein was found to
have
homology to the proteins shown in the BLASTP data in Table SD.
Table SD. Public BLASTP Results for
NOVSa
Protein NOVSa Identities/.
Accession Protein/Organism/Length Residues)SimilaritiesEzpect
for
Number Match the MatchedValue
ResiduesPortion
Q9NPB4 CDNA FLJI 1089 FIS, CLONE 1..225 177/226 3e-92
(78%)
PLACE1005305, HIGHLY SIMILAR TO 1..226 191/226
(84%)
GTP:AMP PHOSPHOTRANSFERASE
MITOCHONDRIAL (EC 2.7.4.10) (CDNA
FLJ10691 FIS, CLONE NT2RP3000359,
HIGHLY SIMILAR TO GTP:AMP
PHOSPHOTRANSFERASE
MITOCHONDRIAL) (CDNA FLJ14628 FIS,
CLONE NT2RP2000329, HIGHLY SIMILAR
_ _
TO GTP:AMP PHOSPHOTRANSFERASE
MITOCHONDRIAL) (HYPOTHETICAL
25.6 KDA PROTEIN) - Homo sapiens
. (Human), 227 aa.
A34442 nucleoside-triphosphate--adenylate1..225 170/226 3e-90
kinase (EC (75%)
2.7.4.10) 3, mitochondria) - bovine, 1..226 188/226
227 aa. (82%)
Q9D7Z1 ADENYLATE KINASE 3 ALPHA LIKE 1..225 172/226 4e-90
- (76%) ~
Mus musculus (Mouse), 227 aa. 1..226 189/226
(83%)
Q9DBM5 ADENYLATE KINASE 3 ALPHA LIKE 1..225 171/226 1e-89
- . (75%)
Mus musculus (Mouse), 227 aa. 1..226 189/226
(82%)
P08760 GTP:AMP phosphotransferase mitochondria!2..225 169/225 1e-89
(75%)
(EC 2.7.4.10) (AK3) - Bos taurus (Bovine),1..225 187/225
(83%)
226 aa.
PFam analysis predicts that the NOVSa protein contains the domain shown in the
Table SE.
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Table 5E. Domain Analysis of NOVSa
Identities/
Pfam Domain NOVSa Match Region Similarities Expect Value
for the Matched Region
adenylatekinase 12..178 77/176 (44%) 1.2e-65
137/176 (78%)
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: 17 X2153 by
NOV6a, GATGCTGGCACTTACATGTGTGTGGCCCAGAACCCGGCTGGTACAGCCTTGGGCAAAA
CG56914-OI DNA T~GTTAAATGTCCAAGTTCCTCCAGTCATTAGCCCTCATCTAAAGGAATATGTTAT
Sequence TGCTGTGGACAAGCCCATCACGTTATCCTGTGAAGCAGATGGCCTCCCTCCGCCTGAC
ATTACATGGCATAAAGATGGGCGTGCAATTGTGGAATCTATCCGCCAGCGCGTCCTCA
GCTCTGGCTCTCTGCAAATAGCATTTGTCCAGCCTGGTGATGCTGGCCATTACACGTG
CATGGCAGCCAATGTAGCAGGATCAAGCAGCACAAGCACCAAGCTCACCGTCCATGTA
CCACCCAGGATCAGAAGTACAGAAGGACACTACACGGTCAATGAGAATTCACAAGCCA
TTCTTCCATGCGTAGCTGATGGAATCCCCACACCAGCAATTAACTGGAAAAAAGACAA
TGTTCTTTTAGCTAACTTGTTAGGAAAATACACTGCTGAACCATATGGAGAACTCATT
TTAGAAAATGTTGTGCTGGAGGATTCTGGCTTCTATACCTGTGTTGCTAACAATGCTG
CAGGTGAAGATACACACACTGTCAGCCTGACTGTGCATGTTCTCCCCACTTTTACTGA
TTCAGGTACTTATGTGTGCACCGCAGAGAACAGCGTTGGCTTTGTGAAGGCAATTGGA
TTTGTTTATGTGAAAGAACCTCCAGTCTT~..AAAGGTGATTATCCTTCTAACTGGATTG
AACCACTTGGTGGGAATGCAATCCTGAATTGTGAGGTGAAAGGAGACt~_C'c:,P~'Ce'~'1~AC
CATCCAGTGGAACAGAAAGGGAGTGGATATTGAAATTAGCCACAGAATCCGGCAACTG
GGCAATGGCTCCCTGGCCATCTATGGCACTGTTAATGAAGATGCCGGTGACTATACAT
GTGTAGCTACCAATGAAGCTGGGGTGGTGGAGCGCAGCATGAGTCTGACTCTGCAAAG
TCCTCCTATTATCACTCTTGAGCCAGTGGAAACTGTTATTAATGCTGGTGGCAAAATC
ATATTGAATTGTCAGGCAACTGGAGAGCCTCAACCAACCATTACATGGTCCCGTCAAG
GGCACTCTATTTCCTGGGATGACCGGGTTAACGTGTTGTCCAACAACTCATTATATAT
TGCTGATGCTCAGAAAGAAGATACCTCTGAATTTGAATGCGTTGCTCGAAACTTAATG
CTTGGGGAACATGCAGCGAAAGTTGTGGGAAAGGTACTCAGACAAGAGCAAGACTTTG
TAATAACCCACCACCAGCGTTTGGTGGGTCCTACTGTGATGGAGCAGAAACACAGATG
CAAGTTTGCAATGAAAGAAATTGTCCAATTCATGGCAAGTGGGCGACTTGGGCCAGTT
GGAGTGCCTGTTCTGTGTCATGTGGAGGAGGTGCCAGACAGAGAACAAGGGGCTGCTC
CGACCCTGTGCCCCAGTATGGAGGAAGGAAATGCGAAGGGAGTGATGTCCAGAGTGAT
TTTTGCAACAGTGACCCTTGCCCAAGTGAGTGTTGGAAATACCCATGGTAACTGGAGT
1~~
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CCTTGGA
ORF Start: ATG at 16 r ORF Stop: TAA at 2137
SEQ ID NO: I8 707 as MW at 76557.7kD
NOV6a, MCVAQNPAGTALGKIKLNVQVPPVISPHLKEYVIAVDKPITLSCEADGLPPPDITWHK
CG56914-OI DGRAIVESIRQRVLSSGSLQIAFVQPGDAGHYTCMAANVAGSSSTSTKLTVHVPPRIR
Protein SequenceSTEGHYTVNENSQAILPCVADGIPTPAINWKKDNVLLANLLGKYTAEPYGELILENW
LEDSGFYTCVANNAAGEDTHTVSLTVHVLPTFTELPGDVSLNKGEQLRLSCKATGIPL
PKLTWTFNNNIIPAHFDSVNGHSELVIERVSKEDSGTYVCTAENSVGFVKAIGFVYVK
EPPVFKGDYPSNWIEPLGGNAILNCEVKGDPTPTIQWNRKGVDIEISHRIRQLGNGSL
AIYGTVNEDAGDYTCVATNEAGVVERSMSLTLQSPPIITLEPVETVINAGGKIILNCQ
ATGEPQPTITWSRQGHSISWDDRVNVLSNNSLYIADAQKEDTSEFECVARNLMGSVLV
RVPVIVQVHGGFSQWSAWRACSVTCGKGIQKRSRLCNQPLPANGGKPCQGSDLEMRNC
QNKPCPVDGSWSEWSLWEECTRSCGRGNQTRTRTCNNPSVQHGGRPCEGNAVEIIMCN
IRPCPVHGAWSAWQPWGTCSESCGKGTQTRARLCNNPPPAFGGSYCDGAETQMQVCNE
RNCPIHGKWATWASWSACSVSCGGGARQRTRGCSDPVPQYGGRKCEGSDVQSDFCNSD
PCPSECWKYPW
SEQ ID NO: 19 15660 by
NOV6b, GATTAGTGGCATAAACTGTAGGTCAGCTGGTGGAGGCAAGCCAGCAAGGGGCTTCATG
~
CG56914-02 GTAACCAGTGGAAACACAAAAATATAAGGGGCTTCTGAGGCGATCGGGCAGTGTCAGT
DNA
Sequence CTTCAGCCGCTAAGCCGAGAAGATCTGGGAAGGAGTCAGTCAGAGAGCCTTGGGCCAG
AGTTCCAGGGGCTCTGGGAGTGGCTGCCAGAAAATACCAGAAAATGAAAGGAATTGAA
ATTAAGAGAAGGGAGAGATTGAAGTGTGGCGCCAAGATTGAAAGGAGAAAGAGGTTGA
AGGATAGGGAGGTTGGAGAAGAGAGTAAAAAGAGGCCACTTACTGGATTTGAAATTGA
ACCACCCAAAGTCACTGTGATGCCCAAGAATCAGTCTTTCACAGGAGGGTCTGAGGTC
TCCATCATGTGTTCTGCAACAGGTTATCCCAAACCAAAGATTGCCTGGACCGTTAACG
ATATGTTTATCGTGGGTTCACACAGGTATAGGATGACCTCAGATGGTACCTTATTTAT
CAAAAATGCAGCTCCCAAAGATGCAGGGATCTATGGTTGCCTAGCAAAAGCCCCTAAG
TTGATGGTAGTTCAGAGTGAGCTCTTGGTTGCCCTTGGGGATATAACCGTTATGGAAT
GCAAAACCTCTGGTATTCCTCCACCTCAAGTTAAATGGTTCAAAGGAGATCTTGAGTT
GAGGCCCTCAACATTCCTCATTATTGACCCTCTCTTGGGACTTTTGAAGATTCAAGAA
ACACAAGATCTGGATGCTGGCGATTATACCTGTGTAGCCATCAATGAGGCTGGAAGAG
CAACTGGCAAGATAACTCTGGATGTTGGCTCACCTCCAGTTTTCATACAAGAACCTGC
TGATGTGTCTATGGAAATTGGCTCAAATGTGACATTACCTTGTTATGTTCAGGGTTAT
CCAGAACCAACAATCAAATGGCGAAGATTAGACAACATGCCAATTTTCTCAAGACCTT
TTTCAGTTAGTTCCATCAGCCAACTAAGAACAGGAGCTCTCTTTATTTTAAACTTATG
GGCAAGTGATAAAGGAACCTATATTTGTGAAGCTGAAAACCAGTTTGGAAAGATCCAG
TCAGAGACAACAGTAACAGTGACCGGACTTGTTGCTCCACTTATTGGAATCAGCCCTT
CAGTGGCCAATGTTATTGAAGGACAGCAGCTTACTTTGCCCTGTAcTCTGTTAGCTGG
AAATCCCATTCCAGAACGTCGGTGGr"~TTAAGA.'vT iy:AC~c:~I~ATG~iutsC'1'(:CAAAAhTCCT
TACATCACTGTGCGCAGTGATGGGAGCCTCCATATTGAAAGAGTTCAGCTTCAGGATG
GTGGTGAATATACTTGTGTGGCCAGTAACGTTGCTGGGACCAATAACAAAACTACCTC
TGTGGTTGTGCATGTTCTGCCAACCATTCAGCATGGGCAGCAGATACTCAGTACAATT
GAAGGCATTCCAGTAACTTTACCATGCAAAGCAAGTGGAAATCCCAAACCGTCTGTCA
TCTGGTCCAAGGTAAATGATACATCTAGTTATATTTCCTGAAGAGCAGAGTGTGAAGT
TCACCTGCAAGTTATCCCTAGTCTTGAGCAGGAGGCTCAGGAGTGGGGCATGGAAAGA
AGATAAGTTAATAAAGGATTTCCTATGTGGCTGGACAGATGTGCTAGGAACCCTCCAA
GAAACCATATAGATGCACCTCAGAAGGCTCCCTCGGCTTTTCGCCGTGTTTTGCAGAA
AGGAGAGCTGATTTCAACCAGCAGTGCTAAGTTTTCAGCAGGAGCTGATGGTAGTCTG
TATGTGGTATCACCTGGAGGAGAGGAGAGTGGGGAGTATGTCTGCACTGCCACCAATA
CAGCCGGCTACGCCAAAAGGAAAGTGCAGCTAACAGTCTATGTAAGGCCCAGAGTGTT
TGGAGATCAACGAGGACTGTCCCAGGATAAGCCTGTTGAGATCTCCGTCCTTGCAGGG
GAAGAGGTAACACTTCCATGTGAAGTGAAGAGCTTACCTCCACCCATAATTACTTGGG
CCAAAGAAACCCAGCTCATCTCACCGTTCTCTCCAAGACACACATTCCTCCCTTCTGG
TTCAATGAAGATCACTGAAACCCGCACTTCAGATAGTGGGATGTATCTTTGTGTTGCC
ACAAATATTGCTGGGAATGTGACTCAGGCTGTCAAATTAAATGTCCATGTTCCTCCAA
AGATACAGCGTGGACCTAAACATCTCAAAGTCCAAGTTGGTCAAAGAGTGGATATTCC
ATGTAATGCTCAAGGGACTCCTCTTCCTGTAATCACCTGGTCCAAAGGTGGAAGCACT
ATGCTGGTTGATGGAGAGCACCATGTTAGCAATCCAGACGGAACTTTAAGCATCGACC
I0I
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TGATGAAACAGAGATAACGCTACATGTCCAAGAACCACCCACAGTGGAAGATCTAGAA
CCTCCATATAACACTACTTTCCAAGAAAGAGTGGCCAATCAACGCATTGAATTTCCAT
GACAGGCAGAGAGCCTGGCATTTCTATCTTGGAAGATGGCACATTGCTGGTTATTGCT
TCTGTTACACCCTATGACAATGGGGAGTACATCTGTGTGGCAGTCAATGAAGCTGGAA
CCACAGAAAGAAAATATAACCTCAAAGTCCATGTTCCTCCAGTAATTAAAGATAAAGA
TCAGCTGACCAATCTCTTCTGTGAAGTG
GAAGGCACTCCATCTCCCATCATTATGTGGTATAAAGATAATGTCCAGGTGACTGAAA
GCAGCACTATTCAGACTGTGAACAATGGGAAGATACTGAAGCTCTTCAGAGCCACTCC
TACTTTAACATTGATGTGCTAGGTACCAACTTCCCAAATGAAGTCTCAGTTGTCCTCA
GTTCAAAGATGGCAATATTAAAGGAGGAAATGTCACCACAGACATATCAGTATTGATC
TGTGAAACACGGGGACTTCCAATGCCTGCCATTACTT
GGTATAAGGACGGGCAGCCAATCATGTCCAGCTCACAAGCACTTTATATTGATAAAGG
ACAATATCTTCATATTCCTCGAGCACAGGTCTCTGATTCAGCAACATATACGTGTCAC
GTAGCCAATGTTGCTGGAACTGCTGP~P.AAATCATTCCATGTGGATGTCTATGTTCCTC
CAATGATTGAAGGCAACTTGGCCACGCCTTTGAATAAGCAAGTAGTTATTGCTCATTC
TCTGACACTGGAGTGCAAAGCTGCTGGAAACCCTTCTCCCATTCTCACCTGGTTGAAA
GATGGTGTACC~T~GuGAAiaGCTA.~~uGACt~.ATATCCGCATAGAAGCTGG'TGGt~AAGAAAG
TCGAAATCATGAGTGCCCAAGAAATTGATCGAGGACAGTACATATGCGTGGCTACCAG
TGTGGCAGGAGAAAAGGAAATCAAATATGAAGTTGATGTCTTGGTGCCACCAGCTATA
GAAGGAGGAGATGAAACATCTTACTTCATTGTGATGGTTAATAACTTACTGGAGCTAG
AATTGATGAAAGGGATGGATTCAAGATTTTATTAAATGGACGCAAACTGGTTATTGCT
CAGGCTCAAGTGTCAAACACAGGCCTTTATCGGTGCATGGCAGCAAATACTGCTGGAG
CCTTTCTGAGAGAGTTGTGGTAAAATACAAGCCTGTCGCCTTGCAGTGCATAGCCAAT
GGGATTCCAAATCCTTCCATTACATGGTTAAAAGATGACCAGCCTGTGAACACTGCCC
AAGGAAACCTTAAAATACAGTCTTCTGGTCGAGTTCTACAAATTGCCAAAACCCTGTT
GGAAGATGCTGGCAGATACACATGTGTGGCTACCAACGCAGCTGGAGAAACACAACAG
CACATTCAACTGCATGTTCATGAACCACCTAGTCTGGAAGATGCTGGAAAAATGCTGA
ATGAGACTGTGTTGGTGAGCAACCCTGTACAGCTGGAGTGTAAGGCAGCTGGAAATCC
TGTGCCTGTTATTACATGGTACAAAGATAATCGTCTACTCTCAGGTTCCACCAGCATG
ACTTTCTTGAACAGAGGACAGATCATTGATATTGAAAGTGCCCAGATCTCAGATGCTG
GCATATATAAATGCGTGGCCATCAACTCAGCTGGAGCTACAGAGTTATTTTACAGTCT
GCAAGTTCATGTGGCCCCATCAATTTCTGGCAGCAATAACATGGTGGCAGTGGTGGTT
AATAACCCGGTGAGGTTAGAATGTGAAGCCAGAGGTATTCCTGCCCCAAGTCTGACCT
GGTTGAAAGATGGGAGTCCTGTTTCTAGTTTTTCTAATGGATTACAGGTTCTCTCTGG
TGGTCGAFiTCCTAGCATTGACCAGTGCACAAATCAGCGACACAGi AAUt~TAC Av:.i'Pr('
GTGGCAGTGAATGCTGCTGGAGAAAAGCAAAGGGACATTGACCTCCGAGTATATGTTC
CGCCAAATATTATGGGAGAAGAACAGAATGTCTCTGTCCTCATTAGCCAAGCTGTGGA
CACCCCTTGCTGAAGAAACCAGGCCTCAGTATATCTGAAAATAGAAGTGTGTTAAAGA
TTGAAGATGCTCAGGTTCAAGACACTGGTCGTTACACTTGTGAAGCAACAAATGTTGC
TGGAAAAACTGAAAA:9AACTACAATGTCAACATTTGGGTCCCCCCAAATATTGGTGGT
TCTGATGAACTTACTCAACTTACAGTCATTGAAGGGAATCTCATTAGTCTGTTGTGTG
GACTGATTCCATGGGGCGAGTTAGAATTTTATCTGGGGGCAGGCAATTACAAATTTCA
ATTGCTGAAAAGTCTGATGCAGCACTCTATTCATGTGTGGCGTCGAATGTTGCTGGGA
CAGCCACCCTACTGAAATTATTGTGACCCGAGGGAAGAGTATCTCCTTGGAGTGTGAG
GTGCAGGGTATTCCACCACCAACAGTGACCTGGATGAAAGATGGCCACCCCTTGATCA
AGGCAAAGGGAGTAGAAATACTGGATGAAGGTCACATCCTTCAGCTGAAGAACATTCA
TGTATCTGACACAGGCCGTTATGTGTGTGTTGCTGTGAATGTAGCAGGAATGACTGAC
AAAAAATATGACTTAAGTGTCCATGGAGGCAGGATGCTACGGCTGATGCAGACCACAA
TGGAAGATGCTGGCCAATATACTTGCGTTGTAAGGAATGCAGCTGGTGAAGAAAGAAA
AATCTTTGGGCTTTCAGTATTAGTACCACCTCATATTGTGGGTGAAAATACATTGGAA
GATGTGAAGGTAAAAGAGAAACAGAGTGTTACGCTGACTTGTGAAGTGACAGGGAATC
CAGTGCCAGAAATTACATGGCACAAAGATGGGCAGCCCCTCCAAGAAGATGAAGCCCA
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TCACATTATATCTGGTGGCCGTTTTCTTCAAATTACCAATGTCCAGGTGCCACACACT
TGTCACTGTCATCCTTAACAGCCCTACATCTTTGGTCTGTGAAGCTTATTCATATCCT
CCAGCTACCATCACCTGGTTTAAGGATGGCACTCCTTTAGAATCTAACCGAAATATTC
AAGATACTCTTGTGTAGCCACGAATGAGGCTGGAGAAATGATAAAGCACTATGAAGTG
ATGATCATGTTAATATTGCTGCGAATGGACACACACTTCAAATAAAGGAGGCTCAAAT
ATCAGACACCGGACGATATACTTGTGTAGCATCTAACATTGCAGGTGAAGATGAGTTG
GATTTTGATGTGAATATTCAAGTTCCTCCAAGTTTTCAGAAACTCTGGGAAATAGGAA
ACATGCTAGATACTGGCAGGAATGGTGAAGCCAAAGATGTGATCATCAACAATCCCAT
TTCTCTTTACTGTGAGACAAATGCTGCTCCCCCTCCTACACTGACATGGTACAAAGAT
GGCCACCCTCTGACCTCAAGTGATAAAGTATTGATTTTGCCAGGAGGGCGAGTGTTGC
AGATTCCTCGGGCTAAAGTAGAAGATGCTGGGAGATACACATGTGTGGCTGTGAATGA
GGCTGGAGAAGATTCCCTTCAATATGATGTCCGTGTACTCGTGCCGCCAATTATCAAG
GGAGCAAATAGTGATCTCCCTGAAGAGGTCACCGTGCTGGTGAACAAGAGTGCACTGA
TAGAGTGTTTATCCAGTGGCAGCCCAGCACCAAGGAATTCCTGGCAGAAAGATGGACA
GCCCTTGCTAGAAGATGACCATCATAAATTTCTATCTAATGGACGAATTCTGCAGATT
CTGAATACTCAAATAACAGATATCGGCAGGTATGTGTGTGTTGCTGAGAACACAGCTG
GGAGTGCCA~AAATATTTTAACCTCAP.TGTTCATGTTCCTCCAAGTGTCATTGGTCC
TAAATCTGAAAATCTTACCGTCGTGGTGAACAATTTCATCTCTTTGACCTGTGAGGTC
TCTGGTTTTCCACCTCCTGACCTCAGCTGGCTCAAGAATGAACAGCCCATCAAACTGA
TGTGCCTGGTGGTCGAACTCTACAGATTATTCGGGCCAAGGT
TACACTTGTATAGCTATCAATCAAGCTGGCGAAAGCAAGAAA
~vU'r'r't°rcc:c'rGAC'rG r t"1'ATGTGCCCCCAAGCATTAAAGACCATGACAGTGAATCTC
TTTCTGTAGTTAATGTAAGAGAGGGAACTTCTGTGTCTTTGGAGTGTGAGTCGAACGC
TGTGCCACCTCCAGTCATCACTTGGTATAAGAATGGGCGGATGATAACAGAGTCTACT
CATGTGGAGATTTTAGCTGATGGACAAATGCTACACATTAAGAAAGCTGAGGTATCTG
ACACAGGCCAGTATGTATGTAGAGCTATAAATGTAGCAGGACGGGATGATAAAAATTT
CCACCTCAATGTATATGTGCCACCCAGTATTGAAGGACCTGAAAGAGAAGTGATTGTG
GAGACGATCAGCAATCCTGTGACATTAACATGTGATGCCACTGGGATCCCACCTCCCA
CGATAGCATGGTTAAAGAACCACAAGCGCATAGAAAATTCTGACTCACTGGAAGTTCG
TATTTTGTCTGGAGGTAGCAAACTCCAGATTGCCCGGTCTCAGCATTCAGATAGTGGA
AACTATACATGTATTGCTTCAAATATGGAGGGAAAAGCCCAGAAATATTACTTTCTTT
CAATTCAAGTTCCTCCAAGTGTTGCTGGTGCTGAAATTCCAAGTGATGTCAGTGTCCT
TCTAGGAGAAAATGTTGAGCTGGTCTGCAATGCAAATGGCATTCCTACTCCACTTATT
CAATGGCTTAAAGATGGAAAGCCCATAGCTAGTGGTGAAACAGAAAGAATCCGAGTGA
GTGCAAATGGCAGCACATTAAACATTTATGGAGCTCTTACATCTGACACGGGGAAATA
CACATGTGTTGCTACTAATCCCGCTGGAGAAGAAGACCGAATTTTTAACTTGAATGTC
TATG T TACACC TACAATTAGG;GGTAA~T~AAAUAuGAA GCAGAGAAACTAATGACTTTAC.
TGGATACTTCAATAAATATTGAATGCAGAGCCACAGGGACGCCTCCACCACAGATAAA
~CTGGCTGAAGAATGGACTTCCTCTGCCTCTCTCCTCCCATATCCGGTTACTGGCAGCA
~GGACAAGTTATCAGGATTGTGAGAGCTCAGGTGTCTGATGTCGCTGTGTATACTTGTG
'TGGCCTCCAACAGAGCTGGGGTGGATAATAAGCATTACAATCTTCAAGTGTTTGCACC
ACCAAATATGGACAATTCAATGGGGACAGAGGAAATCACAGTTCTCAAAGGTAGTTCC
ACCTCTATGGCATGCATTACTGATGGAACCCCAGCTCCCAGTATGGCCTGGCTTAGAG
ATGGCCAGCCTCTGGGGCTTGATGCCCATCTGACAGTCAGCACCCATGGAATGGTCCT
GCAGCTCCTCAAAGCAGAGACTGAAGATTCGGGAAAGTACACCTGCATTGCCTCAAAT
GAAGCTGGAGAAGTCAGCAAGCACTTTATCCTCAAGGTCCTAGAACCACCTCACATTA
ATGGATCTGAAGAACATGAAGAGATATCAGTAATTGTTAATAACCCACTTGAACTTAC
CTGCATTGCTTCTGGAATCCCAGCCCCTAAAATGACCTGGATGAAAGATGGCCGGCCC
CTTCCACAGACGGATCAAGTGCAAACTCTAGGAGGAGGAGAGGTTCTTCGAATTTCTA
CTGCTCAGGTGGAGGATACAGGAAGATATACATGTCTGGCATCCAGTCCTGCAGGAGA
TGATGATAAGGAATATCTAGTGAGAGTGCATGTACCTCCTAATATTGCTGGAACTGAT
GAGCCCCGGGATATCACTGTGTTACGGAACAGACAAGTGACATTGGAATGCAAGTCAG
ATGCAGTGCCCCCACCTGTAATTACTTGGCTCAGAAATGGAGAACGGTTACAGGCAAC
ACCTCGAGTGCGAATCCTATCTGGAGGGAGATACTTGCAAATCAACAATGCTGACCTA
GGTGATACAGCCAATTATACCTGTGTTGCCAGCAACATTGCAGGAAAGACTACAAGAG
AATTTATTCTCACTGTAAATGTTCCTCCAAACATAAAGGGGGGCCCCCAGAGCCTTGT
AATTCTTTTAAATAAGTCAACTGTATTGGAATGCATCGCTGAAGGTGTCCCAACTCCA
103
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TCTTGGAAAATGGATTCCTTCATATTCAATCAGCACATGTCACTGACACTGGACGGTA
TTTGTGTATGGCCACCAATGCTGCTGGAACAGATCGCAGGCGAATAGATTTACAGGTC
CATGTTCCTCCATCTATTGCTCCGGGTCCTACCAACATGACTGTAATAGTAAATGTTC
AAACTACTCTGGCTTGTGAGGCTACTGGGATACCAAAACCATCAATCAATTGGAGAAA
TTATTTCCCCTTCTGTGGATGACACTGCAACCTATGAATGTACTG
TGACAAACGGTGCTGGAGATGATAAAAGAACTGTGGATCTCACTGTCCAAGTTCCACC
TTCCATAGCTGATGAGCCTACAGATTTCCTAGTAACCAAACATGCCCCAGCAGTAATT
TGGTATAA
.TTCTGTCCTCAGGAGCAATTGAAATACT
TGCCACCCAATTAAACCATGCTGGAAGATACACTTGTGTCGCTAGGAATGCGGCTGGC
CAAGTGAACTACACGTCATTCTGAACAATCCTATTTTATTACCATGTGAAGCAACAGG
GACACCCAGTCCTTTCATTACTTGGCAAAAAGAAGGCATCAATGTTAACACTTCAGGC
CAAGTTAAATGTCCAAGTTCCTCCAGTCATTAGCCCTCATCTAAAGGAATATGTTATT
TTACATGGCATAAAGATGGGCGTGCAATTGTGGAATCTATCCGCCAGCGCGTCCTCAG
CTCTGGCTCTCTGCAAATAACATTTGTCCAGCCTGGTGAfiGCTGGCCATTACACGTGC
CACCCAGGATCAGAAGTACAGAAGGACACTACACGGTCAATGAGAATTCACAAGCCAT
TCTTCCATGCGTAGCTGATGGAATCCCCACACCAGCAATTAACTGGAAAAAAGACAAT
GTTCTTTTAGCTAACTTGTTAGGAAAATACACTGCTGAACCATATGGAGAACTCATTT
TAGAAAATGTTGTGCTGGAGGATTCTGGCTTCTATACCTGTGTTGCTAACAATGCTGC
AGGTGAAGATACACACACTGTCAGCCTGACTGTGCATGTTCTCCCCACTTTTACTGAA
CTTCCTGGAGACGTGTCATTAAATAAAGGAGAACAGCTACGATTAAGCTGTAAAGCTA
CTGGTATTCCATTGCCCAAATTAACATGGACCTTCAATAACAATATTATTCCAGCCCA
CTTTGACAGTGTGAATGGACACAGTGAACTTGTTATTGAAAGAGTGTCAAAAGAGGAT
TCAGGTACTTATGTGTGCACCGCAGAGAACAGCGTTGGCTTTGTGAAGGCAATTGGAT
TTGTGTATGTGAAAGAACCTCCAGTCTTCAAAGGTGATTATCCTTCTCACTGGATTGA
ACCACTTGGTGGGAATGCAATCCTGAATTGTGAGGTGAAAGGAGACCCCACCCCAACC
ATCCAGTGGAACAGAAAGGGAGTGGATATTGAAATTAGCCACAGAATCCGGCAACTGG
GCAATGGCTCCCTGGCCATCTATGGCACTGTTAATGAAGATGCCGGTGACTATACATG
TGTAGCTACCAATGAAGCTGGGGTGGTGGAGCGCAGCATGAGTCTGACTCTGCAAAGT
CCTCCTATTATCACTCTTGAGCCAGTGGAAACTGTTATTAATGCTGGTGGCAAAATCA
TATTGAATTGTCAGGCAACTGGAGAGCCTCAACCAACCATTACATGGTCCCGTCAAGG
GCACTCTATTTCCTGGGATGACCGGGTTAACGTGTTGTCCAACAACTCATTATATATT
GCTGATGCTCAGAAAGAAGATACCTCTGAATTTGAATGTGTTGCTCGAAACTTAATGG
GTTCTGTCCTTGTCAGAG T GCCAGVTCATAG'T'CCAGGTTCATt;C_aTGGATTTTCrCprT,_;
GTCTGCATGGAGAGCCTGCAGTGTCACCTGTGGAAAAGGCATCCAAAAGAGGAGTCGT
CTGTGCAACCAGCCCCTTCCAGCCAATGGTGGGAAGCCCTGCCAAGGTTCAGATTTGG
TCTTTGGGAAGAATGCACAAGGAGCTGTGGACGCGGCAACCAAACCAGGACCAGGACT
TCCATCAGTTCAGCATGGTGGGCGGCCATGTGAAGGGAATGCTGTGGAAA
AATAACCCACCACCAGCGTTTGGTGGGTCCTACTGTGATGGAGCAGAAACACAGATGC
AAGTTTGCAATGAAAGAAATTGTCCAGTTCATGGCAAGTGGGCGACTTGGGCCAGTTG
GAGTGCCTGTTCTGTGTCATGTGGAGGAGGTGCCAGACAGAGAACAAGGGGCTGCTCC
GACCCTGTGCCCCAGTATGGAGGAAGGAAATGCGAAGGGAGTGATGTCCAGAGTGATT
TTTGCAACAGTGACCCTTGCCCAACCCATGGTAACTGGAGTCCTTGGAGTGGCTGGGG
AACATGCAGCCGGACGTGTAACGGAGGGCAGATGCGGCGGTACCGCACATGTGATAAC
CCTCCTCCCTCCAATGGGGGAAGAGCTTGTGGGGGACCAGACTCCCAGATCCAGAGGT
GCAACACTGACATGTGTCCTGTGGATGGAAGTTGGGGAAGCTGGCATAGTTGGAGCCA
GTGCTCTGCCTCCTGTGGAGGAGGTGAAAAGACTCGGAAGCGGCTGTGCGACCATCCT
GTGCCAGTTAAAGGTGGCCGTCCCTGTCCCGGAGACACTACTCAGGTGACCAGGTGCA
ATGTACAAGCATGTCCAGGTGGGCCCCAGCGAGCCAGAGGAAGTGTTATTGGAAATAT
TAATGATGTTGAATTTGGAATTGCTTTCCTTAATGCCACAATAACTGATAGCCCTAAC
TCTGATACTAGAATAATACGTGCCAAAATTACCAATGTACCTCGTAGTCTTGGTTCAG
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CAATGAGAAAGATAGTTTCTATTCTAAATCCCATTTATTGGACAACAGCAAAGGAAAT
AGGAGAAGCAGTCAATGGCTTTACCCTCACCAATGCAGTCTTCAAAAGAGAAACY'CAA
GTGGAATTTGCAACTGGAGAAATCTTGCAGATGAGTCATATTGCCCGGGGCTTGGATT
CCGATGGTTCTTTGCTGCTAGATATCGTTGTGAGTGGCTATGTCCTACAGCTTCAGTC
ACCTGCTGAAGTCACTGTAAAGGATTACACAGAGGACTACATTCAAACAGGTCCTGGG
CAGCTGTACGCCTACTCAACCCGGCTGTTCACCATTGATGGCATCAGCATCCCATACA
CATGGAACCACACCGTTTTCTATGATCAGGCACAGGGAAGAATGCCTTTCTTGGTTGA
AACACTTCATGCATCCTCTGTGGAATCTGACTATAACCAGATAGAAGAGACACTGGGT
TTTAAAATTCATGCTTCAATATCCAAAGGAGATCGCAGTAATCAGTGCCCCTCCGGGT
TTACCTTAGACTCAGTTGGACCTTTTTGTGCTGATGAGGATGAATGTGCAGCAGGGAA
TCCCTGCTCCCATAGCTGCCACAATGCCATGGGGACTTACTACTGCTCCTGCCCTAAA
GGCCTCACCATAGCTGCAGATGGAAGAACTTGTCAAGATATTGATGAGTGTGCTTTGG
GTAGGCATACCTGCCACGCTGGTCAGGACTGTGACAATACGATTGGATCTTATCGCTG
TGTGGTCCGTTGTGGAAGTGGCTTTCGAAGAACCTCTGATGGGCTGAGTTGTCAAGAT
ATTAATGAATGTCAAGAATCCAGCCCCTGTCACCAGCGCTGTTTCAATGCCATAGGAA
GTTTCCATTGTGGATGTGAACCTGGGTATCAGCTCAAAGGCAGAAAATGCATGGATGT
GAACGAGTGTAGACAAAATGTATGCAGACCAGATCAGCACTGTAAGAACACCCGTGGT
GGCTATAAGTGCATTGATCTTTGTCCAAATGGAATGACCAAGGCAGAAAATGGAACCT
GTATTGATATTGATGAATGTAAAGATGGGACCCATCAGTGCAGATATAACCAGATATG
TGAGAATACAAGAGGCAGCTATCGTTGTGTATGCCCAAGAGGTTATCGGTCTCAAGGA
GTTGGAAGACCCTGCATGGACATTAATGAATGTGP.A.CAAGTGCCTan,~CCT_rGmr_raC
ATCAGTGCTCCAACACCCCCGGCAGCTTCAAGTGTATCTGTCCACCAGGACAACATTT
ATTAGGGGACGGGAAATCTTGCGCTGGATTGGAGAGGCTGCCAAATTATGGCACTCAA
TACAGTAGCTATAACCTTGCACGGTTCTCCCCTGTGAGAAACAACTATCAACCTCAAC
AGCATTACAGACAGTACTCACATCTCTACAGCTCCTACTCAGAGTATAGAAACAGCAG
AACATCTCTCTCCAGGACTAGAAGGACTATTAGGAAAACTTGCCCTGAAGGCTCTGAG
GCAAGCCATGACACATGTGTAGATATTGATGAATGTGAAAATACAGATGCCTGCCAGC
ATGAGTGTAAGAATACCTTTGGAAGTTATCAGTGCATCTGCCCACCTGGCTATCAACT
CACACACAATGGAAAGACATGCCAAGATATCGATGAATGTCTGGAGCAGAATGTGCAC
TGTGGACCCAATCGCATGTGCTTCAACATGAGAGGAAGCTACCAGTGCATCGATACAC
CCTGTCCACCCAACTACCAACGGGATCCTGTTTCAGGGTTCTGCCTCAAGAACTGTCC
ACCCAATGATTTGGAATGTGCCTTGAGCCCATATGCCTTGGAATACAAACTCGTCTCC
CTCCCATTTGGAATAGCCACCAATCAAGATTTAATCCGGCTGGTTGCATACACACAGG
ATGGAGTGATGCATCCCAGGACAACTTTCCTCATGGTAGATGAGGAACAGACTGTTCC
TTTTGCCTTGAGGGATGAAAACCTGAAAGGAGTGGTGTATACAACACGACCACTACGA
GAAGCAGAGACCTACCGCATGAGGGTCCGAGCCTCATCCTACAGTGCCAATGGGACCA
TTGAATATCAGACCACATTCATAGTTTATATAGCTGTGTCCGCCTATCCATACTAA
GG
_
AACTCTCCAAAGCCTATTCCACATATTTAAACCGCATTAATCATGGCAATCAAGCCCC
CTTCCAGATTACTGTCTCTTGAACAGTTGCAATCTTGGCAGCTTGAAAATGGTGCTAC
ACTCTGTTTTGTGTGCCTTCCTTGGTACTTCTGAGGTATTTTCATGATCCCACCATGG
TCATATCTTGAAGTATGGTCTAGAAAAGTCCCTTATTATTTTATTTATTAC_nCTGGAr
CAGTTACTTCCCAAAGATTATTCTGAACATCTAACAGGACATATCAGTGATGGTTTAC
AGTAGTGTAGTACCTAAGATCATTTTCCTGAAAGCCAAACCAAACAACGAAAAACAAG
AACAACTAATTCAGAATCAAATAGAGTTTTTGAGCATTTGACTATTTTTAGAATCATA
AAATTAGTTACTAAGTATTTTGATCAAAGCTTATAAAATAACTTACGGAGATTTTTGT
AAGTATTGATACATTATAATAGGACTTGCCTATTTTCATTTTTAAGAAGAAAAACCCG
ORF Start: ATG at 1649 ORF Stop:
TAA at 15134
SEQ ID NO: 20 4495 as MW at 488830.SkD
NOV6b, MWLDRCARNPPRNHIDAPQKAPSAFRRVLQKGELISTSSAKFSAGADGSLYWSPGGE
CG569I4-02 ESGEYVCTATNTAGYAKRKVQLTVYVRPRVFGDQRGLSQDKPVEISVLAGEEVTLPCE
Protein Sequence~SLPPPIITWAKETQLISPFSPRHTFLPSGSMKiTETRTSDSGMYLCVATNIAGNVT
QAVKLNVHVPPKIQRGPKHLKVQVGQRVDIPCNAQGTPLPVITWSKGGSTMLVDGEHH
VSNPDGTLSIDQATPSDAGIYTCVATNIAGTDETEITLHVQEPPTVEDLEPPYNTTFQ
ERVANQRIEFPCPAKGTPKPTIKWLFINGRELTGREPGISILEDGTLLVIASVTPYDNG
EYICVAVNEAGTTERKYNLKVH~7PpVIKDKEQVTNVSVLLNQLTNLFCEVEGTPSPII
MWYKDNVQVTESSTIQTVNNGKILKLFRATPEDAGRYSCKAINIAGTSQKYFNIDVLG
TNFPNEVSWLN'RDVALECQVKGTPFPDIHWFKDGNIKGGNVTTDISVLINSLIKLEC
ETRGLPMPAITWYKDGQPIMSSSQALYIDKGQYLHIPRAQVSDSATYTCHVANVAGTA
EKSFHVDVYVPPMIEGNLATPLNKQWIAHSLTLECKAAGNPSPILTWLKDGVPVKAN
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FIVMVNNLLELDCHVTGSPPPTIMWLKDGQLIDERDGFKILLNGRKLVIAQAQVSNTG
LYRCMAANTAGDHKKEFEVTVHVPPTIKSSGLSERVVVICYKPVALQCIANGIPNPSIT
WLKDDQPVNTAQGNLKIQSSGRVLQIAKTLLEDAGRYTCVATNAAGETQQHIQLHVHE
PPSLEDAGKMLNETVLVSNPVQLECKAAGNPVPVITWYKDNRLLSGSTSMTFLNRGQI
IDIESAQISDAGIYKCVAINSAGATELFYSLQVHVAPSISGSNNMVAVVVNNPVRLEC
EARGIPAPSLTWLKDGSPVSSFSNGLQVLSGGRILALTSAQISDTGRYTCVAVNAAGE
KQRDIDLRVYVPPNIMGEEQNVSVLISQAVELLCQSDAIPPPTLTWLKDGHPLLKKPG
LSISENRSVLKIEDAQVQDTGRYTCEATNVAGKTEXZJYNVNIWVPPNIGGSDELTQLT
VIEGNLISLLCESSGIPPPNLIWKKKGSPVLTDSMGRVRILSGGRQLQISIAEKSDAA
LYSCVASNVAGTAKKEYNLQVYIRPTITNSGSHPTEIIVTRGKSISLECEVQGIPPPT
VTWMKDGHPLIKAKGVEILDEGHILQLKNIHVSDTGRYVCVAVNVAGMTDKKYDLSVH
GGRMLRLMQTTMEDAGQYTCWRNAAGEERKIFGLSVLVPPHIVGENTLEDVKVKEKQ
SVTLTCEVTGNPVPEITWHKDGQPLQEDEAHHIISGGRFLQITNVQVPHTGRYTCLAS
SPAGHKSRSFSLNVFVSPTIAGVGSDGNPEDVTVILNSPTSLVCEAYSYPPATITWFK
DGTPLESNRNIRILPGGRTLQILNAQEDNAGRYSCVATNEAGEMIKHYEVKVYTLNAN
IVIIESQPLKSDDHVNIAANGHTLQIKEAQISDTGRYTCVASNIAGEDELDFDVNIQV
PPSFQKLWEIGNMLDTGRNGEAKDVIINNPISLYCETNAAPPPTLTWYKDGHPLTSSD
KVLILPGGRVLQIPRAKVEDAGRYTCVAVNEAGEDSLQYDVRVLVPPIIKGANSDLPE
EVTVLVNKSALIECLSSGSPAPRNSWQKDGQPLLEDDHHKFLSNGRILQILNTQTTDI
GRYVCVAENTAGSAKKYFNLNVHVPPSVIGPKS~~TV~7Vr'!NFT_ ST_.m~crFPPPZ,T.
SWLKNEQPIKLNTNTLIVPGGRTLQIIRAKVSDGGEYTCIAINQAGESKKKFSLTVYV
PPSIKDHDSESLSVVNVREGTSVSLECESNAVPPPVITWYKNGRMITESTHVEILADG
QMLHIKKAEVSDTGQYVCRAINVAGRDDKNFHLNVYVPPSIEGPEREVIVETISNPVT
'LTCDATGIPPPTIAWLKNHKRIENSDSLEVRILSGGSKLQIARSQHSDSGNYTCIASN
MEGKAQKYYFLSIQVPPSVAGAEIPSDVSVLLGENVELVCNANGIPTPLIQWLKDGKP
',IASGETERIRVSANGSTLNIYGALTSDTGKYTCVATNPAGEEDRIFNLNVYVTPTIRG
NKDEAEKLMTLVDTSINIECRATGTPPPQINWLKNGLPLPLSSHTRLLAAGQVIRIVR
AQVSDVAVYTCVASNRAGVDNKHYNLQVFAPPNMDNSMGTEEITVLKGSSTSMACITD
GTPAPSMAWLRDGQPLGLDAHLTVSTHGMVLQLLKAETEDSGKYTCIASNEAGEVSKH
FILKVLEPPHINGSEEHEEISVIVNNPLELTCIASGIPAPKMTWMKDGRPLPQTDQVQ
TLGGGEVLRISTAQVEDTGRYTCLASSPAGDDDKEYLVRVHVPPNIAGTDEPRDITVL
RNRQVTLECKSDAVPPPVITWLRNGERLQATPRVRILSGGRYLQINNADLGDTANYTC
VASNIAGKTTREFILTVNVPPNIKGGPQSLVILLNKSTVLECIAEGVPTPRITWRKDG
AVLAGNHARYSILENGFLHIQSAHVTDTGRYLCMATNAAGTDRRRIDLQVHVPPSIAP
GPTNMTVIVNVQTTLACEATGIPKPSINWRKNGHLLNVDQNQNSYRLLSSGSLVIISP
SVDDTATYECTVTNGAGDDKRTVDLTVQVPPSIADEPTDFLVTKHAPAVITCTASGVP
FPSIHWTKNGIRLLPRGDGYRILSSGAIEILATQLNHAGRYTCVARNAAGSAHRHVTL
HVHEPPVIQPQPSELHVILNNPILLPCEATGTPSPFITWQKEGINVNTSGRNHAVLPS
GGLQISRAVREDAGTYMCVAQNPAGTALGKIKLNVQVPPVISPHLKEYVIAVDKPITL,
~CEADGT PPPDITWHk77GRAZVESIRQR'v'LSSGSLQIT~'T~QPGDAGHYTCMA.~TNVAGS
SSTSTKLTVHVPPRIRSTEGHYTVNENSQAILPCVADGIPTPATNWKKDNVLLANLLG
KYTAEPYGELILENVVLEDSGFYTCVANNAAGEDTHTVSLTVHVLPTFTELPGDVSLN
KGEQLRLSCKATGIPLPKLTWTFNNNIIPAHFDSVNGHSELVIERVSKEDSGTYVCTA
ENSVGFVKAIGFVYVKEPPVFKGDYPSHWIEPLGGNAILNCEVKGDPTPTIQWNRKGV
DIEISHRIRQLGNGSLAIYGTVNEDAGDYTCVATNEAGVVERSMSLTLQSPPIITLEP
VETVINAGGKIILNCQATGEPQPTITWSRQGHSISWDDRVNVLSNNSLYIADAQKEDT
SEFECVARNLMGSVLVRVPVIVQVHGGFSQWSAWRACSVTCGKGIQKRSRLCNQPLPA
MRNCQNKPCPVDGSWSEWSLWEECTRSCGRGNQTRTRTCNNPSVQH
IMCNIRPCPVHGAWSAWQPWGTCSESCGKGTQTRARLCNNPPPAFG
RKCEGSDVQSDFCNSDPCPTHGNWSPWSGWGTCSRTCNGGQMRRYRTCDNPPPSNGGR
ACGGPDSQIQRCNTDMCPVDGSWGSWHSWSQCSASCGGGEKTRKRLCDHPVPVKGGRP
CPGDTTQVTRCNVQACPGGPQRARGSVIGNINDVEFGIAFLNATITDSPNSDTRIIRA
KITNVPRSLGSAMRKIVSILNPIYWTTAKEIGEAVNGFTLTNAVFKRETQVEFATGEI
LQMSHIARGLDSDGSLLLDIVVSGYVLQLQSPAEVTVKDYTEDYIQTGPGQLYAYSTR
LFTIDGISIPYTWNHTVFYDQAQGRMPFLVETLHASSVESDYNQIEETLGFKIHASIS
KGDRSNQCPSGFTLDSVGPFCADEDECAAGNPCSHSCHNAMGTYYCSCPKGLTIAADG
RTCQDIDECALGRHTCHAGQDCDNTIGSYRCVVRCGSGFRRTSDGLSCQDINECQESS
PCHQRCFNAIGSFHCGCEPGYQLKGRKCMDVNECRQNVCRPDQHCKNTRGGYKCIDLC
PNGMTKAENGTCIDIDECKDGTHQCRYNQICENTRGSYRCVCPRGYRSQGVGRPCMDI
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NECEQVPKPCAHQCSNTPGSFKCICPPGQHLLGDGKSCAGLERLPNYGTQYSSYNLAR
FSPVRNNYQPQQHYRQYSHLYSSYSEYRNSRTSLSRTRRTIRKTCPEGSEASHDTCVD
IDECENTDACQHECKNTFGSYQCICPPGYQLTHNGKTCQDIDECLEQNVHCGPNRMCF
QDLIRLVAYTQDGVNgiPRTTFLMVDEEQTVPFALRDENLKGVVYTTRPLREAETYRM12
VRASSYSANGTIEYQTTFIVYIAVSAYPY
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 6B.
Table 6B. Comparison of NOV6a against NOV6b.
Protein Sequence NOV6a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOV6b 1..707 698/707 (98%)
2917..3623 701/707 (98%)
Further analysis of the NOV6a protein yielded the following properties shown
in Table 6C.
Table 6C. Protein Sequence Properties NOV6a
PSort 0.4500 probability located in cytoplasm; 0.3000 probability located in
microbody
analysis: (peroxisome); 0.1000 probability located in mitochondrial matrix
space; 0.1000
probability located in lysosome (lumen)
SignalP No Known Signal Sequence Predicted
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
IdentifierDate] Match the Matched Value
ResiduesRegian
AAB4777IHuman thrombospondin protein,1..707 699/707 (98%)0.0
BTL.012 - Homo sapiens, 184..890702/707 (98%)
1336 aa.
[WO200174852-A2, 11-OCT-2001]
ABG03933Novel human diagnostic protein1..471 471/471 (100%)0.0
#3924 -
Homo sapiens, 1240 aa. 442..912471/471 (100%)
[W0200175067-A2, I I-OCT-2001]
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ABG03933 Novel human diagnostic protein1..471 471/471 (100%) 0.0
#3924 -
Homo sapiens, 1240 aa. 442..912471/471 (100%)
[W0200175067-A2, I I-OCT-2001]
AAG67244 Amino acid sequence of murine395..707246/313 (78%) e-164
thrombospondin 1-like protein - Mus 1..313 280/313 (88%)
musculus, 1068 aa. [W0200109321-Al,
08-FEB-2001
AAB47770 Human thrombospondin protein,471..678208/208 (100%) e-135
BTL.012, fragment 654-861 -Homo 1..208 208/208 (100%)
Sapiens, 208 aa. [W0200174852-A2,
11-
OCT-2001
In a BLAST search of public sequence datbases, the NOV6a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 6E.
Table 6E. Public BLASTP Results for NOV6a
NOV6a Identities/
Protein Residues/SimilaritiesExpect
for
AccessionProtein/Organism/Length Match the MatchedValue
Number ResiduesPortion 1
Q96RW7 HEMICENTIN - Homo Sapiens 1..707 700/707 ~ 0.0
(Human), (99%)
5636 aa. 4058..4764701/707
(99%)
Q96SC3 FIBULIN-6 - Homo sapiens (Human),1..707 698/707 0.0
2673 (98%)
as (fragment). 1095..1801701/707 ~
(98%)
Q96DN3 CDNA FLJ31995 FIS, CLONE 1..460 159/475 8e-64
(33%)
NT2RP7009236, WEAKLY SIMILAR 782..12522351475 ~
TO (49%)
BASEMENT MEMBRANE-SPECIFIC
HEPARAN SULFATE PROTEOGLYCAN
CORE PROTEIN PRECURSOR - Homo
Sapiens (Human), 1252 as (fragment):
T20992 hypothetical protein F15G9.4a-2..511 159/529 1e-59
~ (30%)
Caenorhabditis elegans, 5175 3014..3521241/529
aa. (45%)
076518 HEMICENTIN PRECURSOR- 2..511 159/529 1e-59
(30%)
~ ~ ~
Caenorhabditis elegans, S 3014..3521241 /529
198 aa. (45%)
PFam analysis predicts that the NOV6a protein contains the domains shown in
the Table
6F.
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Table 6F. Domain
Analysis of
NOV6a
Identities/
Pfam DomainNOV6a Match Similarities Expect
Region Value
for the Matched
Region
Ig 37..94 19161 (31 %) 1.1 e-10
43161 (70%)
Ig 127..185 16/62 (26%) 1 e-08
39/62 (63%)
Ig 218..274 20/60 (33%) 9.5e-12
43/60 (72%)
Ig 308..365 20/61 (33%) 2.7e-10
42/6 i (69%)
Ig 398..455 17/61 (28%) ~ 1.6e-09
42/61 (69%)
tsp 1 477..527 28/54 (52%) 1.1 e-16
3 69%)
( 7/54
tsp 1 534..584 25/54 (46%) 5.7e-14
4 I /54 (76%)
tsp 1 591..641 22/54 (41 %) 4e-12
3 67%)
( 6154
tsp 1 648..698 23/54 (43%) 1.9e-14
3 7/54
( 69%)
Example 7.
The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 7A.
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TGGACGCTTCCCGGGCCCAGTTCTTCCTGCACTTGTCCCCAAGCCACTATGCTCTGCTI
TGCAGAAAAGTAGCAGTTCTGAAGATGGGTCAATGGGGAGCTTTTCGGAGAAGTCTAGI
CTCGGCCTGAGCGGCTGTATATCCAGGTGTTCTTGAAGAAGGATGACTCAGTGGGCTAI
CCGGGCTTTGGTGCAGACAGAGGATCATCTGCTACTTTTCCTGCAGCAGTTGGGGAAG
GTGGTGCTGTGGAGCCGTGAGGAGTCCCTGGCAGAAGTGGTGTGCCTAGAGATGGTGG
TTTGGCAAAAAGGCAGAI
TGGCTTGCTGGGGATGTTCCTGAAACGCCTCTCGTCTCAGCTTATCCTGCTGCAAGCAi
GGTGATGGTAACAGCCTCAGGCAAGCTTTTTGGCATTGAGAGCAGCTCTGGCACCATC
CTGTGGAAACAGTATCTACCCAATGTCAAGCCAGACTCCTCCTTTAAACTGATGGTCCI
GTAGCTCCCCCAGTGCTGAAGCGCCCCATCTTGCAGTCCTTGCTTCTCCCAGTCATGG
ATCAAGACTACGCCAAGGTGTTGCTGTTGATAGATGATGAATACAAGGTCACAGCTTT
TCCAGCCACTCGGAATGTCTTGCGACAGCTACATGAGCTTGCCCCTTCCATCTTCTTC
TATTTGGTGGATGCAGAGCAGGGACGGCTGTGTGGATATCGGCTTCGAAAGGATCTCA
CCACTGAGCTGAGTTGGGAGCTGACCATTCCCCCAGAAGTACAGCGGATCGTCAAGGT
GAAGGGGAAACGCAGCAGTGAGCACGTTCATTCCCAGGGCCGTGTGATGGGGGACCGC
TATCATTCACTCCTCTGTGCAGAAGAAAGCCAAAGGCCCTGTCCATATCGTGCATTCAI
GAGAACTGGGTGGTGTACCAGTACTGGAACACCAAGGCTCGGCGCAACGAGTTTACCG
GCCATGGAGGCCACCATCACCGAACGGGGCATCACCAGCCGACACCTGCTGATTGGACI
ORF Start: ATG at 21 ORF Stop: TAA at 2997
SEQ ID NO: 22 992 as MW at 1116S9.1kD
NOV7a, MAAEWASRFWLWATLLIPAAAVYEDQVGKFDWRQQYVGKVKFASLEFSPGSKKLVVAT
CGS7242-O1 EKNVIAALNSRTGEILWRHVDKGTAEGAVDAMLLHGQDVITVSNGGRIMRSWETNIGG
Protein Sequence ~EITLDSGSFQALGLVGLQESVRYIAVLKKTTLALHIiLSSGHLKWVEHLPESDSIH
YQMVYSYGSGVVWALGVVPFSHVNIVKFNVEDGEIVQQVKVSTPWLQHLSGACGVVDE
AVLVCPDPSSRSLQTLALETEWELRQIPLQSLDLEFGSGFQPRVLPTQPNPVDASRAQ
FFLHLSPSHYALLQYHYGTLSLLKNFPQTALVSFATTGEKTVAAVMACRNEVQKSSSS
EDGSMGSFSEKSSSKDSLACFNQTYTINLYLVETGRRLLDTTITFSLEQSGTRPERLY
QAELEGEFGKKADGLLGMFLKRLSSQLILLQAWTSHLWKMFYDARKPRSQIKNEINID'
TLARDEFNLQRN~IVMVTASGKLFGIESSSGTILWKQYLPNVKPDSSFKLMVQRTTAHF
PHPPQCTLLVKDKESGMSSLYVFNPIFGKWSQVAPPVLKRPILQSLLLPVMDQDYAKV'i
LLLIDDEYKVTAFPATRNVLRQLHELAPSIFFYLVDAEQGRLCGYRLRKDLTTELSWEj
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FIGIFLIDGVTGRIIIiSSVQKKAKGPVHIVHSENWWYQYWNTKARRNEFTVLELYEG
TEQYNATAFSSLDRPQLPQVLQQSYIFPSSISAMEATITERGITSRHLLIGLPSGAIL
SLPKALLDPRRPEIPTEQSREENLIPYSPDVQIHAERFINYNQTVSRMRGIYTAPSGL
ESTCLVVAYGLDIYQTRVYPSICQFDVLKDDYDYVLISSVLFGLVFATMITKRLAQVKL
LNR.AWR
Further analysis of the NOV7a protein yielded the following properties shown
in Table 7B.
Table 7B. Protein Sequence Properties NOV7a
PSort 0.4600 probability located in plasma membrane; 0.2800 probability
located in
analysis: endoplasmic reticulum (membrane); 0.2000 probability located in
lysosome
(membrane); 0.1875 probability located in microbody (peroxisome)
SignalP Cleavage site between residues 22 and 23
analysis:
A search of the NOV7a protein against the Geneseq database, a proprieta:y
datr.base u'~at
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 7C.
Table 7C. Geneseq Results
for NOV7a
NOV7a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
Identifier Date] Match the Matched Value
ResiduesRegion
AAB88468 Human membrane or secretoryI ..992 963/993 (96%)0.0
protein
clone PSEC0263 - Homo Sapiens,1..971 967/993 (96%)
971
aa. [EP1067182-A2, 10-JAN-2001)
AAE0707S Human gene 2 encoded secreted508..992~ 485/485 ~ 0.0
protein (100%)
~ 1. d85 485/485 (
HPJCP79, SEQ ID NO:92 - 100,! )
Homo
sapiens, 485 aa. [W0200I54708-Al,
02- '
AUG-2001 ]
AAE07074 Human gene 2 encoded secreted1..354 352/354 (99%)0.0
protein
HPJCP79, SEQ ID N0:91 - 1..354 354/354 (99%)
Homo
Sapiens, 360 aa. [W0200154708-A1,
02-
AUG-2001 ]
ABBS9498 Drosophila melanogaster 440..992252/568 (44%)e-128
polypeptide
SEQ ID NO 5286 - Drosophila363..915350/568 (61%)
melanogaster, 9I S aa. [W0200171042-
A2, 27-SEP-2001
AAY6S107 Human S' EST related polypeptide37..160 114/124 (91%)Se-59
SEQ
ID N0:1268 - Homo Sapiens, 2..125 117/124 (93%)
132 aa.
[W0995305 1-A2, 21-OCT-1999]
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In a BLAST search of public sequence datbases, the NOV7a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 7D.
Table 7D. Public BLASTP Results for NOV7a
NOV7a Identities/
Protein Residues/SimilaritiesExpect
for
AccessionProtein/OrganismlLength Match the Matched Value
Number ResiduesPortion
CAC39832 SEQUENCE 303 FROM PATENT 1..992 963/993 (96%)0.0
EP1067182 - Homo Sapiens 1..971 967/993 (96%)
(Human),
971 aa.
Q14700 KIAA0090 PROTEIN - Homo 89..992 903/905 (99%)0.0
Sapiens (Human), 905 as 1..905 904/905 (99%)
(fragment).
Q9NUCI DJ657E11.5 (KIAA0090 PROTEIN)97..992 ' 81J/928 ' 0.C
(87,') '
- Homo Sapiens (Human), 1..847 815/928 (87%)
847 as
(fragment).
Q9VHY6 CG2943 PROTEIN - Drosophila440..992252/568 (44%)e-127
melanogaster (Fruit fly), 363..915350/568 (61%)
9I5 aa.
Q95TQ6 LD30573P - Drosophila melanogaster470..992233/531 (43%)e-119
(Fruit fly), 521 aa. 6..521 329/531 (61%)
PFam analysis predicts that the NOV7a protein contains the domains shown in
the Table
7E.
Table 7E. Domain Analysis of NOV7a
Identities/
Pfaln Domain NOV7a Match Region Similarities ~ E.xpeci v dine'
for the Matched Region
Bacterial_PQQ 52..89 9/38 (24%) 0.19
24/38 (63%)
Example 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: 23 ~ 1913 by
112
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CG57279-O2 GCGCGCCATGACCGTCGCGCGGCCGAGCGTGCCCGCGGCGCTGCCCCTCCTCGGGGAG
DNA
Sequence CTGCCCCGGCTGCTGCTGCTGGTGCTGTTGTGCCTGCCGGCCGTGTGGGGTGACTGTG
GCCTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGCCGTACAAGTTTTCC
CGAGGATACTGTAATAACGTACAAATGTGAAGAAAGCTTTGTGAAAATTCCTGGCGAG
AAGGACTCAGTGATCTGCCTTAAGGGCAGTCAATGGTCAGATATTGAAGAGTTCTGCA
ATCGTAGCTGCGAGGTGCCAACAAGGCTAAATTCTGCATCCCTCAAACAGCCTTATAT
CACTCAGAATTATTTTCCAGTCGGTACTGTTGTGGAATATGAGTGCCGTCCAGGTTAC
AGAAGAGAACCTTCTCTATCACCAAAACTAACTTGCCTTCAGAATTTAAAATGGTCCA
CAGCAGTCGAATTTTGTAAAAAGAAATCATGCCATAATCCGGGAGAAATACGAAATGG
TCAGATTGATGTACCAGGTGGCATATTATTTGGTGCAACCATCTCCTTCTCATGTAAC
ACAGGGTACAAATTATTTGGCTCGACTTCTAGTTTTTGTCTTATTTCAGGCAGCTCTG
TCCAGTGGAGTGACCCGTTGCCAGAGTGCAGAGGAAAATCTCTAACTTCCAAGGTCCC
ACCAACAGTTCAGAAACCTACCACAGTAAATGTTCCAAATACAGAATTCTCACCAACT
TCTCAGAAAACCACCACAAAAACCACCACACCAAATGCTCAAGCAACACGGAGTACAC
CCGTTTCCAGGACAACCAAGCATTTTCATGAAACAACCCCAAATAAAGGAAGAGGAAC
CACTTCAGGTACTACCCGTCTTCTATCTGGGCACACGTGTTTCACGTTGACAGGTTTG
CTTGGGACGCTAGTAACCATGGGCTTGCTGACTTAGCCAAAGAAGAGTTAAGAAGAAA
ATACACACAAGTATACAGACTGTTCCTAGTTTCTTAGACTTATCTGCATATTGGATAA
AATAAATGCAATTGTGCTCTTCATTTAGGATGCTTTCATTGTCTTTAAGATGTGTTAG
GAATGTCAACAGAGCAAGGAGAAAAAAGGCAGTCCTGGAATCACATTCTTAGCACACC
TACACCTCTTGAAAATAGAACAACTTGCAGnpmTGaGAGTGAmmCr_mmmCCm~ayz~p,GTI
GTAAGAAAGCATAGAGATTTGTTCGTATTTAGAATGGGATCACGAGGAAAAGAGAAGG
AAAGTGATTTTTTTCCACAAGATCTGTAATGTTATTTCCACTTATAAAGGAAATAAAA
AATGAAAAACATTATTTGGATATCAAAAGCAAATAAAAACCCAATTCAGTCTCTTCTA
AGCAAAATTGCTAAAGAGAGATGAACCACATTATAAAGTAATCTTTGGCTGTAAGGCA
TTTTCATCTTTCCTTCGGGTTGGCAAAATATTTTAAAGGTAAAACATGCTGGTGAACC
AGGGGTGTTGATGGTGATAAGGGAGGAATATAGAATGAAAGACTGAATCTTCCTTTGT
TGCACAAATAGAGTTTGGAAAAAGCCTGTGAAAGGTGTCTTCTTTGACTTAATGTCTT
TAAAAGTATCCAGAGATACTACAATATTAACATAAGAAAAGATTATATATTATTTCTG
AATCGAGATGTCCATAGTCAAATTTGTAAATCTTATTCTTTTGTAATATTTATTTATA
TTTATTTATGACAGTGAACATTCTGATTTTACATGTAAAACAAGAAAAGTTGAAGAAG
ATATGTGAAGAAAAATGTATTTTTCCTAAATAGAAATAAATGATCCCATTTTTTGGT
ORF Start: ATG at
66 ORF Stop: TAG
at 1020
SEQ ID NO: 24 318
as MW at 34479.IkD
NOVBa, MTVARPSVPAALPLLGELPRLLLLVLLCLPAVWGDCGLPPDVPNAQPALEGRTSFPED
CGS7279-O2 '~I~CEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQ
Protein Sequencet'T~FPVGTV~YECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCHIdPGEIRNGQI
DVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRGKSLTSKVPPT
VQKPTTVNVPNTEFSPTSQKTTTKTTTPNAQATRSTPVSRTT__rC~-?FFhTTP_rTICGRGTTS
~
GTTRLLSGh rCFTLTGI~LGTL'iiWiGLL~?~
SEQ ID NO: 2S 1962 by
NOV8b, CTGCAAACTTGCATGTCATCTCTTTCAGGTGACTGTGGCCTTCCCCCAGATGTACCTA
CGS7279-04 ATGCCCAGCCAGCTTTGGAAGACGTACAAGTTCCCGAGGATACTGTAATAACGTACAA
DNA
Sequence ATGTGAAGAAAGCTTTGTGAAAATTCCTGGCGAGAAGGACTCAGTGATCTGCCTTAAG
GGCAGTCAATGGTCAGATATTGAAGAGTTCTGCAATCGTAGCTGCGAGGTGCCAACAA
GGCTAAATTCTGCATCCCTCAAACAGCCTTATATCACTCAGAATTATTTTCCAGTCGG
TACTGTTGTGGAATATGAGTGCCGTCCAGGTTACAGAAGAGAACCTTCTCTATCACCA
AAACTAACTTGCCTTCAGAATTTAAAATGGTCCACAGCAGTCGAATTTTGTAAAAAGA
AATCATGCCCTAATCCGGGAGAAATACGAAATGGTCAGATTGATGTACCAGGTGGCAT
ATTATTTGGTGCAACCATCTCCTTCTCATGTAACACAGGGTACAAATTATTTGGCTCG
ACTTCTAGTTTTTGTCTTATTTCAGGCAGCTCTGTCCAGTGGAGTGACCCGTTGCCAG
AGTGCAGAGAAATTTATTGTCCAGCACCACCACAAATTGACAATGGAATAATTCAAGG
GGAACGTGACCATTATGGATATAGACAGTCTGTAACGTATGCATGTAATAAAGGATTC
ACCATGATTGGAGAGCACTCTATTTATTGTACTGTGAATAATGATGAAGGAGAGTGGA
GTGGCCCACCACCTGAATGCAGAGGAAAATCTCTAACTTCCAAGGTCCCACCAACAGT
TCAGAAACCTACCACAGTAAATGTTCCAACTACAGAAGTCTCACCAACTTCTCAGAAA
ACCACCACAAAAACCACCACACCAAATGCTCAAGCAACACGGAGTACACCTGTTTCCA
. GGACAACCAAGCATTTTCATGAAACAACCCCAAATAAAGGAAGTGGAACCACTTCAGG
I13
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TACTACCCGTCTTCTATCTGGGCACACGTGTTTCACGTTGACAGGTTTGCTTGGGACG
CTAGTAACCATGGGCTTGCTGACTTAGCCAAAGAAGAGTTAAGAAGAAAATACACACA
AGTATACAGACTGTTCCTAGTTTCTTAGACTTATCTGCATATTGGATAAAATAAATGC
AATTGTGCTCTTCATTTAGGATGCTTTCATTGTCTTTAAGATGTGTTAGGAATGTCAA
CAGAGCAAGGAGAAAAAAGGCAGTCCTGGAATCACATTCTTAGCACACCTACACCTCT
TGAAAATAGAACAACTTGCAGAATTGAGAGTGATTCCTTTCCTAAAAGTGTAAGAAAG
CATAGAGATTTGTTCGTATTTAGAATGGGATCACGAGGAAAAGAGAAGGAAAGTGATT
TTTTTCCACAAGATCTGTAATGTTATTTCCACTTATAAAGGAAATAAAAAATGAAAAA
CATTATTTGGATATCAAAAGCAAATAAAAACCCAATTCAGTCTCTTCTAAGCAAAATT
GCTAAAGAGAGATGAACCACATTATAAAGTAATCTTTGGCTGTAAGGCATTTTCATCT
TTCCTTCGGGTTGGCAAAATATTTTAAAGGTAAAACATGCTGGTGAACCAGGGGTGTT
GATGGTGATAAGGGAGGAATATAGAATGAAAGACTGAATCTTCCTTTGTTGCACAAAT
AGAGTTTGGAAAAAGCCTGTGAAAGGTGTCTTCTTTGACTTAATGTCTTTAAAAGT
A
T
_
_
CCAGAGATACTACAATATTAACATAAGAAAAGATTATATATTATTTCTGAATCGAGAT
GTCCATAGTCAAATTTGTAAATCTTATTCTTTTGTAATATTTATTTATATTTATTTAT
GACAGTGAACATTCTGATTTTACATGTAAAACAAGAAAAGTTGAAGAAGATATGTGAA
GAAAAATGTATTTTTCCTAAATAGAAATAAATGATCCCATTTTTTGGT
ORF Start: ATG at
13 ORF Stop: TAG
at 1069
SEQ ID NO: 26 (3S2
as ~MW at 38279.8kD
NOVBb, MSSLSGDCGLPPDVPNAQPALEDVQVPEDTVITYKCEESFVKIPGEKDSVICLKGSQW
CGS7279-04 SDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTC
PIOtelri SequeriCeLQ~'KWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSF
CLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIG
EHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTK
TTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTM
GLLT
SEQ ID NO: 27 978
by
NOVBC, GGATCCGACTGTGGCCTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGCC
CGS7279-OS GTACAAGTTTTCCCGAGGATACTGTAATAACGTACAAATGTGAAGAAAGCTTTGTGAA
DNA
SequeriCe ~TTCCTGGCGAGAAGGACTCAGTGATCTGCCTTAAGGGCAGTCAATGGTCAGATATT
GAAGAGTTCTGCAATCTCGGTACTGTTGTGGAATATGAGTGCCGTCCAGGTTACAGAA
GAGAACCTTCTCTATCACCAAAACTAACTTGCCTTCAGAATTTAAAATGGTCCACAGC
AGTCGAATTTTGTAAAAAGAAATCATGCCCTAATCCGGGAGAAATACGAAATGGTCAG
ACTGATGTACCAGGTGGCATATTATTTGGTGCAACCATCTCCTTCTCATGTAACACAG
GGTACAAATTATTTGGCTCGACTTCTAGTTTTTGTCTTATTTCAGGCAGCTCTGTCCA
GTGGAGTGACCCGTTGCCAGAGTGCAGAGAAATTTATTGTCCAGCACCACCACAAATT
GACAATGGAATAATTCAAGGGGAACGTGACCATTATGGATATAGACAGTCTGTAACGT
ATGCATGTAATAAAGGATTCACCATGATTGGAGAGCACTCUATTTATTGTAW
UzTGAH
TAE1T('~~lTGi-~AGGAGAGTGC~AG~t
GGCCCAt~CACC T csAATGI:AGAGGAAAATCTCTAACT
TCCAAGGTCCCACCAACAGTTCAGAAACCTACCACAGTAAATGTTCCAACTACAGAAG
TCTCACCAACTTCTCAGAAAACCACCACAAAAACCACCACACCAAATGCTCAAGCAAC
ACGGAGTACACCTGTTTCCAGGACAACCAAGCATTTTCATGAAACAACCCCAAATAAA
GGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTATCTGGGCACACGTGTTTCACGT
TGACAGGTTTGCTTGGGACGCTAGTAACCATGGGCTTGCTGACTCTCGAG
ORF Start: at 7 ORF Stop:
at 973
SEQ ID NO: 28 322 as MW at 34931.OkD
NOVBC, DCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEE
CGS7279-OS FCNLGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQTD
PTOteiri SequenceVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDN
GIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSK
VPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGS
GTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT
SEQ ID NO: 29 978 by
NOV$(1, . GGATCCGACTGTGGCCTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGCC
175070639 DNA GTACAAGTTTTCCCGAGGATACTGTAATAACGTACAAATGTGAAGAAAGCTTTGTGAA
Sequence AATTCCTGGCGAGAAGGACTCAGTGATCTGCCTTAAGGGCAGTCAATGGTCAGATATT
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Sequence comparison of the above .protein sequences yields the following
sequence
relationships shown in Table 8B.
Table 8B. Comparison of NOVBa against NOVBb through NOVBd.
Protein SequenceNOYBa Residues/Identities!
Match ResiduesSimilarities for the Matched
Region
NOVBb 34..284 215/317 (67%)
~ 6..318 219/317 (68%)
NOVBc X35..284 ~192/316(600~)
288 ~ i77i316
1 6i%
. . (
.. )
NOVBd 35..302 196/334 (58%)
3..308 201/334 (59%)
Further analysis of the NOVBa protein yielded the following properties shown
in Table 8C.
Table 8C. Protein Sequence Properties NOVBa
PSort 0.7571 probability located in outside; 0.1000 probability located in
endoplasmic
analysis: reticulum (membrane); 0.1000 probability located in endoplasmic
reticulum (lumen);
0.1000 probability located in lysosome (lumen)
SignalP Cleavage site between residues 35 and 36
analysis:
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A search of the NOV8a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 8D.
Table 8D. Geneseq Results for NOVBa
NOVBa Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAW73505 Decay accelerating factor 1..318 300/381 (78%)e-165
protein -
Homo Sapiens, 38I aa. [JP10313865-A,1..381 301/381 (78%)
02-DEC-1998]
AAY31740 Human CD55 and 791Tgp72 1..318 300/381 (78%)[ e-165
tumour
z Z/~ , ma,
associated antigen - Homo I ..~ moi~
sapiens, 381 81 J~ 11 J V
1 ', V .
V J
aa. [W09943800-A1, 02-SEP-1999]
AAR66683 Decay accelerating factor- 1..318 300/381 (78%)e-165
Homo
sapiens, 381 aa. [IJS5374548-A,1..381 301/381 (78%)
20-
DEC-1994]
AAW27483 Human glycophosphatidylinositol1..307 282/370 (76%)e-154
anchored DAF - Homo sapiens,1..370 284/370 (76%)
440 aa.
[W09735886-Al, 02-OCT-1997]
AAR66684 Decay accelerating factor 1..307 282/370 (76%)e-154
- Homo
Sapiens, 440 aa. [US5374548-A,1..370 284/370 (76%)
20-
DEC-1994]
In a BLAST search of public sequence datbases, the NOV 8a protein was found to
have
homology to the proteins shown in the BLASTP data in Table, 8E.
Table 8E. Public BLASTP Results for NOVBa
NOV8a Identities/
Protein Residues/ SimilaritiesExpect
for
Accession Protein/Organism/Length Match the Matched Value
Number Residues Portion
CAC07712 SEQUENCE 1 FROM PATENT 1..318 300/381 (78%) e-165
W09943800 - Homo Sapiens 1..381 301/381 (78%)
(Human),
3 81 aa.
P08174 Complement decay-accelerating1..318 299/381 (78%) e-164
factor
precursor (CDSS antigen) 1..381 300/381 (78%)
- Homo
sapiens (Human), 381 aa.
CAA03840 SEQUENCE I FROM PATENT 1..307 282/370 (76%) e-153
WO9735886 - unidentified, 1..370 284/370 (76%)
440 aa.
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Q9MYJ5 DECAY-ACCELERATING FACTOR 35..318 241/307 (78%) e-136
- Pan troglodytes (Chimpanzee), 305 as 1..305 251/307 (81%)
(fragment).
CAC39504 SEQUENCE 31 FROM PATENT 35..294 242/323 (74%) e-130
W00132901 - unidentified, 611 aa. 289..611 243/323 (74%)
PFam analysis predicts that the NOVBa protein contains the domains shown in
the Table
8F.
Table 8F. Domain
Analysis of
NOVBa
Identities/
Pfam DomainNOVBa Match RegionSimilarities. Expect
Value
for the Matched
Region
sushi 36..94 14/67 (21%) 1.4e-1
1
45/67 (67%)
sushi 98..158 18/67 (27%) 1.6e-09
40/67 (60%)
sushi 163..220 24/64 (38%) ' 8.8e-13
45/64 (70%)
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: 31 ~ 1266 Up _ _____._~
CG9463O-OI DNA 'GGGCGGGCTCCCACTCCATGAGGTATTTCAGCACCGCCGTTTCCTGGCCGGGCCGCGG
Sequence 'jGGAGCCCAGCTTCATTGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGGTCGAC
r,,..,..T
~.~,r.r~r.mr.TP.mnmnnr_r_~rmr_nar_rrr_~r_r_r_nr_rr~mr_r~mr~r~arc~ar..ra~c~hc';(
'~
CGGAGTATTGGGACCTACAGACACTGGGCGCCAAGGCCCAGGCACAGACTGACCGAGT
GAACCTGCGGACCCTGCTCCGCTACTACAACCAGAGCGAGGCGGGGTATCACATCCTC
CAGGGAATGTTTGGCTGCGACCTGGGGCCCGACGGGCGTCTCCTCCGCGGGTATGAGC
AGTATGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCCTGGAC
GAGCAAAGGAGAGCCTACCTGGAGGGCACCTGCATGGAGTGGCTCCGCAGACACCTGG
CTACCCTGCGGAGATCACATTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGAC
CTGTGATGTGGAGGAAGAAGAGCTCAGATAGAAAAGGAGGGAGCTACTCTCAGGCTGC
117
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AATTTCCCTCCCTTGACTCCATCAACATCGGCACCTGCCAGACGCCCACCACCCACCA
TCGAAGTGCTGAGAAGAAGTGCAAGGTACTCAACCTGCTCTGGGGATACAGCAGGAAA
GCAGAGTGTTTACGGATTTCACATTCCATCAAAGAAAATCCATTTTGA
ORF Start: ATG at 1 ORF Stop:
TGA at 1264
SEQ ID NO: 32 421 as MW at 47476.7kD
NOV9a, MAPRTLLLLLSGTLALAETWAGSHSNB2YFSTAVSWPGRGEPSFIAVGYVDDTQFVR.VD
CG9463O-O1 SDAVSLRMKTRARWVEQEGPEYWDLQTLGAKAQAQTDRVNLRTLLRYYNQSEAGYHIL
Protein SequenceQGMFGCDLGPDGRLLRGYEQYAYDGKDYIALNEDLRSWTAADTAAQITQRKYEAANVA
EQRRAYLEGTCMEWLRRHLENGKETLQRAGITRSWVLGFYPAEITLTWQRDGEDQTQD
MELVETRPTGDGTFQKWAVVVVPSGEEQRYTCHVQHKGLPKPLILRWEPSPQPTIPIV
GIIAGLVLLGAVVTGAVVTAVMWRKKSSDRKGGSYSQAAKNIIKVKTEKFLALWCIRS
RCKLVQPAALGLRVA12DSFEFPS?~DSINIGTCQTPTTHHRSAEKKCKVLNLLWGYSRK
AECLRISHSIKENPF
Further analysis of the NOV9a protein yielded the following properties shown
in Table 9B.
Table 9B. Protein Sequence Properties NOV9a
PSort 0.4600 probability located in plasma membrane; 0.1335 probability
located in
analysis: microbody (peroxisome); O.I000 probability located in endoplasmic
reticulum
(membrane); 0.1000 probability located in endoplasmic reticulum (lumen)
SignalP ~ Cleavage site between residues I8 and 19
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 9C.
Table 9C. Geneseq Results
for NOV9a
NOV9a Identities/
Geneseq Protein/Organisr.~./Length ~ Residues/Simi:ar idea ~ Eapect
~Da:ent #, for
IdentifierBatej Match the Matched Value
ResiduesRegion
AAB36874 MHC class I protein - Unidentified,1..331 2661347 (76%)e-153
365
aa. [US6140305 A, 31-OCT-2000]~ 285/347 (81%)
4..350
AAB58683 HLA-A2/A28 protein #4 - 1..331 265/347 (76%)e-153
Unidentified,
365 aa. [L1S6153408-A, 28-NOV-2000]4..350 285/347 (81%)
AAY52922 HLA-A2/A28 family peptide 1..331 265/347 (76%)e-153
A2 (Lee)
SEQ ID NO:100 - Mammalia, 4..350 285/347 (81%)
365 aa.
[US5976551-A, 02-NOV-1999]
AAY68268 Human leukocyte antigen I ..331 265/347 (76%)e-153
A2/A28 family
protein SEQ ID NO:100 - 4..350 285/347 (81
Homo sapiens, %)
365 aa. [US60I 1146-A, 04-JAN-2000]
AAB58687 HLA-A2/A28 protein #8 - 1..331 264/347 (76%)e-153
Unidentified,
365 aa. [US6153408-A, 28-NOV-2000]4..350 284/347 (81%)
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In a BLAST search of public sequence datbases, the NOV9a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 9D.
Table 9D. Public BLASTP Results for NOV9a
NOV9a Identities/
Protein Residues/SimilaritiesExpect
for
AccessionProtein/Organism/Length Match the Matched Value
Number ResiduesPortion
I54493 MHC class I histocompatibility1..331 273/347 (78%),e-158
antigen
HLA-A alpha chain precursor4..350 292/347 (83%)
- human,
365 aa.
Q9MXI8 MHC CLASS I ANTIGEN - Pan 1..331 274/347 (78%)e-158
troglodytes (Chimpanzee), 4..350 292/347 (83%)
365 aa.
Q9MXL0 MHC CLASS I ANTIGEN - Pan 1..331 274/347 (78%)e-I 58
troglodytes (Chimpanzee), 4..350 291/347 (82%)
365 aa.
Q9MXI7 MHC CLASS I ANTIGEN - Pan 1..331 274/347 (78%)e-158
troglodytes (Chimpanzee), 2..348 291/347 (82%)
363 as
(fragment).
Q9TPL3 MHC CLASS I ANTIGEN I ..331 274/347 (78%)e-158
(LYMPHOCYTE ANTIGEN) - Pan 4..350 290/347 (82%)
troglodytes (Chimpanzee),
365 aa.
PFam
analysis
predicts
that
the
NOV9a
protein
contains
the
domains
shown
in the
Table
9E.
Table 9E.
Domain
Analysis
of NOV9a
Identities/
Pfam DomainNOV9a Match RegionSimilarities Expect
' Value
for the Matched
Region
MHC_I 22..200 140/180 (78%) 3.4e-131
170/180 (94%)
ig 212..266 12/56 (21%) 0.00049
41/56 (73%)
Example 10.
The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 10A.
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----..~.....
Table 10A.
NOV10 Sequence
Analysis
SEQ ID NO: 33 717 by
NOVlOa, GCAGCATGGGGAGCTTCCACGCGGGCATACGGTGCATCAAGTACATGCTGGTTGGCTT
CG94831-O1 C~CCTGCTCTTCTGGCTGGCTGGATCGGCCGTCATTGCTTTTGGACTATGGTTTCGG
DNA
Sequence TTCGGAGGTGCCATAAAGGAGTTATCATCAGAGGACAAGTCCCCAGAGTATTTCTATG
TGGGTGGGCTGTATGTTCTGGTTGGAGCCGGGGCCCTGATGATGGCCGTGGGGTTCTT
CGGGTGCTGCGGAGCCATGCGGGAGTCGCAATGTGTGCTTGGATCATTTTTTACCTGC
CTCCTGGTGATATTTGCTGCTGAAGTAACCACTGGAGTATTTGCTTTTATAGGCAAGG
CTATCCGACATGTTCAGACCATGTATGAAGAGGCTTACAATGATTACCTTAAAGACAG
GGGAAAAGGCAATGGGACACTCATCACCTTCCACTCAACATTTCAGTGCTGTGGAAAA
GAAAGCTCCGAACAGGTCCAACCTACATGCCCAAAGGAGCTTCTAGGACACAAGAATT
GCATCGATGAAATTGAGACCATAATCAGTGTTAAGCTCCAGCTCATTGGAATTGTCGG
TATTGGAATTGCAGGTCTGACGGTGATCTTTGGCATGATATTCAGCATGGTCCTCTGC
TGTGCGATACGAAACTCACGAGATGTGATATGAAGCTACTTCTACATGAAAATTGCAA
TCTAAAGCTTTCATACCAAAT
ORF Start: ATG at
6 ORF Stop: TGA
at 669
SEQ ID NO: 34 X221
as MW at 24077.1kD
NOVlOa, MGSFHAGIRCIKYMLVGFNLLFWLAGSAVIAFGLWFRFGGAIKELSSEDKSPEYFYVG
CG94831-O1 GLYVLVGAGALMMAVGFFGCCGAMRESQCVLGSFFTCLLVIFAAEVTTGVFAFIGKAI
Protein Sequence~Q~EAYNDYLKDRGKGNGTLITFHSTFQCCGKESSEQVQPTCPKELLGHKNCI
DEIETIISVKLQLIGIVGIGIAGLTVIFGMIFSMVLCCAIRNSRDVI
SEQ ID NO: 35 742
by
NOVIOb, TGTAGGTCTTCTTGATCCGCAGCATGGGGCGCTTCCGCGGGGGCCTGCGGTGCATCAA
CG94831-O2 GTACCTGCTGCTTGGCTTCAACCTGCTCTTCTGGCTGGCTGGATCGGCCGTCATTGCT
DNA
Sequence TTTGGACTATGGTTTCGGTTCGGAGGTGCCATAAAGGAGTTATCATCAGAGGACAAGT
CCCCAGAGTATTTCTATGTGGGGCTGTATGTTCTGGTTGGAGCCGGGGCCCTGATGAT
GGCCGTGGGGTTCTTCGGATGCTGCGGAGCCATGCGGGAGTCGCAATGTGTGCTTGGA
TCATTTTTTACCTGCCTCCTGGTGATATTTGCTGCTGAAGTAACCACTGGAGTATTTG
CTTTTATAGGCAAGGGGGTAGCTATCCGACATGTTCAGACCATGTATGAAGAGGCTTA
CAATGATTACCTTAAAGACAGGGGAAAAGGCAATGGGACACTCATCACCTTCCACTCA
ACATTTCAGTGCTGTGGAAAAGAAAGCTCCGAACAGGTCCAACCTACATGCCCAAAGG
AGCTTCTAGGACACAAGAATTGCATCGATGAAATTGAGACCATAATCAGTGTTAAGCT
CCAGCTCATTGGAATTGTCGGTATTGGAATTGCAGGTCTGACGATCTTTGGCATGATA
TTCAGCATGGTCCTCTGCTGTGCGATACGAAACTCACGAGATGTGATATGAAGCTACT
TCTACATGAAAATTGCAATCTAAAGCTTTCATACACAAATAAGGGC
OItF Start: ATG at
24 ORi StnY: T O
A at 687
SEQ ID NO: 36 221 MW at 24147.2kD
as
NOVlOb, MGRFRGGLRCIKYLLLGFNLLFWLAGSAVIAFGLWFRFGGAIKELSSEDKSPEYFYVG
CG9483I-02 LYVLVGAGALN1MAVGFFGCCGAMRESQCVLGSFFTCLLVIFAAEVTTGVFAFIGKGVA
Protein SequenceT~Q~'M~~~~~RGKGNGTLITFHSTFQCCGKESSEQVQPTCPKELLGHKNC
IDEIETIISVKLQLIGIVGIGIAGLTIFGMIFSMVLCCAIRNSRDVI
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 10B.
Table 10B. Comparison of NOVlOa against NOVlOb.
Protein Sequence NOVlOa Residues/ Identities/
Match Residues Similarities for the Matched Region
NOV 1 Ob 1..221 ~ 194/223 (86%)
1..221 195/223 (86%)
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Further analysis of the NOV 10a protein yielded the following properties shown
in Table
10C.
Table 10C. Protein Sequence Properties NOVlOa
PSort 0.6400 probability located in plasma membrane; 0.4000 probability
Located in GoIgi
analysis: body; 0.3000 probability located in endoplasmic reticulum
(membrane); 0.0300
probability located in mitochondria) inner membrane
SignalP Cleavage site between residues 42 and 43
analysis:
A search of the NOV 10a 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 OD.
Table )OD. Geneseq Results
for NOVlOa
NOVlOa Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the MatchedValue
ResiduesRegion
AAG73745 Human colon cancer antigen 1..221 213/223 e-119
protein SEQ (95%)
ID N0:4509 - Homo sapiens, 9..229 216/223
229 aa. (96%)
[W0200122920-A2, OS-APR-2001]
AAW61623 Clone HAIDQ59 5'of TM4SF 1..221 213/223 e-119
(95%)
superfamily - Homo sapiens,1..221 216/223
221 aa. (96%)
[W09831799-A2, 23-JUL-1998]
ABB57234 Mouse ischaemic condition 7..221 103/223 2e-50
related (46%)
protein sequence SEQ ID 6..226 137/223
N0:6I2 - Mus (6I%)
. . ., musculus, 226 aa. [W0200188188-A2,
22-NOV-ZOO I J
ABB44580 Mouse wound healing related7..221 103/223 2e-50
polypeptide (46%)
SEQ 1D NO 37 - Mus musculus,6..226 1371223
226 aa. (61%)
[CA2325226-A 1, 17-MAY-2001
]
AAG75156 Human colon cancer antigen 7..221 103/225 1 e-49
protein SEQ (45%)
ID N0:5920 - Homo Sapiens, 53..275 137/225
275 aa. (60%)
[W0200122920-A2, OS-APR-2001]
In a BLAST search of public sequence datbases, the NOV 1 Oa protein was found
to have
homology to the proteins shown in the BLASTP data in Table 10E.
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Table 10E. Public BLASTP Results for NOVlOa
NOVlOa
Protein Identities/
Accession Protein/Organism/Length Residues/Similarities Expect
for the
Number Matched PortionValue
Residues
Q8WU05 HYPOTHETICAL 24.1 KDA I..221 213/223 (95%) e-I
19
PROTEIN - Homo sapiens 1..221 216/223 (96%)
(Human),
221 aa.
Q9JJW 1 TSPAN-2 PROTEIN - Rattus 1..221 202/223 (90%) e-114
norvegicus (Rat), 221 1..221 213/223 (94%)
aa.
Q922J6 RIKEN CDNA 6330415F13 1..221 200/223 (89%) e-113
GENE -
Mus musculus (Mouse), 1..221 210/223 (93%)
221 aa.
Q9D39'7 6330415F13RIK PROTEIN 1..221 199/223 (89%) e-113
- Mus
musculus (Mouse), 221 1..221 ~
aa. 210/223 (93%)
060636 Tetraspanin 2 (Tspan-2) 1..221 206/224 (91%) e-112
- Homo
' Sapiens (Human), 222 aa. 1..222 209/224 (92%)
PFam analysis predicts that the NOV 1 Oa protein contains the domains shown in
the Table
1 OF.
Table 10F. Domain Analysis of NOVlOa
Identities/
Pfam Domain NOVlOa Match Region Similarities Expect Value
for the Matched Region
transmembrane4 12..214 68/268 (25%) S.l e-62
168/268 (63%)
Examule 11.
The NOV 11 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are showmin Table 1 1A.
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CACCTGTCATAAAATCCAGTGGAAGATTTCTATGGATTAAATTTTTTGCTGATGGAGA
GACCTTGGAGCTTTGAAACCATTACCAGCGTGTGAGTTTGAGATGGGCGGTTCCGAAG
CATGCTACGCACGGGTCTTGGGGTGATCCGCATGTGGGCAGATGAGGGCAGTCGAAAC
AGCCGATTTCAGATGCTCTTCACATCCTTTCAAGAACCTCCTTGTGAAGGCAACACAT
TCTTCTGCCATAGTAACATGTGTATTAATAATACTTTGGTCTGCAATGGACTCCAGAA
GAGTTATGCACTCTCAGAGGGACAGGAGCTACAGCTGACTTTGCAGATGTGGCAGATG
ACTTTGAAAATTACCATAAACTGCGGAGGTCATCTTCCAAATGCATTCATGACCATCA
CTGTGGATCACAGCTGTCCAGCACTAAAGGCAGCCGCAGTAACCTCAGCACAAGAGAT
GCTTCTATCTTGACAGAGATGCCCACACAGCCAGGAAAACCCCTCATCCCACCCATGA
ACAGAAGAAATATCCTTGTCATGAAACACAGCTACTCGCAAGATGCTGCAGATGCCTG
TGACATAGATGAAATCGAAGAGGTGCCGACCACCAGTCACAGGCTGTCCAGACACGAT
OIZF Start: ATG at 98 ORF Stop:~TGA at 1676
SEQ ID NO: 38 526 as MW at 59333.81cD
NOVIIa, MIHGRSVLHIVASLIILHLSGATKKGTEKQTTSETQKSVQCGTWTKHAEGGIFTSPNY
CG94892-Ol PSKYPPDRECIYIIEAAPRQCIELYFDEKYSIEPSWECKFDHIEVRDGPFGFSPIIGR
Protein Sequence FCGQQNPPVIKSSGRFLWIKFFADGELESMGFSARYNFTPDPDFKDLGALKPLPACEF
EMGGSEGIVESIQIMKEGKATASEAVDCKWYIRAPPRSKIYLRFLDYEMQNSNECKRN
FVAVYDGSSSVEDLKAKFCSTVANDVMLRTGLGVIRMWADEGSRNSRFQMLFTSFQEP
PCEGNTFFCHSNMCINNTLVCNGLQNCVYPWDENHCKEKRKTSLLDQLTNTSGTVIGV
TSCIVIILIIISVIVQIKQPRKKYVQRKSDFI7QTVFQEVFEPPHYELCTLRGTGATAD
FADVADDFENYHKLRRSSSKCIHDHHCGSQLSSTKGSRSNLSTRDASILTEMPTQPGK
PLIPPMNRRNILVMKHSYSQDAADACDIDEIEEVPTTSHRLSRHDKAVQRSVSIDFLM
TTIT
Further analysis of the NOV 1.1 a protein yielded the f~iloWing pr~pert:es
sho~,hrr: in ?'able
11B.
Table I1B. Protein Sequence Properties NOVlla
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); O.I000 probability located in outside
SignaIP ~ Cleavage site between residues 23 and 24
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.
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Table 11C. Geneseq Results
for NOVlla
NOVlIa Identities/
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAB47296 PR04401 polypeptide - Homo 10..520 301 /519 (57%)e-180
Sapiens,
525 aa. [W0200140465-A2, 14..525 387/519 (73%)
07-JUN-
2001]
AAU12228 Human PR04401 polypeptide 10..520 3011519 (S7%)e-180
sequence -
Homo Sapiens, S2S aa. [W0200140466-14..525 387/519 (73%)
A2, 07-JUN-2001 J
AAM93946 Human polypeptide, SEQ ID 10..520 301/519 (57%)e-180
NO: 4135 -
Homo Sapiens, S2S aa. [EP1130094-A2,14..525 387/519 (73%)
OS-SEP-2001 J
AAU 18670Renal and cardiovascular-associated66..520 282/463 (60%)e-16t"s
protein, Seq ID 109 - Homo 19..474 356/463 (7S%)
Sapiens, 474 ,~
aa. [W0200155328-A2, 02-AUG-2001]
ABBSS774 Human polypeptide SEQ ID 133..520234/396 (S9%)e-135
NO 154 -
Homo Sapiens, 389 aa. [L152001039335-1..389 296/396 (74%)
A 1, 08-NOV-2001 J
In a BLAST search of public sequence datbases, the NOV 11 a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 11 D.
Table 11D. Public BLASTP
Results for NOVlla
Protein NOVlIa Identities/
Accession Protein/Organism/Length Residues/~ Expect
Similarities '
for
Number . _ _ . Match ; the Matchedvalve
ResiduesPortion
AAL48461 GH11189P - Drosophila 55..363 107/318 (33%)1e-47
melanogaster
(Fruit fly), 677 aa. 154..448163/318 (SO%)
Q96SP4 CDNA FLJ14724 FIS, CLONE 327..52091/201 (45%) 9e-41
NT2RP3001716 - Homo sapiens8..201 130/201 (64%)
(Human), 201 aa.
061 849 K03E5.1 PROTEIN - Caenorhabditis52..287 82/246 (33%) 3e-32
elegans, 321 aa. 75..31 ~
S 126/246 (S0%)
Q96RU9 INTRINSIC FACTOR-VITAMIN 37..295 77/265(29%) 3e-28
B 12 RECEPTOR - Homo sapiens2084..2326138/265 (S2%)
(Human), 3494 as (fragment).
060494 INTRINSIC FACTOR-B 12 37..295 77/265 (29%) 3e-28
.
RECEPTOR PRECURSOR - Homo2213..2455138/265 (52%)
Sapiens (Human), 3623
aa.
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PFam analysis predicts that the NOV 11 a protein contains the domains shown in
the Table
11 E.
Table 11E. Domain Analysis of NOVlla
Identities/
Pfam DomainNOVlla Match Similarities Expect
Region Value
for the Matched
Region
CUB 41..152 43/I 18 (36%) 6.3e-29~
84/118 (71%)
CUB 172..284 27/124 (22%) 0.0003
64/124 (52%)
ldl_recept 290..328 13/43 (30%) 0.00073
a
2 5/43 (58%)
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 NO: 39 X2302 by
NOV 12a,
CG95227-O1 DNA
Sequence
GCCCCTCAGGCATTACTATCCCTGGAAAACCAGGTGCCCAAGGGGTGCCAGGGCCCCC
CTCAAGGGGGATAATGGAGTGGGCCAGCCCGGGCTGCCTGGGGCCCCAGGGCAGGGGG
TGGTCCCCCAGGGCTCCCTGGCAAGGTCGGGCCACCAGGGCAGCCGGGGCTTCGGGGG
GAGCCAGGAATACGAGGGGACCAGGGCCTCCGGGGACCCCCAGGACCCCCTGGCCTCC
CGGGCCCCTCAGGCATTACTATCCCTGGAAAACCAGGTGCCCAAGGGGTGCCAGGGCC
GGCCTCAAGGGGGATAATGGAGTGGGCCAGCCCGGGCTGCCTGGGGCCCCAGGGCAGG
GGGGTGCCCCCGGCCCCCCCGGCCTCCCTGGTCCAGCTGGCTTAGGCAAACCTGGTTT
ACAGGGGTGAGCCAGGGGAGGATGGGGAGCCAGGGGAGCAGGGCCCACAGGGTCTTGG
CCTAAGGGTGAGGCAGGGCCTGGAGGACCCCCAGGAGTGCCTGGCATTCGAGGTGACC
GGCCCATGGACCCCCTGGACCAACTGGGCCCAAGGGTGAGCCGGGTTTCACGGGTCGC
CCTGGAGGACCAGGGGTGGCAGGAGCCCTGGGGCAGAAAGGTGACTTGGGGCTCCCTG
GGCAGCCTGGCCTGAGGGGTCCCTCAGGAATCCCAGGACTCCAGGGTCCAGCTGGCCC
TATTGGGCCCCAAGGCCTGCCGGGCCTGAAGGGGGAACCAGGCCTGCCAGGGCCCCCT
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GGAGAGGGGAGAGCAGGGGAACCTGGCACGGCTGGGCCCACGGGGCCCCCAGGGGTCCi
CTGGCTCCCCTGGAATCACGGGCCCTCCGGGGCCTCCCGGGCCCCCGGGACCCCCTGG
TGCCCCTGGGGCCTTCGATGAGACTGGCATCGCAGGCTTGCACCTGCCCAACGGCGGT
GTGGAGGGTGCCGTGCTGGGCAAGGGGGGCAAGCCACAGTTTGGGCTGGGCGAGCTGT
CTGCCCATGCCACACCGGCCTTCACTGCGGTGCTCACCTCGCCCTTCCCCGCCTCGGG
CATGCCCGTGAAATTTGACCGGACTCTCTACAATGGCCACAGCGGCTACAACCCAGCC
ACTGGCATCTTCACCTGCCCTGTGGGCGGCGTCTACTACTTTGCTTACCATGTGCACG
TCAAGGGCACCAACGTGTGGGTGGCCCTGTACAAGAACAACGTGCCGGCCACCTATAC
CTACGATGAGTACAAGAAGGGCTACCTGGACCAGGCATCTGGTGGGGCCGTGCTCCAG
CTGCGGCCCAACGACCAGGTCTGGGTGCAGATGCCGTCGGACCAGGCCAACGGCCTCT
ACTCCACGGAGTACATCCACTCCTCCTTTTCAGGATTCTTGCTCTGCCCCACATAA
CC
_
CGCGGGGGGGGTCCTGCTGCCCTGGCCTCCTCCCCTTTAGTGGTAGAGCGACCTTTTC
AATTACAAAGAACCTCCTGGp~~AAAAAAACAAAAGCTNNN
ORF Start: ATG at ORF Stop:
1 TAA
at 2200
SEQ ID NO: 40 733 as MW at 6999S.4kD
NOVl2a, MDYWVPPHPVIFLFLFFLVETGFHHVGQAGLKLLTSSNPPPGLPGKVGPPGQPGLRGE
CG9S227-O1 PGIRGDQGLRGPPGPPGLPGPSGITIPGKPGAQGVPGPPGFQGEPGPQGEPGPPGDRG
Protein SequenceLKGDNGVGQPGLPGAPGQGGAPGPPGPAGPPGFSRMGKAGPPGLPGKVGPPGQPGLRG
EPGIRGDQGLRGPPGPPGLPGPSGITIPGKPGAOGVPGPPGFOGEPGPOGEPGPPGDR
GLKGDNGVGQPGLPGAPGQGGAPGPPGLPGPAGLGKPGLDGLPGAPGDKGESGPPGVP
GPRGEPGAVGPKGPPGVDGVGVPGAAGLPGPQGPSGAKGEPGTRGPPGLIGPTGYGMP
GLPGPKGDRGPAGVPGLLGDRGEPGEDGEPGEQGPQGLGGPPGLPGSAGLPGRRGPPG
PKGEAGPGGPPGVPGIRGDQGPSGLAGKPGVPGERGLPGAHGPPGPTGPKGEPGFTGR
PGGPGVAGALGQKGDLGLPGQPGLRGPSGIPGLQGPAGPIGPQGLPGLKGEPGLPGPP
GEGRAGEPGTAGPTGPPGVPGSPGITGPPGPPGPPGPPGAPGAFDETGIAGLHLPNGG
VEGAVLGKGGKPQFGLGELSAHATPAFTAVLTSPFPASGMPVKFDRTLYNGHSGYNPA
TGIFTCPVGGVYYFAYHVHVKGTNVWVALYKNNVPATYTYDEYKKGYLDQASGGAVLQ
LRPNDQVWVQMPSDQANGLYSTEYIHSSFSGFLLCPT
SEQ ID NO: 4I 1950
by
NOVl2b, TGACTGCCCCTTTCTCTTTCTTTCTCAGAAATGCCTCTACCGCTGCTGCCGATGGACC
CG9S227-02 TGAAGGGAGAGCCCGGCCCCCCTGGGAAGCCCGGGCCCTGGGGTCCCCCTGGCCCCCC
DNA
Sequence TGGCTTCCCAGGAAAACCAGGCCATGGAAAGCCAGGACTCCATGGGCAGCCTGGCCCT
GCTGGGCCCCCTGGCTTCTCCCGGATGGGCAAGGCTGGTCCCCCAGGGCTCCCTGGCA
ACGTCGGGCCACCAGGGCAGCCGGGGCTTCGGGGGGAGCCAGGAATACGAGGGGACCA
GGGCCTCCGGGGACCCCCAGGACCCCCTGGCCTCCCGGGCCCCTCAGGCATTACTATC
CCTGGAAAACCAGGTGCCCAAGGGGTGCCAGGGCCCCCAGGATTCCAGGGGGAACCAG
GGCCCCAGGGCGAGCCTGGGCCCCCAGGTGATCGAGGCCTCAAGGGGGATAATGGAGT
GGGCCAGCCCGGGCTGCCTC~GGCCC~,.AGGGCAGGGGGGTGCCCCCGGCCCCCCCGGC
CTCCCTGGTCCAGC T
GGCTTAGGCAAACC T
GGTTTGGA'i'GGG W
WCC~i~i;GGGi:CCC:AG
GAGACAAGGGTGAGTCTGGGCCTCCTGGAGTTCCAGGCCCCAGGGGGGAGCCAGGAGC
TGTGGGCCCAAAAGGACCTCCTGGAGTAGACGGTGTGGGAGTCCCAGGGGCAGCAGGG
TTGCCAGGACCACAGGGCCCATCAGGGGCCAAAGGGGAGCCAGGAACCCGGGGCCCCC
CTGGGCTGATAGGCCCCACTGGCTATGGGATGCCAGGACTGCCAGGCCCCAAGGGGGA
CAGGGGCCCAGCTGGGGTCCCAGGACTCTTGGGGGACAGGGGTGAGCCAGGGGAGGAT
GGGGAGCCAGGGGAGCGGGGCCCACAGGGTCTTGGGGGTCCCCCCGGACTTCCTGGGT
CTGCAGGGCTTCCTGGCAGACGTGGGCCCCCTGGGCCTAAGGGTGAGGCAGGGCCTGG
AGGACCCCCAGGAGTGCCTGGCATTCGAGGTGACCAGGGGCCTAGTGGCCTGGCTGGG
AAACCAGGGGTCCCAGGTGAGAGGGGACTTCCTGGGGCCCATGGACCCCCTGGACCAA
CTGGGCCCAAGGGTGAGCCGGGTTTCACGGGTCGCCCTGGAGGACCAGGGGTGGCAGG
AGCCCTGGGGCAGAAAGGTGACTTGGGGCTCCCTGGGCAGCCTGGCCTGAGGGGTCCC
TCAGGAATCCCAGGACTCCAGGGTCCAGCTGGCCCTATTGGGCCCCAAGGCCTGCCGG
G,CCTGAAGGGGGAACCAGGCCTGCCAGGGCCCCCTGGAGAGGGGAGAGCAGGGGAACC
TGGCACGGCTGGGCCCACGGGGCCCCCAGGGGTCCCTGGCTCCCCTGGAATCACGGGC
CCTCCGGGGCCTCCCGGGCCCCCGGGACCCCCTGGTGCCCCTGGGGCCTTCGATGAGA
CTGGCATCGCAGGCTTGCACCTGCCCAACGGCGGTGTGGAGGGTGCCGTGCTGGGCAA
GGGGGGCAAGCCACAGTTTGGGCTGGGCGAGCTGTCTGCCCATGCCACACCGGCCTTC
ACTGCGGTGCTCACCTCGCCCTTCCCCGCCTCGGGCATGCCCGTGAAATTTGACCGGA
CTCTCTACAATGGCCACAGCGGCTACAACCCAGCCACTGGCATCTTCACCTGCCCTGT
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relationships shown in Table 12B.
Table 12B. Comparison of NOVl2a against NOVl2b.
Protein Sequence ~ NOVl2a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOVl2b ~ 565..733 ~ 155/169 (91%)
470..638 156/169 (91%)
Further analysis of the NOV 12a~ protein yielded the following properties
shown in Table
12C.
Table 12C. Protein Sequence Properties NOVl2a
PSort 0.5899 probability located in outside; 0.1000 probability located in
endoplasmic
analysis: reticulum (membrane); 0.1000 probability located in endoplasmic
reticulum (lumen);
0.1000 probability located in lysosome (lumen)
SignaIP - Cleavage site between residues 22 and 23
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.
12?
Sequence comparison of the above protein sequences yields the foiiowing
sequence
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Table 12D. Geneseq Results for NOVl2a
NOVl2a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesEzpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAM79782 Human protein SEQ ID NO 96..733 607/640 (94%)0.0
3428 -
Homo sapiens, 644 aa. 16..644 609/640 (94%)
[W0200157190-A2, 09-AUG-2001]
AAM78798 Human protein SEQ 1D NO 96..733 607/640 (94%)0.0
1460 -
Homo sapiens, 635 aa. 7..635 609/640 (94%)
[WO200157190-A2, 09-AUG-2001
AAM39127 Human polypeptide SEQ ID 100..732369!649 (56%)0.0
NO 2272 -
Homo sapiens, 744 aa. 117..743418/649 (63%)
[W0200153312-Al, 26-JUL-2001]
AAM40913 Human polypeptide SEQ ID i 00.. 368/649 (~6io)' 0.0
NO 5844 - 732
Homo Sapiens, 755 aa. 128..754417/649 (63%)
[W0200153312-A1, 26-JUL-2001]
AAW57673 Collagen-like polymer - ~ 24..621~ 317/629 ~ e-159
Synthetic, 829 (50%)
aa. (US5773249-A, 30-JUN-1998]42..662 341/629 (53%)
In a BLAST search of public sequence datbases, the NOV 12a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 12E.
Table 12E. Public BLASTP Results for NOVl2a
Protein ~ NOVl2aIdentities/
AccessionProtein/Organisnn/Length Residues/SimilaritiesE$pect
for
Match the Matched Value
Number ResiduesPortion
BAB84955 FLJ00201 PROTEIN - Homo 68..733 623/670 (92%)0.0
Sapiens
(Human), 705 as (fragment).51..705 631/670 (93%)
P25067 Collagen alpha 2(VIII) chain96..733 603/640 (94%)0.0
(Endothelial collagen) - 7..635 6081640 (94%)
Homo Sapiens
(Human), 635 as (fragment).
A24450 collagen alpha 2(VIII) chain96..570 422/477 (88%)0.0
- bovine,
469 as (fragment). 1..469 432/47? (90%)
Q9D2V4 ~ PROCOLLAGEN, TYPE VIII, 5..732 400/760 (52%)0.0
ALPHA 1 - Mus musculus (Mouse),3..743 460/760 (59%)
744 aa.
Q92I S8 PROCOLLAGEN, TYPE VIII, 5..732 399/760 (52%)0.0
ALPHA 1 - Mus musculus (Mouse),3..743 460/760 (60%)
744 aa.
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PFam analysis predicts that the NOV I2a protein contains the domains shown in
the Table
12F.
Table 12T. Domain Analysis of NOVl2a
Identities/
Pfam DomainNOVl2a Match Similarities Expect
Region Value
for the Matched
Region
Collagen 25..83 28/60 (47%) 0.00092
37/60 (62%)
Collagen 86..144 34/60 (57%) 2.4e-05
51/60 (85%)
Collagen 151..208 33/60 (55%) 0.0002
46/60 (77%)
Collagen 209..267 35/60 (58%) 0.00054
47/60 (78%)
Collagen 270..328 31/60 (52%) 5.2e-05
46/60 (77%)
Collagen 329..387 29/60 (48%) 0.00048
44/60 (73%)
Collagen 388..447 35/60 (58%) 4.4e-1
1
48/60 (80%)
Collagen 448..507 34/60 (57%) 8.9e-1
1
46/60 (77%)
Collagen 508..566 39/60 (65%) 9.3e-05
50/60 (83%)
Clq 606..730 68/137 (50%) ~ 3.4e-75
123/137 (90%)
Example 13.
S The NOV 13 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 13A.
Table 13A. NOV13 Sequence Analysis
SEQ ID NO: 43 [ 789 by
NOVl3a, ATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATGTGCCTGTGG
CG96384-O1 DNA ~CCTTGCCCTGGGCATCGCTGCTGCTGCCAGCGCCCTTCACCCATGCTTCAGCATGG
SeqllenCe TGCCCATACGCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAGCACAGGAGA
GATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCTTAGTGTGCT
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CAGGAGATCGCTCTTCAGCAAATAATGTCTCAGACTGTGAATGTGAAAA,ACGATATGA
TTACTTTGGAGAAGAGTGAATTTTCAGCCCTCAGAGCAGAACGTGAGAAAATAAAACT
CAAACTACATCAGTTAAAACAAGTAATGGATGAAGTGATTAAAGTCCGAACAGATACT
AAATTAGACTTCAACCTAGAAAAGAGCAGAGTAAAAGAATTGTATTCGTTGAATGAAA
GGAAGCTGCTGGAATTGAGAACAGAAATAGTGACATTGCATGCCCAGCAAGATTGGGC
CGTCACCCAGAGAGATAGGAAGATAGAAACTGAGGATGCTGGCCCCAAAACCATGCTT
GAGTCATACAAGCTTGATAATATTAAATATTTAGCAGGGTCTATATTTACGTGCCTAA
CAGTAGCTCTGGGATTTTATCACCTGTGGATCTAA
ORF Start: ATG at
1 OItF Stop: TAA
at 787
SEQ ID NO: 44 262
as MW at 30251.7kD
NOVl3a, MPWVCRPTGWTKRRVDVPVGPCPGHRCCCQRPSPMLQHGAHTHFLQESAGYLQLEHRR
CG96384-O1 DFSSSGSRKLSFDTRSLVCFLEDHGFATQQAEIIVSALVQVLEANVDIVYIIC)MATKNIK
Protein SequenceQEIALQQIMSQTVNVKNDMITLEKSEFSALRAEREKIKLKLHQLKQVMDEViKVRTDT
KLDFNLEKSRVKELYSLNERKLLELRTEIVTLHAQQDWAVTQRDRKIETEDAGPKTML
ESYKLDNIKYLAGSIFTCLTVALGFYHLWI
SEQ ID NO: 45 789 by
NOVl3b, ATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATGTGCCTGTGG
~
CG96384-02 GGCCTTGCCCTGGGCATCGCTGCTGCTGCCAGCGCCCTTCACCCATGCTTCAGCATGG
DNA
Sequence TGCCCATACGCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAGCACAGGAGA
GATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCTTAGTGTGCT
TTCTGGAAGACCATGGGTTTGCTACTCAGCAAGCAGAAATCATTGTGTCTGCATTGGT
CCAGGTACTGGAGGCCAACGTGGACATCGTCTACAAAGATATGGCCACCAAGATGAAG
CAGGAGATCGCTCTTCAGCAAATAATGTCTCAGACTGTGAATGTGAAAAACGATATGA
TTACTTTGGAGAAGAGTGAATTTTCAGCCCTCAGAGCAGAACGTGAGAAAATAAAACT
CAAACTACATCAGTTAAAACAAGTAATGGATGAAGTGATTAAAGTCCGAACAGATACT
AAATTAGACTTCAACCTAGAAAAGAGCAGAGTAAAAGAATTGTATTCGTTGAATGAAA
GGAAGCTGCTGGAATTGAGAACAGAAATAGTGACATTGCATGCCCAGCAAGATTGGGC
CGTCACCCAGAGAGATAGGAAGATAGAAACTGAGGATGCTGGCCCCAAAACCATGCTT
GAGTCATACAAGCTTGATAATATTAAATATTTAGCAGGGTCTATATTTACGTGCCTAA
CAGTAGCTCTGGGATTTTATCACCTGTGGATCTAA
ORF Start: ATG at ORF Stop: TAA at 787
1
SEQ ID NO: 46 262 as MW at 30251.7kD
NOVl3b, MPWVCRPTGWTKRRVDVPVGPCPGHRCCCQRPSPMLQHGAHTHFLQESAGYLQLEHRR
CG96384-02 DFSSSGSRKLSFDTRSLVCFLEDHGFATQQAEIIVSALVQVLEANVDIVYKDMATKMK
PrOteln SequenceQEIALQQIMSQTVNVKNDMITLEKSEFSALRAEREKIKLKLHQLKQVMDEVIKVRTDT
KLDFNLEKSRVKELYSLNERKLLELRTEIVTLHAQQDWAVTQRDRKIETEDAGPKTML
ECvKLDNIKI'LAGS T_FTCLTVALGFYUT..~WI
SEQ ID NO: 47 285 by
NOV13C, GGATCCACCATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATG
209749131 TGCCTGTGGGGCCTTGCCCTGGGCATCGCTGCTGCTGCCAGCGCCCTTCACCCATGCT
DNA
Sequence TCAGCATGGTGCCTATACTCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAG
CACAGGAGAGATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCT
TAGTGTGCTTTCTGGAAGACCATGGGTTTGCTACTCAGCAAGCAGAACTCGAG
ORF Start: at 1 ORF
Stop: end of sequence
SEQ ID'NO: 48 95
as MW at 10768.1kD
NOV13C, GSTMPWVCRPTGWTKRRVDVPVGPCPGHRCCCQRPSPMLQHGAYTHFLQESAGYLQLE
209749131 ~FSSSGSRKLSFDTRSLVCFLEDHGFATQQAELE
Protein
Sequence .
SEQ ID NO: 49 801
by
NOVl3d, GGATCCACCATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATG
209749O3O TGCCTGTGGGGCCTTGCCCTGGGCATCGCTGCTGCTGCCAGCGCCCTTCACCCATGCT
DNA
Sequence TCAGCATGGTGCCTATACTCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAG
CACAGGAGAGATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCT
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relationships shown in Table 13B.
Table 13B.
Comparison
of NOVl3a
against NOVl3b
through NOVl3d.
Protein Sequence( NOVl3a Residues/Identities/
Match ResiduesSimilarities for the Matched Region
NOVl3b 1..262 262/262 (100%)
1..262 262/262 (100%)
NOV 13c 1..91 89/91 (97%)
4..94 91/91 (99%)
NOV 13d 1..262 261 /262 (99%)
4..265 262/262 (99%)
Further analysis of the NOV 13a protein yielded the following properties shown
in Table
13C.
Table 13C. Protein Sequence Properties NOVl3a
PSort 0.7000 probability located in plasma membrane; 0.2000 probability
located in
analysis: endoplasmic reticulum (membrane); 0.1000 probability located in
mitochondria) inner
membrane; 0.0000 probability located in endoplasmic reticulum (lumen)
SignalP ~ No Known Signal Sequence Predicted
analysis:
A search of the NOV 13 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 13D.
131
Sequence comparison of the above protein sequences yields the following
sequence
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Table 13D. Geneseq Results
for NOVl3a
NOVl3a Identities/
Geneseq Protein/Organism/Length [ Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAM40693 Human polypeptide SEQ ID [ 45..262185/220 (84%)2e-9S
NO 5624 -
Homo Sapiens, 246 aa. [WO200I27..246 197/220 (89%)
53312-
AI, 26-JUL-2001) ~
AAM38907 Human polypeptide SEQ ID 104..262138/160 (86%)2e-70
NO 2052 -
Homo Sapiens, 160 aa. [W0200I53312-~ 1..160145/160 (90%)
A1, 26-JUL-2001)
AAG73708 Human colon cancer antigen 142..262110/122 (90%)8e-54
protein
SEQ ID NO:4472 - Homo Sapiens,8..129 I 141122
129 (93%)
- aa. [W0200122920-A2, OS-APR-2001]
AAG787S0 Human calthrin light chain 111..26170/1 S2 (46io)' Se-34
17 - Homo '
sapiens, 153 aa. [W020017S04S-A2,I..1S2 104/152 (68%)
I I-
OCT-2001 )
AAB28214 Novel human protein # 12 s 59..19767/140-(47%)2e-30
- Homo
sapiens, 1 S6 aa. [WO2000S216S-A2,17..1 99/140 (69%)
08- S6
SEP-2000]
....
In a BLAST search of public sequence datbases, the NOV 13a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 13E.
Table 13E. Public BLASTP
Results for NOVl3a
Protein NOVl3a ~ Identities/
AccessionProtein/Organism/Length Residues/SimilaritiesExpect
for
Number Maxch ~ the Matched! Value
Residues Portion
Q96JS7 SIMILAR TO RIKEN CDNA 45..262 186/220 (84%)1e-96
6230416A05 GENE - Homo 102..321 198/220 (89%)
Sapiens
(Human), 321 as (fragment).
Q96AQ8 SIMILAR TO RIKEN CDNA 45..262 185/220 (84%)Se-9S
6230416A05 GENE - Homo 140..359 197/220 (89%)
Sapiens
(Human), 359 aa.
Q9NUI2 DJSOOL14.I (NOVEL PROTEIN)45..262 185/220 (84%)Se-95
-
Homo sapiens (Human), I ..220 197/220 (89%)
220 as
(fragment).
132
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Q9CXD6 6230416AOSRIK PROTEIN - Mus musculus (Mouse), 45..262 163/220 (74%) 2e-
340 aa. ' 121..340 188/220 (85%) 83
Q9GZT6 MDSO11 (MDS025) (HYPOTHETICAL 29.5 KDA 59..261 96/204 (47%) 6e-
PROTEIN) - Homo Sapiens (Human), 254 aa. 50..253 138/204 (67%) 48
PFam analysis predicts that the NOV I3a protein contains the domains shown in
the Table
13F.
Table 13F. Domain Analysis of NOVl3a
Identities/
Pfam Domain NOVl3a Match Region Similarities Expect Value
for the Matched Region
Example 14.
The NOV 14 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 14A.
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CGTGTTCTTCACCATCTGGATCATTGGAGGAGGCACGACACCCATGTTGTCATGGCTT
AATATCAGAGTTGGTGTTGACCCTGATCAAGATCCACCACCCAACAATGACAGCTTTC
AAGTCTTACAAGGGGACAGCCCAGATTCTGCCAGAGGAAACTGGACAAAACAGGAGAG
CACATGGATATTCAGGCGGTGGTACAGCTTTGATCACAATTACCTGAAGCCCATCCTC
ACACACAGCGGCTCCCCGCTAACCACCACTCTCCCGCCCGCCTGGTGTAGCTTGCTAG
CTCGATGTCTGACCAGTCCCCAGGTGTACGATAACCAAGAGCCACTGAGAGAGGGAAA
CTCTGATTTTATTCTGACTGAAGGCGACCTCACATTGACCTATGGGGACAGCACAGTG
ACTGCAAATGGCTTCTCAGGTTCCCACACTGCCTCCACGAGTCTGGAGGGCAGCTGGA
GAATGAAGAGCAGCTCAGAGGAAGTGCTGGAGCAGGACGTGGGAP.TGGGAAACCAGAA
GGTTTCGAACCAGGGTACCCGCCTAGTGTTTCCTCTGGAAGATAATGTTTGACTTTCC
CTGCAAACCCTGGCACGATGGGGTAGGCTCCCAATGGGGTGAGGATGGCTTCAAGCCC
TAATGTTGCTTGAGGTGGGGCAGTGACTAGATTGAATTAACTCTTCTATTTTATTGGG
GTCTGAAGTTATTGTAACACTTAAAATTTAACTCATGATGCAGATGGTGAGGCAAAAG
TGTCTCTAAATTCAGACAAATGTAGACCTATTTCTACTTTTTTTCACACAGTAGTGCG
CTGTTTCAGAGTTAAACAAACAAAAAAATAGCATACTTTAATGGTCTCTTAATTCATT
CACCTGCAGTGTCTGAACAAGGCAGGGCAGGTGCTGAGTGGGGGGCTTCCTCTTACAA
GAGGCTGCATCTCAGTACACAGTGGTGCAAGTCAAGCTGACCATAGAAATATCAAGTT
AGGGGAGAACAGGCTGGAAGAAAGATTGAGAAGGAAGGCAATTGAGATAGGACCTCCA
AGGATTATGGGAAACTGTTGTTAAATGGAACAGAAAACATGAAAAAATAATATGAGTG
GAGGCTCTGGCAAGGAAGGCTGTGTGACTGCAACCTCATATCAGGATTCCTGACTTTT
ATGCTACCTGTGTTTCTTCTAGACTGA~l.GATTTG_A Z1.A pTnmnTr_Cn
TrzLn_CAmTTCAA
CATGAAACAAAGAATTATAGTTCCTTCTCTGGAGATGTCCATAAAGAAGTAATTATGA
TATGTTTAAAACCAGACCGGGTGTGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGA
GGCCGAGGCCGGCGCATCATCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGG
TGAAACCCCGTCTCTACTAP.AAAATACAAATATTAGCCAGGCGTGGTGGCAAGCACCT
TAATCCCAGCTACTTGGGAGGCAGAGGCAGGAGAATCGTTTGAACCCAGGAGGCTGAG
GTTGCAGTGAGCCCAGATAAAGCCACTGCACTCAAACGTGGGGAACAAGAGTGAGACT
TCTCTCAAAAAATAAATAAATAAATAAAATAATATAAAATAAACCAAAGGCAAATAGT
GTTACACTGTTAATTTTTAGTTAATCTGAAGAAAGGAGGTATTTAGAAACTGATGATG
GTATCACTGCAAAAAGCATTAAACTTTTGAGGGCTACCATATGAGCTGACAGCCTAGG
AAATAATTAGTAAGACTGAGGTATCTACTGTGGGTTTAGAAATAACACCAAATTTTGT
AGGATGCTATTATCTGGGGAAGAGAAGGCAGCAAGAAACCTACAAGGCAACAGGCTAG
AAATCAGAGGGAAAGAAATCTCTATGCAAAAAATTAACTGCAAAGAAATACAAGATGG
TAGTCTCAAGACCGGGTAGAATTCTGAACTTTGAGCTGTCGAATGCATGAGATTTCAG
TGGCCACATGAGGAATCAGTGGGAAGTCAATGGAACGTTAAGTATTTTCAACCCACTA
GAAGGTCCTGTCTTCTATAAGTTTAAGAATCTAAGTGTCTTTATGCCATTGAGTGCGG
TGCAGAGAAGGGTCATTTTCCCTTTATCTGGGGAGGCTGCTTCACCAGCCTACCATGT
GGGTGTGATTTGAAAGTTAGGTTTTCAGTTTGGGTTCTTTCTGGATGAGCTGTTCTGT
CCGCCCACACCTGTAGTGCTGAAATACTGAAAGATCTCTCCGGAAAAGTTTGAGTTTC
TCCCCATGTTTCTGTGCTTCAGCAATAGCATTTTTT_TGGCAACCCAATTTCTAAAAA.A
TGCi1TCAATt'1ATGTGGGCATTTTCTTATTATGGCAG~~HlziWt~.W.AUJa;.TA
GTCTACCATAATGAAATCAGCCATTTAATCTTCTCAATTTGCATGTTTAATGGTTAAT
TTTTAAATAAGACATTGCTTCACATCTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGT
CTTGCTCTTTCGCCCGGGCTGGAGTGCAGTCGTGCAACCTCGGCTCGCTGCAATCTCT
GCGCCCCCAGGTTCACGTGATTCTCCTGCCTCAGC_CTCCCTAGTAGCTGGGATTGCAG
GTGCCCACCACCACACCTGGCTAATTTTTTATTTTTAGTAGAGATGGGGTTTTGCCAT
GTTGGCCAGGCTGGTCTTGAACTCCTGACCTTCAGGTGATCCACCTGCCTTGGCCTCC
CGAAGTGCTAGGATTACAGGCATGAGCCACCATGCCCGGCCTGCGTCATAACTTTGTG
TTTGAATTGATAATTTGTGCAAATCAGGAAAATATATATTTAATTAAGTTGAGCCATA
TGAATTTGCTGATATTCAACCATTTTGTAAAAACAGGAGTGGCAATTTCATATGGTTC
AATAAAATAAAATTGAGGCCGGGCACAGTGGCTTACACCCATAATCCCAACACTTTGG
GAGGCTGAAGCAGGAGGATCGCTTGAGCTCAGGAGTTTGAGACCAGACTGAGCAACAT
GGCAGAACTCTGTCTCTACAAAAATACAAAAATGAGACAGGCATGGTGGCACATACCT
TTAGTTCAGTTCTAGGTGATTGGGAGGCTGAGGTGGGAGGATCGCTTGAACCCAAGAG
GCAGAGGATGCAGTGAGCCAAGATCATACCACTGGAAACCAGCCTGGGCAACAGAGTG
AGAACCTGTCTCAAACAAACAAACAAACTGGAATTTATTTTTATGTATGGTATGAGAA
AGGGATTTGTTTTTTTTGTTTTTTTTTTTTTTTTTTTGATATGGAGTCTTACTCTGTT
GCCCAGGCTGGAGTGCAGTGGCGCCATCTTGGCTCACTGCAACTTCTGCCTCCTTTCC
TTTGTTCAAGCGATTCTCTTGTCCCTGAGTAGCTGGGATT_ACAGGCACCGGCCACCAC
GCCCAGCTATTTTTTGTATTTTTAGTAGAGACAGGGTTTC_ACCATGTTGGCCAGGCTG
GTCTCAAACTCCTGACCTCAGGCATTCTGCCCCCCTTGGCCTCCGAAAGTGCTGGGAT
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CACAGGCATGAGCCACTGTGCCCAGTCTGAGAAAGGGATTTAATTTACTTTTTTTCTT
CCAAATGGATAGCTCCTTGTCCCAACACTCTCTATTAGTCTGTCATTTCCCAAGTGAT
TTTAAATGTCTCCTTTAACATATACTAAGATTACACACACACACACACACACACACAT
ACACACAAATGGACACATATATGTGTGTGTGTGTATATATATTTTTCTGGACTTTTAA
OIZF Start: ATG at 73 ORF Stop: TGA at 2080
SEQ ID NO: 52 ~ 669 as MW at 73935.6kD
~NOVl4a, MEPGDAALPCPGRVAQAPPRRLLLLLPLLLGRGLRVTAEASASSSGAAVENSSAMEEL
CG96432-OI VTEKEAEESHRPDSVSLLTFILLLTLAILTIWLFKYCRVHFLHETGLAMICGLIVGVI
'Protein SequenceLRYGTPGTRGRDKLLNCTQEDQAFSTLVVTFDPEVFFNILLPPVIFHAGYSLKRHFFR
NLGSLLGHSLGTAVSCFRIGNLRYGMVKLMKIMRQLSDKFYYTHCLFFRAIISATDPV
i TVLVIINELHADMDLYVLLFGESILNDVVMWLSSSIVGYQPAGLNTHAFDAAAFLKS
VGIFLGIFSGCFTMGAVTGVVTALVTKFTKLDCFPLLETALFFLMSWSTFLLAEACGF
TGWAVLFCGITQAHYTFNNLSVESRSRSKQLFEAENFIFSCMILALFTFQKHVFSPV
FIIGAFVAVFLGRAAHIYPLSFFLSLGRRHKIGWNFQHTMMFSGLRGAMAFALAICDT
ASYARQMTFPTTPFIVFFTIWIIGGGTTPMLSWLNIRVGVDPDQDPPPNNDSFQVLQG
DSPDSARGNWTKQESTWIFRRWYSFDHNYLKPILTHSGSPLTTTLPPAWCSLLARCLT
SPQVYDNQEPLREGNSDFILTEGDLTLTYGDSTVTANGFSGSHTASTSLEGSWRMKSS
SEEVLEQDVGMGNQKVSNQGTRLVFPLEDNV
Further analysis of the NOV 14a protein yielded the following properties shown
in Table
14B.
Table I4B. Protein Sealuence P~-operiies hT~Vi_4a
PSort 0.8000 probability located in plasma membrane; 0.4000 probability
located in Golgi
analysis: body; 0.3000 probability located in endoplasmic reticulum
(membrane); 0.0300
probability located in mitochondria) inner membrane
SignalP Cleavage site between residues 39 and 40
analysis:
A search of the NOV I 4a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 14C.
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Table 14C. Geneseq Results for NOVl4a
NOVl4a Identities/
Geneseq Protein/OrganismlLength Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAE16770 Human transporter and ion SS..668 S46/673 (81%)0.0
channel-7
(TRICH-7) protein - Homo 1..672 570/673 (84%)
Sapiens, 673
aa. [WO200192304-A2, 06-DEC-2001]
AAB90637 Human secreted protein, 43..517 412/514 (80%)0.0
SEQ ID NO:
.
180 - Homo sapiens, 526 6..519 431/514 (83%)
aa.
[W0200121658-Al, 29-MAR-2001]
AAB90SSS Human secreted protein, SS..S 402/502 (80%)0.0
SEQ ID NO: 93 17
- Homo Sapiens, 509 aa. 1..502 420/502 (83%)
[W0200121658-Al, 29-MAR-2001]
AAU02883 Human HsNHE-6 polypeptide 13..645 379/641 (S9%)t 0.0
- Homo
sapiens, 664 aa. [W0200133945-A1,13..631 450/641 (70%)
17-
MAY-2001 J
AAB90591 Human secreted protein, 335..6683021339 (89%)e-174
SEQ ID NO:
129 - Homo Sapiens, 339 1..338 314/339 (92%)
aa.
[W0200121658-Al, 29-MAR-2001]
_ . .. _._. ~
In a BLAST search of public sequence datbases, the NOV I4a protein Was
found,to have
homology to the proteins shown in the BLASTP data in Table 14D.
Table I4D. Public BLASTP Results for NOVl4a
Protein NOVl4a Identities/
Accession~ Residues/~ Expect
Protein/Organism/Length Similarities
for
Number Match she lwiaici~edV apnne
ResiduesPortion
Q96T83 NONSELECTIVE SODIUM 1..668 584/727 (80%)0.0
POTASSIUM/PROTON EXCHANGER 1..724 609/727 (83%)
- Homo sapiens (Human), 725
aa.
075827 DJ71L16.S (KIAA0267 LIKE 111..668494/617 (80%)0.0
PUTATIVE NA(+)/H(+) 1..615 517/617 (83%)
EXCHANGER) - Homo Sapiens
(Human), 616 as (fragment).
Q92581 Sodium/hydrogen exchanger 19..668 414/657 (63%)0.0
6
(Na(+)/H(+) exchanger 6) 19..654 492/657 (74%)
(NHE-6) -
Homo sapiens (Human), 669
aa.
Q9U624 SODIUM-HYDROGEN EXCHANGER 52..620 287/654 (43%)e-128
NHE3 - Drosophila melanogaster17..659 378/654 (56%)
(Fruit
fly), 687 aa.
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_-
.._......_.........................._.._.~._...._..._................._....._..
..._........_.._... 3 _ . ,~",~"",
Q9VM99 NHE3 PROTEIN - Drosophila 52..620 287/654 (43%) e-128
melanogaster (Fruit fly), 727 aa. 57..699 378/654 (S6%)
PFam analysis predicts that the NOV 14a protein contains the domains shown in
the Table
14E.
Table 14E. Domain Analysis of NOVI4a
Identities/
Pfam Domain NOVl4a Match Region Similarities Expect Value
for the Matched Region
Na_H_Exchanger 75..502 143/472 (30%) 8.I e-103
3 S 11472 (74%)
Example 15.
The NOV 1 S clone was analyzed, and the nucleotide and encoded poIypeptide
sequences
are shown in Table 15A.
Table ISA.
NOV15 Sequence
Analysis
SEQ ID NO: 53 766
by
NOVlSa TATTTGGCGCCCGCTCTCTCTCTCTGTCCCTTTGCCTGCCTCCCTCCCTCCGGATCCC
, ~CTCTCTCCCCGGAGTGGCGCGTCGGGGGCTCCGCCGCTGGCCAGGCGTGATGTTGC
CG96S4S-02
DNA
Sequence ACGTGGAGATGTTGACGCTGGTGTTTCTGGTGCTCTGGATGTGTGTGTTCAGCCAGGA
CCCGGGCTCCAAGGCCGTCGCCGACCGCTACGCTGTCTACTGGAACAGCAGCAACCCC
AGATTCCAGAGGGGTGACTACCATATTGATGTCTGTATCAATGACTACCTGGATGTTT
TCTGCCCTCACTATGAGGACTCCGTCCCAGAAGATAAGACTGAGCGCTATGTCCTCTA
CATGGTGAACTTTGATGGCTACAGTGCCTGCGACCACACTTCCAAAGGGTTCAAGAGA
TGGGAATGTAACCGGCCTCACTCTCCAAATGGACCGCTCAAGTTCvCTvT"~TTCC
AGCTCTTCACTCCCTTTTCTCTAGGATTTGAATTCAGGCCAGGCCGAGAATATTTCTA
CATCTCCTCTGCAATCCCAGATAATGGAAGAAGGTCCTGTCTAAAGCTCAAAGTCTTT
GTGAGACCAACAAATGACACCGTACATGAGTCAGCCGAGCCATCCCGCGGCGAGAACG
CGGCACAAACACCAAGGATACCCAGCCGCCTTTTGGCAATCCTACTGTTCCTCCTGGC
GATGCTTTTGACATTATAGCACAGTCTCCTCCCATCACTTGTCACAGAAAACATCAGG
GTCTTGGAACAC
' ORF Start: ATG at ORF Stop:
110 TAG
at 713
SEQ ID NO: 54 201 as MW at 23295.41eD
NOVISa, MLHVEMLTLVFLVLWMCVFSQDPGSKAVADRYAVYWNSSNPRFQRGDYHIDVCINDYL
CG96S4S-O2 DVF'CPHYEDSVPEDKTERYVLYMVNFDGYSACDHTSKGFKRWECNRPHSPNGPLKFSE
PTOtelri SequenceKFQLFTPFSLGFEFRPGREYFYISSAIPDNGRRSCLKLKVFVRPTNDTVHESAEPSRG
ENAAQTPRIPSRLLAILLFLLAMLLTL
SEQ ID NO: SS 764 by
NOVISb TATTTGGCGCCCGCTCTCTCTCTGTCCCTTTGCCTGCCTCCCTCCCTCCGGATCCCCG
, CCCTCTCCCCGGAGTGGCGCGTCGGGGGCTCCGCCGCTGGCCAGGCGTGATGTTGCAC
CG96S4S-O3
DNA
Sequence GTGGAGATGTTGACGCTGGTGTTTCTGGTGCTCTGGATGTGTGTGTTCAGCCAGGACC
CGGGCTCCAAGGCCGTCGCCGACCGCTACGCTGTCTACTGGAACAGCAGCAACCCCAG
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GTTCCAGAGGGGTGACTACC~P.TATTGATGTCTGTATCAATGACTACCTGGATGTTTTC
TGCCCTCACTATGAGGACTCCGTCCCAGAAGATAAGACTGAGCGCTATGTCCTCTACA
TGGTGAACTTTGGTGGCTACAGTGCCTGCGACCACACTTCCAAAGGGTTCAAGAGATG
GGAATGTAACCGGCCTCACTCTCCAAATGGACCGCTGAAGTTCTCTGAAAAATTCCAG
CTCTTCACTCCCTTTTCTCTAGGATTTGAATTCAGGCCAGGCCGAGAATATTTCTACA
TCTCCTCTGCAATCCCAGATAATGGAAGAAGGTCCTGTCTAAAGCTCAAAGTCTTTGT
GAGACCAACAAATGACACCGTACATGAGTCAGCCGAGCCATCCCGCGGCGAGAACGCG
GCACAAACACCAAGGATACCCAGCCGCCTTTTGGCAATCCTACTGTTCCTCCTGGCGA
TGCTTTTGACATTATAGCACAGTCTCCTCCCATCACTTGTCACAGAAAACATCAGGGT
CTTGGAACAC
ORF Start: ATG at ORF Stop:
108 TAG
at 711
SEQ ID NO: S6 201 as MW at 23237.3kD
NOVlSb, MLHVEMLTLVFLVLWMCVFSQDPGSKAVADRYAVYWNSSNPRFQRGDYHIDVCINDYL
CG96S4S-03 DVFCPHYEDSVPEDKTERYVLYMVNFGGYSACDHTSKGFKRWECNRPHSPNGPLKFSE
Protein SequenceKFQLFTPFSLGFEFRPGREYFYISSAIPDNGRRSCLKLKVFVRPTNDTVHESAEPSRG
ENAAQTPRIPSRLLAILLFLLAMLLTL
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 1 SB.
Table 15B. Comparison of NOVlSa against NOVlSb.
Protein Sequence NOVlSa Residues/ Identities/
Match Residues Similarities for the Matched Region
NOV l Sb 1..186 ' 18S/186 (99%)
1..186 18S/186 (99%)
Further analysis of the NOV 1 Sa protein yielded the following properties
shown in Table
1 SC.
''able 15C. Protein Sequence Properties NtW' iSa
PSort 0.9190 probability located in plasma membrane; 0.2212 probability
located in
analysis: microbody (peroxisome); 0.2000 probability located in lysosome
(membrane); 0.1000
probability located in endoplasmic reticulum (membrane)
SignalP Cleavage site between residues 21 and 22
analysis:
S A search of the NOV 15a 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 SD.
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Table 15D. Geneseq Results for NOVlSa
NOVlSa Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAW00035 HEK4 binding protein - Homo1..201 201/228 (88%)e-115
sapiens,
228 aa. [W09623000-A1, Ol-AUG-1..228 201/228 (88%)
1996]
AAW02586 Lerk-7 protein - Homo Sapiens,1..201 201/228 (88%)e-115
228 aa.
[W09617925-A1, 13-JLTN-1996]1..228 201/228 (88%)
AAR97854 Human AL-1, a ligand for 1..201 201/228 (88%)e-115
eph-related "
tyrosine kinase receptor 1..228 201/228 (88%)
REK7 - Homo
Sapiens, 228 aa. [W09613518-A1,
09-
MAY-1996]
ABG27837 Novel human diagnostic protein4..201 i 98/225 ' e-113
#27828 - (88%)
Homo Sapiens, 335 aa. [W0200175067-111..335198/225 (88%)
A2, I1-OCT-2001]
ABG27837 Novel human diagnostic protein4..201 198/225 (88%)e-113
#27828 -
Homo sapiens, 335 aa. [W0200175067-1 11..335198/225 (88%'0)
A2, 11-OCT-2001]
In a BLAST search of public sequence datbases, the NOV 15a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 15E.
Table 15E. Public BLASTP Results for NOVlSa
Protein NOVISa Identities/
AccessionProtein/OrganismlLength Residues/SimilaritiesExpect
for
Number . _ Match s the MatchesValue
~
ResiduesPortion
P52803 Ephrin-AS precursor (EPH-related1..201 20I/228 (88%)e-115
receptor tyrosine kinase 1..228 201/228 (88%)
ligand 7) CLERK-
7) (AL-1 ) - Homo sapiens
(Human), 228
as.
P97605 Ephrin-AS precursor (EPH-related1..201 199/228 (87%)e-114
receptor tyrosine kinase 1..228 200/228 (87%)
ligand 7) CLERK-
7) (AL-1 ) - Rattus norvegicus
(Rat), 228
as.
008543 Ephrin-AS precursor (EPH-related1..201 199/228 (87%)e-114
receptor tyrosine kinase 1..228 200/228 (87%)
ligand 7) CLERK-
7) (AL-1 ) - Mus musculus
(Mouse), 228
as.
P52804 Ephrin-AS precursor (EPH-related1..201 181/28 (79%)e-102
1..228 186/228 (81%)
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7) (RAGS protein) - Gallus gallus
(Chicken), 228 aa.
P79728 Ephrin-AS precursor (EPH-related 1..201 152/229 (66%) 3e-85
receptor tyrosine kinase ligand 7) CLERK- 1..228 173/229 (75%)
7) (AL-1) (ZFEPHL4) - Brachydanio rerio
(Zebrafish) (Zebra danio), 228 aa.
PFam analysis predicts that the NOV 1 Sa protein contains the domains shown in
the Table
1 SF.
Table 15F. Domain Analysis of NOVlSa
Identities!
Pfam Domain NOVlSa Match Region Similarities ~ Ezpect Value
for the Matched Region
Ephrin 26..164 86/148 (58%) 7.8e-91
138/148 (93%)
Egamule 16.
The NOV 16 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 16A.
Table 16A.~NOVl6 Sequence Analysis
SEQ ID NO: 57 ~ ~~ 369
bp~
~~~
NOVl6a, CCCAGGTGAAGATCTGGAGGCACTCCTATGATGTCCCACCACCTCCAATGGAGCCCAA
CG97101-OI CCGCAGGAGGCCTCGGTGAGGAAGGGGCTGGGCAGTGGACTGGAGGAAGTGAGGGGCG
DNA
Sequence GCCCCTCCTCAGAGCAGCTGCCGCAGCCCGAGGTAATCTCGGCCCCGCGCCGGGGCTG
GCTGGGCAGCACCCGAC,~~,C
; ;r,TmnTCAG ;ATVGCAGGTVAGAGATGGCAAiiC-rG
CAGGCACCGCAGGGGCGAATCAGGTAGGCTACCCCAGCCAGGATTGTTCTTGTACAAG
TGTTTGTATGACCAGATGCTTTCAGTTCTCTTGAACATATACCTAGAAGTAGAATTTC
TGGGTCATATGGTAATTTTAT
ORF Start: ATG at ORF
28 Stop:
TGA
at
322
SEQ ID NO: 58 98 as MW at 10060.2kD
NOVl6a, MMSHHLQWSPTAGGLGEEGAGQWTGGSEGRPLLRAAAAARGNLGPAPGLAGQHPSTGI
CG97101-O1 I~AGQRWQTAGTAGANQVGYPSQDCSCTSVCMTRCFQFS
Protein Sequence
Further analysis of the NOV 16a protein yielded the following properties shown
in Table
16B.
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Table 16B. Protein Sequence Properties NGVl6a
PSort 0.8061 probability located in lysosome (lumen); 0.6027 probability
located in
analysis: microbody (peroxisome); 0.4500 probability located in cytoplasm;
0.1000 probability
located in mitochondria) matrix space
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.
Table 16C. Geneseq Results for NOVl6a
NOVl6a ~ Identities/t
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
Residues Region
AAUI Spider natural silk protein13..79 26167 (38%) 0.011
1781 Spidroin 1 -
Nephila clavipes, 651 aa. 90..151 29/67 (42%)
[W0200190389-A2, 29-NOV-2001]
AAY59070N. clavipes spider silk 13..79 26/67 (38%) 0.011
protein 1 -
Nephila clavipes, 718 aa. 90..151 29/67 (42%)
[LJS5989894-
A, 23-NOV-1999]
AAY40097Spider silk protein spidroine13..79 26/67 (38%) 0.01
major 1 - I
Nephila clavipes, 651 aa. 90..151 29/67 (42%)
[FR2774588-
A1, 13-AUG-1999]
AAW53346Nephila clavipes spider 13..79 26/67 (38%) 0.011
silk protein -
Nephila clavipes, 718 aa. 90..151 29/67 (42%)
[US5728810-
A, 17-MAR-1998]
AAR14308N.clavipes dragline silk 13.:79 26/67 (38%) 0.011
protein-1 - ~
Nephilia clavipes, 718 aa. 90..151 29/67 (42%)
[EP452925-A,
23-OCT 1991
In a search of public sequence NOV 16a
BLAST datbases, the protein
was found
to have
S 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 MatchedValue
ResiduesPortion
Q9UGU4 DJ526I14.1 (PERIPHERAL 2..94 48/94 (51%)4e-18
BENZODIAZEPINE RECEPTOR 1..94 54/94 (57%)
RELATED PROTEIN (ISOFORM 2))
-
Homo sapiens (Human), 102
aa.
Q13849 PERIPHERAL BENZODIAZEPINE 2,.94 47/94 (50%)3e-17
RECEPTOR RELATED PROTEIN - 1..94 53/94 (56%)
Homo sapiens (Human), 102
aa.
A36068 major ampullate fibroin protein' 13..7926/67 (38%)' 0.025
- orb spider '
(Nephila clavipes), 718 as 90..151 29/67 (42%)
(fragment).
Q8WSW4 DRAGLINE SILK PROTEIN -Nephila13..79 31/67 (46%)0.025
clavipes (Orb spider), 644 47..104 35/67 (51%)
as (fragment).
046172 DRAGLINE SILK PROTEIN SPIDROIN13..79 31/67 (46%)0.025
~
1 - Nephila clavipes (Orb 44..1 35/67 (51
spider), 617 as Ol %)
(fragment).
PFam analysis predicts that the NOV 16a protein contains the domains shown in
the Table
I6E.
Table 16E. Domain Analysis of NOVl6a
Identities/
Pfam Domain NOVl6a Match Region Similarities ~ ~~_E_ xoect value
for the Matched Region
Example 17.
The NOV 17 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 17A.
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TACTATATTATAGTATTTGTTTTCATTTTTTTGTTAAGTTCCATCTTGAACGGATTAA
GTATATATCTATTAACTAGAGGCGAAAATATAATTTATTCTCTAACTAACAAAGTTTG
GAATCATATTTTAAGATTAAAAACGTCTTTTTTTGATAAAAATAGTAATGGTGAACTA
TTAAGTAGAATTATAGATGACACTAAATCAATAAACAGTTTCATTACAGAAATTATAC
CATCTTTTTTTCCATCAATAATTGTACTATTTGGATCAATCTTTTTTTTATTTATGCT
AGATTGGGAAACAGCTTTAATTGCTCTTATTTCAATACCTATGTATGTCATTTTAATA
ATACCAATAAGCAACGTAATGCAAAAACTTTCTTATAAAACACAACTTGAAACTGCTA
AGGTTAGTGGTGTAATAGCTCATGTTTTATCTAAAATCAAATTAGTTAAACTTTCAAA
TTCAATTAATAAAGAGTTTCGTCAAACTAATTCATATTTACGAAATATATATTATTTG
GGGGTGAAAGAAGGTGTTATCAATTCAATTGTAGTACCTCTTTCTACGTTAATTATGC
TTGTTTCAATGGGGGGTGTATTAGGTTTTGGAGGATATAGAGTGGCATCTGGAGCCAT
ATCTCCTGGCACGCTTATTGCTCTTATTTTTTATATGACTCAATTAACTGACCCTATT
GAAAAP,ATATCTAGTCTCTTTACAGGATATAAAAAAACTATAGGTGCAAGTCAAAGAC
TTTCTGAAATATTAAGTGAAGAAAAAGAAAATTTACAAAATAATAATCTTAATATTTT
AAATTCAGTAGATTTATCATTTAATAACGTATCTTTCAGTTATGATGAAAACAATCAT
GTTTTTACTAATTTATCCTTTACTATACCTAAAAATAAAATAACTGCTATAGTAGGTC
CTTCCGGTTCTGGTAAAACAACTATTCTTAATTTGATTTCAAGACTATATGAAATTCA
AAGTGGTTCAATTAAGTATGGAACTAATTCTATTTATGACTATTCTTTAGTTAATTGG
AGAAAAAATTTAGGATATGTTATGCAAAACTCTGGTGTATTGAATAGAACAGTC.~AAAA
GCAATATTACTTATTCGCTACAAGAGACACCATGTATAGAAGATATCATTTATTATTC
TAAGCTAGCGTC_n n rGCamr_ammmmAm~pTGp~AT~p~CCTAF~TGP'I'T~~~
~TnCGC-,..,
ATTGGAGAAAAAGGAATTAATTTATCTGGCGGTGAAAAACAAAGGTTAGATATAGCTA
GAAACTTTATTAAAACACCTGGGATTTTGTTGTTAGATGAAGCTACTTCAAATTTAGA
TAGCGAAAGTGAACACAAAATACAAGAATCTATAAAAAATGTTAGCAACGATAGAACA
ACAATAATAGTAGCGCATCGTCTTTCCACTGTACTAAAAGCTGATAAAATAATTTTTA
TCGATAATGGTGAAATTACAGGAATGGGTACTCATGAAGAGTTATTAGCTAGACATTC
AAAATATAAAAATATGATTGAGCTACAACAATTAAAGTAAGATATTCAGAATTATATG
ACATATACT
OIRF Start: ATG at 7 ORF
Stop: TAA at 1720
SEQ ID NO: 60 571 as MW at 64349.O1eD
NOVl7a, MKQKNPVLHLVNEIEIPKWLLFFSVLLSIIGSTFQLIVPLFTQNIVDNFSEVIKNKYY
CG97168-O1 IIVFVFIFLLSSILNGLSIYLLTRGENIIYSLTNICVWNHILRLKTSFFDKNSNGELLS
Protein SequenceRIIDDTKSINSFITEIIPSFFPSIIVLFGSIFFLFMLDWETALIALISIPMYVILIIP
ISNVMQKLSYKTQLETAKVSGVIAHVLSKIKLVKLSNSINKEFRQTNSYLRNIYYLGV
KEGVINSIWPLSTLIMLVSMGGVLGFGGYRVASGAISPGTLIALIFYMTQLTDPIEK
ISSLFTGYKKTIGASQRLSEILSEEKENLQNNNLNILNSVDLSFNNVSFSYDENNHVF
TNLSFTIPKNKITAIVGPSGSGKTTILNLISRLYEIQSGSIKYGTNSIYDYSLVNWRK
NLGYVMQNSGVLNRTVKSNITYSLQETPCIEDIIYYSKLASTHDFIMKLPNDYNTLIG
EKGINLSGGEKQRLDIARNFIKTPGILLLDEATSNLDSESEHKIQESIKNVSNDRTTI
IVAHI2LSTVLKADKi I FIiu7Gr;1
T'GInGT"~~Li~H$T~Y ~.~'1'NiiELSlc,~Ln
Further analysis of the NOV 17a protein yielded the following properties shown
in Table
17B.
Table 17B. Protein Sequence Properties NOVl7a
PSort 0.6400 probability located in plasma membrane; 0.4600 probability
located in Golgi
analysis: body; 0.3700 probability located in endoplasmic reticulum
(membrane); 0.1000
probability located in endoplasmic reticulum (lumen)
SignalP Cleavage site between residues 36 and 37
analysis:
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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 17C.
Table I7C. Geneseq Results
for NOVl7a
NOVl7a Identities/
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent #, for
IdentifierDate) Match the Matched Value
ResiduesRegion
ABB48756Listeria monocytogenes protein19..571 200/554 (36%)e-116
#1460 -
Listeria monocytogenes, 23..575 349/554 (62%)
575 aa. ~
[W0200177335-A2, 18-OCT-2001
]
AAU36908Staphylococcus aweus cellular~ 18..570~ 180/569 ~ 7e-81
(31%)
proliferation protein #107813-.~78 3n9/569153~f1
-
Staphylococcus aureus, 578
aa.
[W0200170955-A2, 27-SEP-2001]1
AAU36908Staphylococcus aureus cellular18..570 180/569 (31%)7e-81
~
proliferation protein #107813..578 309/569 (53%)
-
Staphylococcus aureus, 578
aa.
[W0200170955-A2, 27-SEP-2001i
]
AAE02437Human ATP binding cassette,29..570 172/557 (30%)~ I
ABCB9 e-76
transporter protein - Homo ; 196..743296/557 (52%)
sapiens, 766
aa. [W0200140305-AI, 07-JUN-2001]
AAG79246Amino acid sequence'of a i 29..570172/557 (30%)1e-76
human TAP-
Like (HUTAPL) polypeptide 196..743296/557 (52%)
- Homo
sapiens, 766 aa. [W0200173018-A2,
04-
OCT-2001
In a BLAST search of public sequence datbases, the NOV 17a protein was found
to have
S . - - >romology to the proteins shown in the BLASTP data it T able 17 D.
Table 17D. Public BLASTP
Results for NOVI7a
Protein NOVl7a Identities/
AccessionProtein/Organism/Length Residues/SimilaritiesExpect
for
Number Match the Matched Yalue
ResiduesPortion
Q93GF4 AURT - Staphylococcus aureus,I..570 283/572 (49%)e-164
571 aa.
1..571 414/572 (71%)
Q99VX4 ATP-BINDING CASSETTE 1..570 2731574 (47%)e-160
TRANSPORTER A - Staphylococcus1..573 413/574 (71
%)
aureus (strain Mu50 / ATCC
700699),
and, 575 aa.
P72354 ATP-BINDING CASSETTE 1..570 272/574 (47%)e-159
I..573 413/574 (71%)
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aureus, 575 aa. ~ ~ ~~,~~~-~~-~~~
Q54121 PEPT - Staphylococcus epidermidis,278/571 (48%) e-157
571 1..570
aa. 1..570 401/571 (69%)
Q53614 ABCA - Staphylococcus aureus, 268/574 (46%) e-156
575 aa. 1..570
1..573 407/574 (70%)
PFam analysis predicts that the NOV 17a protein contains the domains shown in
the Table
17E.
Table 17E. Domain Analysis of NOVl7a
Identities/
Pfam Domain NOVl7a Match RegionSimilarities Expect
Value
for the Matched
Region
transmembrane4~ 18..69 17/52 (33%) 0.078
40/52 (77%)
ABC_membrane21..288 82/285 (29%) 3.4e-32
187/285 (66%)
PRK ~ 360..378 8/19 (42%) 0.22
- ~ I 4/ 19 (74%)
ABC_tran 358..543 61/199 (31%) 8.1e-49
145/199 (73%)
Example 18.
The NOV 18 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 18A.
Table 18A. NOV18 Sequence Analysis
~ SEQ ID NO: 61 ( 103 8 by
NOVIBa, CAGGCACCGGCGTTAGCGGGTCGCCGACCCGCAATCCCCGCC
CG97420-O1 DNA CGGAGTGTGCGCCGGCACCTGCCGCCGGAGACATGTTGCAAA
Sequence CCGCTCTGGCGGCCAGGCCGAGAGGGACAGAGACTGGAGCCA
CGAGCACGTCCCGCGGGCCGGGCGGCTCGCAGGGGTCGCAGGGCCCCTCGCCTCAGGG
CTTGAACCCAAGAGCAACACTTACATCCTCATCAACACCCTGGAGCCTCCTGTGGAGG
CTTAGGGCTCATCTTTATGAAGGGCAACACCATCAAGGAAACTGAAGCCTGGGACTTT
CTGCGGCGCTTAGGGGTCTACCCCACCAAGAAGCATTTAATTTTCGGAGATCCAAAGA
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AAGATGAAAGTTCTTAAGTTTGTGGCCAAGGTCCATAATCAAGACCCCAAGGACTGGC
CAGCGCAGTACTGTGAGGCTTTGGCAGATGAGGAGAACAGGGCCAGACCTCAGCCTAG
TGGCCCAGCTCCATCCTCTTGAAAGGTGGATTCAGAGGGACCCCCGGGACAA
ORF Start: ATG at ORF Stop:
91 TGA
at 1006
SEQ ID NO: 62 305 as MW at 34404.8kD
NOVl8a, MLQKPRNRGRSGGQAERDRDWSHSGNPGASRAGEDARVLRDGFAEEAPSTSRGPGGSQ
CG97420-O1 GSQGPSPQGARRAQAAPAVGPRSQKQLELKVSELVQFLLIKDQKKIPIKRADILKHVI
Protein SequenceGDYKDTFPDLFKRAAERLQYVFGYKLVELEPKSNTYILINTLEPPVEEDAEMRGDQGT
PTTGLLMIVLGLIFMKGNTIKETEAWDFLRRLGVYPTKKHLIFGDPKKLITEDFVRQR
YLEYRRIPHTDPVDYEFQWGPRTNLETSKMKVLKFVAKVWQDPKDWPAQYCEALADE
ENRARPQPSGPAPSS
SEQ ID NO: 63 727 by
NOVI8b, AGACATGTTGCAAAAACCGAGGAACCGGGGCCGCTCTGGCGGCCAGGCCGAGAGGGAC
CG9742O-02 AGAGACTGGAGCCATAGCGGAAACCCCGGGGCTTCGCGGGCCGGGGAAGACGCCCGGG
DNA
Sequence TTCTCAGAGACGGCTTTGCCGACATACTGAAGCACGTCATCGGGGACTACAAGGACAT
CTTCCCCGACCTCTTCAAACGGGCCGCCGAGCGCCTCCAGTACGTCTTCGGGTATAAG
CTGGTGGAACTTGAACCCAAGAGCAACACTTACATCCTCATCAACACCCTGGAGCCTG
TGGAGGAGGATGCCGAGATGAGGGGTGACCAAGGCACGCCCACTACGGGCCTCCTGAT
GATCGTCTTAGGGCTCATCTTTATGAAGGGCAACACCArCAAGGAAACTGAAGCCTGG
GACTTTCTGCGGCGCTTAGGGGTCTACCCCACCAAGAAGCATTTAATTTTCGGAGATC
CAAAGAAACTCATTACTGAGGACTTTGTGCGACAGCGTTACCTGGAATACCGGCGGAT
ACCCCACACCGACCCCGTCGACTACGAATTCCAGTGGGGCCCGCGAACCAACCTGGAA
ACCAGCAAGATGAAAGTTCTTAAGTTTGTGGCCAAGGTCCATAATCAAGACCCCAAGG
ACTGGCCAGCGCAGTACTGTGAGGCTTTGGCAGATGAGGAGAACAGGGCCAGACCTCA
GCCTAGTGGCCCAGCTCCATCCTCTTGAAAG
ORF Start: ATG at ORF'
5 Stop:
TGA
at 722
SEQ ID NO: 64 239 as MW at 27463.9kD
NOVlBb, MLQKPRNRGRSGGQAERDRDWSHSGNPGASRAGEDAR.VI~RDGFADILKHVIGDYKDIF
CG9742O-O2 PDLFKRAAERLQYVFGYKLVELEPKSNTYILINTLEPVEEDAEMRGDQGTPTTGLLMI
Protein Sequence~GLIFMKGNTIKETEAWDFLRRLGVYPTKKHLIFGDPKKLITEDFVRQRYLEYRRIP
HTDPVDYEFQWGPRTNLETSKMKVLKFVAKVHNQDPKDWPAQYCEALADEENRARPQP
SGPAPSS
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 18B.
Table 188. Comparison of NOVl8a against NOVl8b.
Protein Sequence NOVl8a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOVl8b 109..305 196/197 (99%)
44..239 196/197 (99%)
Further analysis of the NOV 18a protein yielded the following properties shown
in Table
18C.
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Table 18C. Protein Sequence Properties NOVlBa
PSort 0.4500 probability located in cytoplasm; 0.1000 probability located in
mitochondria)
analysis: matrix space; 0.1000 probability located in lysosome (lumen); 0.0806
probability
located in microbody (peroxisome)
SignalP No Known Signal Sequence Predicted
~alysis:
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 18D.
Table 18D. Geneseq Results
for NOVl8a
f NOVl8aIdentities/ 1
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent #, for
IdentifierDatej Match the Matched Value
ResiduesRegion
ABB 11541Human melanoma Ag homologue,90..305 213/216 (98%)e-122
~SEQ
ID N0:1911 - Homo Sapiens, 1..215 213/216 (98%)
215 aa.
[W0200157188-A2, 09-AUG-2001]
AAB60476 Human cell cycle and proliferationI..302 151/304 (49%)1e-79
protein CCYPR-24, SEQ ID 1..293 203/304 (66%)
N0:24 -
Homo Sapiens, 308 aa. [W0200107471-
A2, O 1-FEB-2001 ]
AAY79141 Human haemopoietic stem 46..287 109/243 (44%)1 e-55
cell regulatory
protein SCMI 13 - Homo Sapiens,240..479160/243 (64!)
606 aa.
[W0200008145-A2, 17-FEB-2000]
AAB94174 Human protein sequence SEQ 79..294 102/217 (47%)2e-52
ID
NO:14482 - Homo Sapiens, 438..650150/217 (69%)
706 aa.
[EPI074617-A2, 07-FEB-2001]
AAB92822 Human protein sequence SEQ 79..294 102/217 (47%)3e-52
ID
NO:11353 - Homo sapiens, 297..509150/217 (69%)
565 aa.
[EP I 074617-A2, 07-FEB-2001
]
In a BLAST search of public sequence datbases, the NOV 18a protein was found
to have
homology to the proteins shown in the BLASTP data in Table I 8E.
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Table 18E. Public BLASTP
Results for NOVl8a
Protein NOVl8a Identities/
AccessionProtein/OrganismlLength Residues/SimilaritiesExpect
for
Number Match the MatchedValue
ResiduesPortion
Q96MG7 CDNA FLJ32395 FIS, CLONE 1..305 304/305 e-177
(99%)
SKMUS2000117, MODERATELY 1..304 304/305
(99%)
SIMILAR TO HOMO SAPIENS
MAGEF 1 MRNA - Homo Sapiens
(Human), 304 aa.
Q9CPR8 5730494G16RIK PROTEIN IMAGE-G1)1..305 233/306 e-128
(76%)
- Mus musculus (Mouse), 279 1..279 250/306
aa. (81%)
Q9D378 5730494G16RIK PROTEIN - Mus ' 1..3 ' 2321306 i e-i
VJ ( 75 io) 2 i
musculus (Mouse), 279 aa. 1..279 249/306
(80%)
BAB84964 FLJ00211 PROTEIN - Homo Sapiens85..290 205/206 e-116
(99%)
(Human), 213 as (fragment). 1..205 205/206
(99%)
Q99PB1 MAGE-G2 -Mus musculus (Mouse),1..305 201/306 e-106
294 (65%)
aa. 9..294 226/306
(73%)
PFam analysis predicts that the NOV I 8a protein contains the domain shown in
the Table
18F.
Table 18F. Domain Analysis of NOVl8a
Identities/
Pfam Domain NOVl8a Match Region Similarities Expect Value
for the Matched Region ~
MAGE 1..209 781262 (30%) 1.5e-33
144/262 (55%)
Example 19.
The NOV 19 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 19A.
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GCTGAACCTGTGTGAGGATGGTCCATGTCACAAACGGCGGGCAAGCATCTGCTGTACC
CAGCTGGGGTCCCTGTCGGCCCTGAAGCATGCTGTCCTGGGGCTCTACCTGCTGGTCT
CGACCTGAAGGCCCTGACTCGCAATGTGAACCGGCTGAATGAGAGCTTCCGGGACTTG
GTGGCCCTGCTGCGGGACCGCACGGGCCAGCAGAGCGACACGGCGCAGCTGGAGCTCT
GCTGGACGGGCTGGCGCGCAGGGTGGGCATCCTGGGCGAGGAGCTGGCCGACGTGGGC
CCCTCGCGAAAGGGCCCCCGGGACCCAAAGGTGA
CAGAGCTGGGGATGCCAGTGGCGTGGAGGCCCCGATGATGATCCGCCTGGTGAATGGC
TCAGGTCCGCACGAGGGCCGCGTGGAAGTGTACCACGACCGGCGCTGGGGCACCGTGT
CGGTGTGGAGGAGGTGTACCGCACAGCTCGATTCGGGCAAGGCACTGGGAGGATCTGG
CTGAAAGTGGGCAGA
ORF Start: ATG at 17 ORF Stop: TGAat 1568
SEQ ID NO: 66 517 aa~~MW at 56256.2kD
NOVl9a, MLRSEGTRLYSLSQGHSAVAGCDGEQTMYLHTVSDCDTSSICEDSFDGRSLSKLNLCE
CG97430-OI DGPCHKRRASICCTQLGSLSALKHAVLGLYLLVFLILVGIFILAVSRPRSSPDDLKAL
Protein Sequence TLNESFRDLQLRLLQAPLQADLTEQVWKVQDALQNQSDSLLALAGAVQRLEGA
LWGLQAQAVQTEQAVALLRDRTGQQSDTAQLELYQLQVESNSSQLLLRRHAGLLDGLA
VTEDLRLKDWEHSIALRNISLAKGPPGPKGDQGDEGKEGRPGIP
SGVEAPMMIRLVNGSGPHEGRVEVYHDRRWGTVCDDGWDKKDGDWCRMLGFR~
YRTARFGQGTGRIWMDDVACKGTEETIFRCSFSKWGVTNCGHAEDASWCNRH
SEQ ID NO: 67 903 by
NOVl9b, CCACCATGGGAAATGCTGAGATCTGAGGGGACAAGGCTCTACAGCCTCAGCCAGGCGC
CCn'~~~C l'~ DNA
~p'CTCAGCTGTTGCAGGGTGTGirivGAVT~CAi~c~i~i:TATGTACCTACACACCGTc:iW CG
J l'TJ -V~.
Sequence ACTGTGACACCAGCCCCATCTGTGAGGATTCCTTTGATGGCAGGAGCCTGTCCAAGCT
GAACCTGTGTGAGGATGGTCCATGTCACAAACGGCGGGCAAGCATCTGCTGTACCCAG
CTGGGGTCCCTGTCGGCCCTGAAGCATGCTGTCCTGGGGCTCTACCTGCTGGTCTTCC
TGATTCTTGTGGGCATCTTCATCTTAGCAGGGCCACCGGGACCCAAAGGTGATCAGGG
GGATGAAGGAAAGGAAGGCAGGCCTGGCATCCCTGGATTGCCTGGACTTCGAGGTCTG
CCCGGGGAGAGAGGTACCCCAGGATTGCCCGGGCCCAAGGGCGATGATGGGAAGCTGG
GGCGCTGGGGCACCGTGTGTGACGACGGCTGGGACAAGAAGGACGGAGACGTGGTGTG
CCGCATGCTCGGCTTCCGCGGTGTGGAGGAGGTGTACCGCACAGCTCGATTCGGGCAA
GGCACTGGGAGGATCTGGATGGATGACGTTGCCTGCAAGGGCACAGAGGAAACCATCT
TCCGCTGCAGCTTCTCCAAATGGGGGGTGACAAACTGTGGACATGCCGAAGATGCCAG
CGTGACATGCAACAGACACTGAAAGTGGGCAGA
Start: ATG at 80 ORF Stop: TGA at 890
SEQ ID NO: 68 270 as ~MW at 28880.SkD
NOVl9b, ~MENFtAMXYLHTVSDCDTSPICEDSFDGRSLSKLNLCEDGPCHKRRASICCTQLGSLSAL
CG97430-02 KHAVLGLYLLVFLILVGIFILAGPPGPKGDQGDEGKEGRPGIPGLPGLRGLPGERGTP
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Protein Sequence ~GLPGPKGDDGKLGATGPMGMRGFKGDRGPKGEKGEKGDRAGDASGVEAPMMIRLVNGS
DDVACKGTEETIFRCSFSKWGVTNCGHAEDASVTCNRH
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 19B.
Table 19B. Comparison of NOVl9a against NOVl9b.
Protein Sequence NOVl9a Residues/ Identities/
Match Residues Similarities for the Matched Region
NOVl9b 329..517 141/189 (74%)
82..270 141/189 (74%)
Further analysis of the NOV 19a protein yielded the following properties shown
in Table
19C.
Table 19C. Protein Sequence Properties NOVl9a
PSort 0.8000 probability located in mitochondria) inner membrane; 0.6500
probability
analysis: located in plasma membrane; 0.3000 probability located in microbody
(peroxisome);
0.3000 probability located in Golgi body
SignalP ~ 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 19D.
Table 19D. Geneseq Results
for NOVl9a
NOVl9a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate) Match the Matched Value
ResiduesRegion
AAE08824Human scavenger receptor 98..517 399/420 (95%)0.0
like protein -
Homo Sapiens, 435 aa. [W0200157260-16..435 399/420 (95%)
A1, 09-AUG-2001]
AAE08846Human scavenger receptor 105..517394/413 (95%)0.0
like protein
mature sequence - Homo Sapiens,1..413 394/413 (95%)
413
aa. [W0200157260-Al, 09-AUG-2001]
AAE08823Human partial scavenger 329..455126/127 (99%)6e-75
receptor like
protein - Homo Sapiens, 1..127 127/127 (99%)
127 aa.
[W0200157260-A1, 09-AUG-2001]
150
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AAW 19708Macrophage scavenger receptor protein164/500 (32%)I e-69
- 25..514
Homo Sapiens, 451 aa. [US5624904-A,247/500 (48%)
2..449
29-APR-1997]
AAR27036 Bovine sol. scavanger receptor - 161/482 (33%)1 e-68
Bos 37..514
taurus, 453 aa. [W09214482-A, 03-SEP-249/482 (51%)
13..451
1992)
In a BLAST search of public sequence datbases, the NOV 19a protein was found
to have
homology to the proteins shown in the BLASTP data in Table 19E.
Table 19E. Public BLASTP
Results for NOVl9a
Protein NOVl9a Identities/
AccessionProtein/Organism/Length ~ SimilaritiesExpect
Residues/for
Number Match she i~~aichedV aiue
i
ResiduesPortion
Q91 WD6 SIMILAR TO RIKEN CDNA 26..514 430/489 (87%)0.0
4933425F03 GENE - Mus musculus4..488 449/489 (90%)
(Mouse), 491 aa.
Q9CUC3 4933425F03RIK PROTEIN - Mus 26..397 329/372 (88%)0.0
musculus (Mouse), 375 as 4..375 344/372 (92%)
(fragment).
Q9D4G8 4932433F15RIK PROTEIN - Mus 130..403243/274 (88%)e-139
musculus (Mouse), 280 aa. 1..274 258/274 (93%)
P21758 Macrophage scavenger receptor37..514 166/482 (34%)1 e-70
types I
and II (Macrophage acetylated13..451 249/482 (51%)
LDL
receptor I and II) - Bos
taurvs (Bovine),
453 aa.
Q05585 Macrophage scavenger receptor77..514 161/450 (35%)3e-70
types I
and II (Macrophage acetylated46..452 236/450 (51%)
LDL
receptor I and II) - Oryctolagus
cuniculus
(Rabbit), 454 aa.
PFam analysis predicts that the NOV 19a protein contains the domains shown in
the Table
19F.
Table 19F. Domain Analysis of NOVl9a
Identities/
Pfam Domain NOVl9a Match Region Similarities Expect Value
for the Matched Region
Collagen 337..396 28/60 (47%) 9.1e-13
43/60 (72%)
SRCR 418..515 ~ 44/114 (39%) 2e-33
81/114 (71%)
151
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Example 20.
The NOV20 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 20A.
Table 20A. NOV20 Sequence Analysis
SEQ ID NO: 69 ~ 1538 by
NOV20a, TGGCGCCCAGCGGGGTCATGGTGCCCGGCGCCCGCGGCGGCGGCGCACTGGCGCGGGC
CG97440-O1 DNA TGCCGGGCGGGGCCTCCTGGCTTTGCTGCTCGCGGTCTCCGCCCCGCTCCGGCTGCAG
Sequence GCGGAGGAGCTGGGTGATGGCTGTGGACACCTAGTGACTTATCAGGATAGTGGCACAA
TGACATCTAAGAATTATCCCGGGACCTACCCCAATCACACTGTTTGCGAAAAGACAAT
CAGACCTGTGCTTCTGACTATCTTCTCTTCACCAGCTCTTCAGATCAATATGGTCCAT
ACTGTGGAAGTATGACTGTTCCCAAAGAACTCTTGTTGAACACAAGTGAAGTAACCGT
CCGCTTTGAGAGTGGATCCCACATTTCTGGCCGGGGTTTTTTGCTGACCTATGCGAGC
TATGGTAGATGGATATAGAGATACCTCTTTAfiTGTGCAAAGCTGCCATCCATGCAGGA
ATAATTGCTGATGAACTAGGTGGCCAGATCAGTGTGCTTCAGCGCAAAGGGATCAGTC
CATTACAACGGfiGGCTATTCCATTGGTGCTCCTTGTTGTCCTGGTGTTTGCTGGAATG
GGGATCTTTGCAGCCTTTAGAAAGAAGAAGAAGAAAGGAAGTCCGTATGGATCAGCAG
AGGCTCAGAAAACAGACTGTTGGAAGCAGATTAAATATCCCTTTGCCAGACATCAGTC
AGCTGAGTTTACCATCAGCTATGATAATGAGAAGGAGATGACACAAAAGTTAGATCTC
ATCACAAGTGATATGGCAGATTACCAGCAGCCCCTCATGATTGGCACCGGGACAGTCA
CGAGGAAGGGCTCCACCTTCCGGCCCATGGACACGGATGCCGAGGAGGCAGGGGTGAG
CTGCCCCTGGCGCCCCCGGAGCCCGAGTACGCCACGCCCATCGTGGAGCGGCACGTGC
Start: ATG at ? 8 1 ORS' Stap: TG a at 1 s36
ID NO: 70 1506 as BMW at 54247.6kD
Sequence VPKELLLNTSEVTVRFESGSHISGRGFLLTYASSDHPDLITCLERASHYLKTEYSKFC
PAGCRDVAGDISGNMVDGYRDTSLLCKAAIHAGIIADELGGQISVLQRKGISRYEGIL
ANGVLSRESSLGINITTVAIPLVLLVVLVFAGMGIFAAFRKKKKKGSPYGSAEAQKTD
CWKQIKYPFARHQSAEFTISYDNEKEMTQKLDLITSDMADYQQPLMIGTGTVTRKGST
FRPMDTDAEEAGVSTDAGGHYDCPQRAGRHEYALPLAPPEPEYATPIVERHVLRAHTF
SAQSGYRVPGPQPGHHIiSLSSGGFSPVAGVGAQDGDYQRPHSAQPADRGYDRPKAVSA
SEQ ID NO: 71 X636 by
199652779 DNA ~fiGACATCTAAGAATTATCCCGGGACCTACCCCAATCACACTGTTTGCGAAAAGAC
Sequence ~~TTACAGTACCAAAGGGGAAAAGACTGATTCTGAGGTTGGGAGATTTGGATATCGAA
CATACTGTGGAAGTATGACTGTTCCCAAAGAACTCTTGTTGAACACAAGTGAAGTAAC
CGTCCGCTTTGAGAGTGGATCCCACATTTCTGGCCGGGGTTTTTTGCTGACCTATGCG
152
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CAGAATACAGCAAATTCTGCCCAGCTGGTTGTAGAGACGTAGCAGGAGACATTTCTGG
GAATATGGTAGATGGATATAGAGATACCTCTTTATTGTGCAAAGCTGCCATCCATGCA
GGAATAATTGCTGATGAACTAGGTGGCCAGATCAGTGTGCTTCAGCGCAAAGGGATCA
GTCGATATGAAGGGATTCTGGCCAATGGTGTTCTTTCGAGGGAGTCTTCTCTCGAG
ORF Start: at 1 ORF Stop:
end
of sequence
SEQ ID NO: 72 212 as MW at 22920.41cD
NOV20b, GTEELGDGCGHLVTYQDSGTMTSKNYPGTYPNHTVCEKTITVPKGKRLILRLGDLDIE
199652779 SQTCASDYLLFTSSSDQYGPYCGSMTVPKELLLNTSEVTVRFESGSHISGRGFLLTYA
Protein
Sequence SSDHPDLITCLERASHYLKTEYSKFCPAGCRDVAGDISGNMVDGYRDTSLLCKAAIHA
GIIADELGGQISVLQRKGISRYEGILANGVLSRESSLE
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 35..243 209/209 (100%)
3..211 209/209 ( 100%)
Further analysis of the NOV20a protein yielded the following properties shown
in Table
20C.
Table 20C. Protein Sequence Properties NOV20a
PSort 0.8500 probability located in endoplasmic reticulum (membrane); 0.4400
probability
analysis: located in plasma membrane; 0.3500 probability located in nucleus;
0.1000
probability located in mitochondria) inner membrane
SignalP Cleavage site between residues 35 and 36 -
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.
Table 20D. Geneseq Results for
NOV20a
NOV20a Identities/
Geneseq Protein/Organism/Length Residues/Similarities for
(Patent #, Expect
Identifier Date] Match the Matched Value
Residues Region
AAB19126 Polypeptide isolated from14..506 382/503 (75%) 0.0
lymph node
stromal cells of fsn -/- mice - 5..503 415/503 (81 fo)
Mus sp,
503 aa. (W0200058463-Al, OS-OCT-
2000]
153
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AAU00670 Human TANGO 229 polypeptide238..506266/269 (98%) e-157
- Homo
Sapiens, 715 aa. [W0200129088-A1, 447..715267/269 (98%)
26-
APR-2001
AAU00630 ' Novel human protein (NHP)1..243 241/243 (99%) e-138
sequence #3
- Homo Sapiens, 539 aa. [W0200129219-1..243 2421243 (99%)
A1, 26-APR-2001)
AAU00629 Novel human protein (NHP) 1..243 241/243 (99%) e-138
sequence #2
- Homo Sapiens, 586 aa. [W0200129219-48..290242/243 (99%)
A 1, 26-APR-2001 )
AAU00628 Novel human protein (NHP) 53..243189/191 (98%) e-107
sequence #1
- Homo Sapiens, 487 aa. [W0200129219-1..191 190/191 (98%)
A1, 26-APR-2001)
In a BLAST search of public sequenceNOV20a
datbases, the protein
was
found
to
have
homology to the proteins shown in the BLASTP data in Table 20E.
Table 20E. Public BLASTP
Results for NOV20a
Protein NOV20a Identities/
AccessionProtein/Organism/Length Residues/SimilaritiesExpect
for
Number Match the Matched Value
ResiduesPortion
Q9D4J3 4631413K11 RIK PROTEIN - 14..506 384/503 (76%)0.0
Mus
musculus (Mouse), 503 aa. 5..503 416/503 (82%)
Q9D696 4631413K11RIK PROTEIN - 53..506 353/464 (76%)0.0
Mus
musculus (Mouse), 460 aa. 1..460 383/464 (82%)
Q96NH2 CDNA FLJ30900 FIS, CLONE 352..506155/155 (100%)1e-89
FEBRA2005752 - Homo Sapiens1..155 155/155 (100%)
(Human), 155 aa.
Q96PD2 ENDOTHELIAL AND SMOOTH 20..249 110/2361,46%)4e-51
MUSCLE CELL-DERIVED 51..286 148/236 (62%)
NEUROPIL1N-LIKE PROTEIN
- Homo
Sapiens (Human), 775 aa.
Q91ZV3 ENDOTHELIAL AND SMOOTH 3..249 117/271 (43%)Se-50
MUSCLE CELL-DERIVED 13..283 154/271 (56%)
NEUROPILIN-LIKE PROTEIN
- Mus
musculus (Mouse), 769 aa.
PFam analysis predicts that the NOV20a protein contains the domains shown in
the Table
20F.
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Table 20F. Domain Analysis of NOV20a
Identities/
Pfam Domain NOV20a Match Region Similarities Expect Value
for the Matched Region
CUB 41..147 34/117 (29%) 2.8e-20
73/117 (62%)
Examule 21.
The NOV21 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 21A.
Table 21A. NOV21 Sequence Analysis
SEQ ID NO: 73 X2570 by
~NOV21 a,
~CG97451-O1 DNA
Sequence
GAAGTGGAAGTGGAGGTGGTGGGGGAGGTGGTCTCCTGGGGGGCCTGCTTGGTGGTGGi
GGGTGGAGGGGGTGGCAGTGATCTGTTGGGTGGAGGCTTACTGGGTGGCAGTGGCAGC~
TACAGACGCATTGAATTCCCCCGAGGTGTTGGTGATATTCCCTACAATGACTTCCATGI
GAGATCCTTGAGTCCGAGGGAAGCATCAGGGACCTCCGAAACAGTGGCTATCGCAGTG
CCGAGAATGCATATGGAGGCCACAGGGGCCTCGGGCGATACAGGGCAGCACCTGTGGG
TCGCAGGCCAAGGTGGCCTGCTCGGCGGAGGTGGTCTCCTTGGTGATGGAGGACTTCT
TGGAGGAGGGGGTGTCCTGGGCGTGCTCGGCGAGGGTGGCATCCTCAGCACTGTGCAA~
GAACCGAGTCCTGGCCGACGTCCTCCCTGACTTGCTCTGCCCCATCGTGGATGTGGTGI
CCAAGTCCCAGCTGGCCATGTCTGCCAACTTCCTGGGCTCAGTGCTGACTCTACTGCA~
GAAGCAGCATGCTCTAGACCTGGATATCACCAATGGCATGTTTGAAGAGCTTCCTCCA
CCTGCCCACTTATCA
CAGAACCTCPAACGTGGGCAACTTTGATATTGGCCTCATGGAGGTGCTGGTGGAGAAG~
155
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ATTTTTGACCTGGCATTCATGCCCGCAATGAACGCTGTGCTGGGTTCTGGCGTCCCTC
TCCCCAAAATCCTCAACATCGACTTTAGCAATGCAGACATTGACGTGTTGGAGGACCT
TTTGGTGCTGAGCGCATGAGTGACAGAGGCAGAGATGCTGCTGCAACTGGAAGAAGCT
GGAACCAGTCCCAGAGAGGCTCGGCCTGGAAACAGTCCCCTGCCCAGAGTCCCCTCAG
CCTCCATGACAGGTCCCTCCCTGGCCCCCCAACCCTCTTCCTCCCTTGCCCCAACCCT
GAGAAAGGGTCCAGCCACTACCCTGTTGGCAAACATTCCCTTCCATGGTCAGCCTGCC
AGGAGGAGGGGAGTCACCTTGGGGCTGGAGGCCTCTCAGACCCCATCCTGACAGCAGG
TTGAGTATTCCCACTTTCAATAAAAGACTCCACTTTCCCGGCACTTGTGACGAGTTTC
CATGAAGGACCCTCCTGA
ORF Start: ATG at ORF Stop: TGA at 2221
130
SEQ ID NO: 74 697 as MW at 71241.3kD
NOV2la, ~ MLQQSDALHSALREVPLGKARGDGGGPLLGGLLGGSGSGGGGGGGLLGGLLGGGGGGG
GG97451-O1 GSDLLGGGLLGGSGSSGGELLGGGGGSGGGLLGGSGGGLLGGSGGGLLGGSRGGLLGG
Protein SequenceSGGGLLGGGRHHYNDYRRIEFPRGVGDIPYNDFHVRGPPPVYTNGKKLDGIYQYGHIE
TNDNTAQLGGKYRYGEILESEGSIRDLRNSGYRSAENAYGGHRGLGRYRAAPVGRLHR
RELQPGEIPPGVATGAVGPGGLLGTGGMLAADGILAGQGGLLGGGGLLGDGGLLGGGG
VLGVLGEGGILSTVQGITGLRIVELTLPRVSVRLLPGVGVYLSLYTRVAINGKSLIGF
LDIAVEVNITAKVRLTMDRTGYPRLVIERCDTLLGGIKVKLLRGLLPNLVDNLVNRVL
ADVLPDLLCPIVDVVLGLVNDOLGLVDSLIPLGILGSVO__YTFSSLPLVTGEFLELDLN
TLVGEAGGGLIDYPLGWPAVSPKPMPELPPMGDNTKSQLAMSANFLGSVLTLLQKQHA
LDLDITNGMFEELPPLTTATLGALIPKVFQQYPESCPLIIRIQVLNPPSVMLQKDKAL
VKVLATAEVMVSQPKDLETTICLIDVDTEFLASFSTEGDKLMIDAKLEKTSLNLRTSN
VGNFDIGLMEVLVEKIFDLAFMPAMNAVLGSGVPLPKILNIDFSNADIDVLEDLLVLS
A
Further analysis of the NOV21 a protein yielded the following properties shown
in Table
21B.
Table Z1B. Protein Sequence Properties NOVZla
PSort 0.8500 probability located in endoplasmic reticulum (membrane); 0.4400
probability
analysis: located in plasma membrane; 0.3033 probability located in microbody
(peroxisome);
0.1000 probability located in mitochondria) inner membrane
SignalP No Known Signal Sequence Predicted
analysis:
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.
Table 21C. Geneseq Results for NOV2la
NOV2la Identities/
Geneseq Protein/Organism/Length [PatentResidues/Similarities Expect
#, for
Identifier Date] Match the Matched Value
ResiduesRegion
AAM05009 Peptide #3691 encoded by 1..188 ~ 188/188 e-110
probe for (100%)
measuring breast gene expression 4..191 188/188 (100%)
-
Homo Sapiens, 191 aa. [WO200157270-
A2, 09-AUG-2001 ]
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AAM69485 Human bone marrow expressed1..188 I 88/188 ( e-1
probe 100%) I
0
encoded protein SEQ ID NO: 29791 4..191 188/188 (100%)
-
Homo Sapiens, 191 aa. [WO200157276-
A2, 09-AUG-2001 ]
AAM57094 Human brain expressed single1..188 188/188 (100%)e-110
exon
probe encoded protein SEQ ID NO: 4..191 188/188 (100%)
29199 - Homo sapiens, 191 aa.
[W0200157275-A2, 09-AUG-2001]
ABB21687 Protein #3686 encoded by 1..188 ~ 188/188 (100%)e-110
probe for
measuring heart cell gene expression4..191 188/188 (100%)
-
Homo sapiens, 191 aa. [W0200157274-
A2, 09-AUG-2001
AAG77922 Human new lipid binding 278..696165/420 (39%) 1 e-77
protein 3 -
Homo Sapiens, 472 aa. [W0200179492- 62..469~
252/420 (59%)
A2, 25-OCT-2001]
In a BLAST search of public sequence~dO
datbases, the V 21
a p.roWi~~
vYa~
found
iv
itav2
homology to the proteins shown in the BLASTP data in Table 21D.
Table 21D. Public BLASTP Results for NOV2la
Protein NOV2la Identities/
AccessionProtein/Organism/Length Residues/Similarities Expect
for
Number Match the Matched Value
ResiduesPortion
CAD121S0 SEQUENCE 3 FROM PATENT 96..697 573/602 (95%)0.0
W00179269 - Homo sapiens 57..637 573/602 (95%)
(Human),
637 aa.
CAD12149 SEQUENCE 1 FROM PATENT 136..697558/562 (99%)0.0
W00179269 - Homo Sapiens 53..614 559/562 (99%)
(Human),
614 as (fragment).
CAClBoe DJ726C3.5 (ORTHOLOG OF ~ 229..697469/469 (100%)0.0
7 ~
POTENTIAL LIGAND BINDING 1..469 469/469 (100%)
PROTEIN RY2G5 (RAT)) - Homo
Sapiens (Human), 469 as
(fragment).
Q05704 POTENTIAL LIGAND-BINDING 229..696426/469 (90%)0.0
PROTEIN - Rattus rattus 1..469 451/469 (95%)
(Black rat),
470 as (fragment).
Q05701 POTENTIAL LIGAND-BINDING 243..696I 87/470 (39%)8e-83
PROTEIN - Rattus rattus 13..470 275/470 (57%)
(Black rat),
473 aa.
PFam analysis predicts that the NOV21 a protein contains the domains shown in
the Table
21 E.
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Table 21E. Domain Analysis of NOV2la
Identities/
Pfam Domain NOV2la Match Region Similarities Expect Value
for the Matched Region
Collagen 245..304 19/61 (31%) 0.72
24/61 (39%)
LBP_BPI_CETP_C 512..697 51/197 (26%) 2.7e-11
111/197 (S6%)
Example 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: 7S 11
SO by
NOV22a, GACTGCCTGGCAGGTGTGAAAGGCAGCGGTGGCCACAGAGGCGGTGGAGATGGCCTTC
CG97~S2-OI AGCGGTTCCCAGGCTCCCTATCTGAGCCCAGCCGTCCCCTTTTCTGGGACTATCCAAG
DNA
SequeriCe GGGGTCTCCAGGACGGATTTCAGATCACTGTCAATGGGGCCGTTCTCAGCTCCAGTGG
AACCAGGTTTGCTGTGGACTTTCAGACGGGCTTCAGTGGAAACGACATTGCCTTCCAC
TTCAACCCTCGGTTTGAAGACGGAGGGTATGTGGTGTGCAACACGAGGCAGAAAGGAA
GATGGGGGCCCGAGGAGAGGAAGATGCACATGCCCTTCCAGAAGGGGATGCCCTTTGA
CCTCTGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTAATGGTGAACGGGAGCCTCTTC
GTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCATCTCCGTCAATGGCT
CTGTGCAGCTGTCCTACATCAGCTTCCAGAATCCCCGCACAGTCCCCGTTCAGCCTGC
CTTCTCCACGGTGCCGTTCTCCCAGCCTGTCTGTTTCCCACCCAGGCCCAGGGGGCGC
AGACAAAAACCTCCCAGCGTGCGGCCTGCCAACCCAGCTCCCATTACCCAGACAGTCA
TCCACACGGTGCAGAGCGCCTCTGGACAGATGTTCTCTACTCCCGCCATCCCACCTAT
GATGTACCCCCACCCTGCCTATCCGATGCCTTTCATCACCACCATTCCGGGAGGGCTG
TACCCATCCAAGTCCATCATCCTGTCAGGCACTGTCCTGCCCAGTGCTCAGAGGTTCC
ACATCAACCTGTGCTCTGGGAGCCACATCGCCTTCCACATGAACCCCCGTTTTGATGA
_
~
GAATGCTGTGGTCCGTAACACCCAGATCAACAACTCTTGGGGGTCTGAGGAGCGAAGT
CTGCCCCGAAAAATGCCCTTCGTCCGAGGCCAGAGCTTCTCGGTATGGATCTTGTGTG
AAGCTCACTGCCTCAAGGTGGCCGTGGATGGTCAGCACGTGTTTGAATACTACCATCG
CCTGAGGAACCTGCCCACCATCAACAAACTGGAAGTGGGTGGCGACATCCAGCTGACC
CACGTGCAGACATAGGCGGCTCCCTGGCCCTGGGGCCGGGGGCTGGGG
OItF Start: ATG at
SO ORF Stop: TAG
at 111 S
SEQ 1D NO: 76 3SS as MW at 39S32.OIcD
NOV22a, MAFSGSQAPYLSPAVPFSGTIQGGLQDGFQITVNGAVLSSSGTRFAVDFQTGFSGNDI
CG978S2-OI A~~RFEDGGYWCNTRQKGRWGPEERKMHNIPFQKGMPFDLCFLVQSSDFKVMVNG
Protein SequenceSLFVQYFHRVPFHRVDTISVNGSVQLSYISFQNPRTVPVQPAFSTVPFSQPVCFPPRP
RGRRQKPPSVRPANPAPITQTVIHTVQSASGQMFSTPAIPPMMYPHPAYPMPFITTIP
GGLYPSKSIILSGTVLPSAQRFHINLCSGSHTAFHMNPRFDENAVVRNTQINNSWGSE
ERSLPRKMPFVRGQSFSVWILCEAHCLKVAVDGQHVFEYYHRLRNLPTINKLEVGGDI
QLTHVQT .
SEQ ID NO: 77 1460 by
NOV22b, CTACAAAGGACTTCCTAGTGGGTGTGAAAGGCAGCGGTGGCCACAGAGGCGGCGGAGA
CG978S2-03 _GATGGCCTTCAGCAGTTCCCAGGCTCCCTACCTGAGTCCAGCTGTCCCCTTTTCTGGG
DNA
ACTATTCAAGGAGGTCTCCAGGACGGACTTCAGATCACTGTCAATGGGACCGTTCTCA
1S8
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Sequence GCTCCAGTGGAACCAGGTTTGCTGTGAACTTTCAGACTGGCTTCAGTGGAAATGACAT
TGCCTTCCACTTCAACCCTCGGTTTGAAGACGGAGGGTATGTGGTGTGCAACACGAGG
CAGAAAGGAACATGGGGGCCCGAGGAGAGGAAGACACACATGCCTTTCCAGAAGGGGA
TGCCCTTTGACCTCTGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTGATGGTGAACGG
GATCCTCTTCGTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCATCTCC
GTCAATGGCTCTGTGCAGCTGTCCTACATCAGCTTCCAGCCTCCCGGCGTGTGGCCTG
CCAACCCGGCTCCCATTACCCAGACAGTCATCCACACAGTGCAGAGCGCCCCTGGACA
GATGTTCTCTACTCCCGCCATCCCACCTATGGTG'PACCCCCACCCCGCCTATCCGATG
ACCAGCTGTCTGCTCCTGGTGGGAGGTGGCCCTCCTCAGCCCCTCCTCTCTGACCTTT
AACCTCACTCTCACCTTGCACCGTGCACCAACCCTTCACCCCTCCTGGAAAGCAGGCC
TCCTTTCCCAGTGTCCTTAAAATAAAGAAATGAAAATGCTTGTTGG
OItF Start: ATG at 60 ORF Stop: TGA at 798
SEQ ID NO: 78 246 as MW at 26802:6kD
NOV22b, MAFSSSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFSGNDI
CG97852-O3 ~FNPRFEDGGYWCNTRQKGTWGPEERKTHMPFQKGMPFDLCFLVQSSDFKVMVNG
Pi'Oteln Sequence ILFVQYFHRVPFHRVDTISVNGSVQLSYISFQPPGVWPANPAPITQTVIHTVQSAPGQ
MFSTPAIPPMVYPHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRCGSCVKLTASR
WPWMVSTCLNTTIA
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 22B.
Table 22B. Comparison of NOV22a against NOV22b.
Protein ~eqr,~ence I NOV22a Residues/ Identities/ E
Match Residues Similarities for the Matched Region
NOV22b 1..253 208/253 (82%)
1..221 211/253 (83%)
Further analysis of the NOV22a protein yielded the following properties shown
in Table
22C.
Table 22C. Protein Sequence Properties NOV22a
PSort 0.6400 probability located in microbody (peroxisome); 0.3267 probability
located in
analysis: Iysosome (lumen); 0.3000 probability located in nucleus; 0.1000
probability located
in mitochondria) matrix space
SignaIP No Known Signal Sequence Predicted
analysis:
I59
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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 22D.
Table 22D. Geneseq Results for NOV22a
NOV22a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAE13847 Human lung tumour-specific1..355 ~ 338/355 0.0
protein (95%)
21871 - Homo Sapiens, 378 24..378 345/355 (96%)
aa.
[W0200172295-A2, 04-OCT-2001]
AAE13847 Human lung tumour-specific1..355 338/355 (95%)0.0
protein
21871 - Homo sapiens, 378 24..378 ~
aa. 345/355 (96%)
[W0200172295-A2, 04-OCT-2001]
AAY06997 Galectin-9 protein sequence1..355 338/355 (95%)0.0
- Homo
Sapiens, 355 aa. [W09904265-A2,1..355 345/355 (96%)
28-
JAN-1999] ~
AAW85664 Galectin-9 like protein 1..355 ~ 338/355 0.0
- Homo sapiens, (95%)
355 aa. [W099I0490-A1, 1..355 ~ 345/355
04-MAR- (96%)
1999]
AAY56802 Human eosinophil chemotactic1..355 [ 305/355 e-179
factor (85%)
(ecalectin)~- Homo Sapiens,1..323 ~ 312/355
323 aa. (86%)
[W09962556-Al, 09-DEC-1999]
In a BLASTsearch of public sequence
datbases, the NOV22a protein
was found to have
homology to the proteins shown in the BLASTP data in Table 22E.
_~.. . Fable 22E. Public BLASTP Results for NO~%2~a
NOV22a
Protein Identities/
Accession Protein/Organism/Length Residues/Similarities Expect
for the
Number Matched PortionValue
Residues
Q9NQ58 GALECTIN-9 - Homo Sapiens 1..355 344/355 (96%) 0.0
(Human), 355 aa. 1..355 348/355 (97%)
000182 Galectin-9 (HOM-HD-21) .1..355 338/355 (95%) 0.0
(Ecalectin) - Homo sapiens (Human),1..355 345/355 (96%)
355 aa.
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Q9XSM9 URATE TRANSPORTER/CHANNEL PROTEIN, 1..345263/345 e-
(76%)
ISOFORM~(UATP,I) - Sus scrofa (Pig), 349 1..345292/345 160
aa. (84%)
P97840 Galectin-9 (36 kDa beta-galactoside 1..355251/355 e-
binding lectin) (70%)
(Urate transporter/channel) (UAT) - Rattus 1..354286/355 151
norvegicus (79%)
(Rat), 354 aa.
008573 Galectin-9 - Mus musculus (Mouse), 1..355244/355 a
353 aa. 68%)
(
1..353~ 285/355 146
(79%)
PFam analysis predicts that the NOV22a protein contains the domains shown in
the Table
22F.
Table 22F. Domain Analysis of NOV22a
Identities/ T .
Pfam Domain NOV22a Match Region Similarities ' .~uR~r2Ci i'aiu2
for the Matched Region
Gal-bind_lectin 16..147 49/139 (35%) 1.2e-43
106/139 (76%)
Gal-bind_lectin 226..355 ~ 5I/142 (36%) 7.3e-39
102/142 (72%)
Example 23.
The NOV23 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 23A.
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ORF Start: at 2 ORF Stop: TGA at _989 _
SEQ ID NO: 80 329 as MW at 37I30.91cD
NOV23a, QFLLLALLLPGGDNADASQEHVSFFiAIQIFSFVNQSWARGQGSGWLDELQTHGWDSES
CG99S7S-O1 GTIIFLHNWSKGNFSNEELPDLELLFRFYLFGLTREIQDHASQDYSKYPFEVQVKAGC
Protein SequenceE~SGKSPEGFFQVAFNGLDLLSFQNTTWVPSPGCGSLAQSVCHLLNHQYEGVTETVY
NLIRSTCPRFLLGLLDAGKMYVHI2QVRPEAWLSSRPSLGSGQLLLVCHASGFYPKPVW
VTWNn2NEQEQLGTKHGDILPNADGTWYLQVILEVASEEPAGLSCRVRHSSLGGQDIIL
YWGHFiFSMNWIALVVIVPLVILIVLVLWFKKHCSYQDIL
SEQ ID NO: 81 1116 by
NOV23b, ACAGAGATCAGCAAACAGCTTTTCTGAGAGAAAGAAACATCTGCAAATGACATGCTGT
CG99S7S-O2 TTCTGCAGTTTCTGCTGCTAGCTCTTCTTCTCCCAGGTGGTGACAATGCAGACGCATC
DNA
Sequence CCAGGAACACGTCTCCTTCCATGTCATCCAGATCTTCTCATTTGTCAACCAATCCTGG
GCACGAGGTCAGGGCTCAGGATGGCTGGACGAGTTGCAGACTCATGGCTGGGACAGTG
CAAGACCATGCAAGTCAAGATTACTCGAAATATCCCTTTGAAGTACAGGTGAAAGCGG
GCTGTGAGCTGCATTCTGGAAAGAGCCCAGAAGGCTTCTTTCAGGTAGCTTTCAACGG
ATTAGATTTACTGAGTTTCCAGAATACAACATGGGTGCCATCTCCAGGCTGTGGAAGT
TTTGGGTGACATGGA
TCTTCCTAATGCTGA
TCCTCTACTGGGCTCATATCAGGACATCCTGTGAGACTCTTCCCCCTGACTCCCCCAT
TGTGTTAAGAACCCAGCAACCCAGGAGCCTAGTACAATATAGTGATGCCATCCCGTCG
ACTCTCCATTTAAATTGTTTCTCTTTCTGCATAATAAACATTTGTTAATAAAAACCAA
ORF Start: ATG at S2 ORF Stop: TAA at 1090
SEQ 1D NO: 82 346 as MW at 38907.71eD
NOV23b, MLFLQFLLLALLLPGGDNADASQEHVSFHVIQIFSFVNQSWARGQGSGWLDELQTHGW
CG99S7S-02 DSESGTIIFLHNWSKGNFSNEELSDLELLFRFYLFGLTREIQDHASQDYSKYPFEVQV
Protein Sequence ~GCELHSGKSPEGFFQVAFNGLDLLSFQNTTWVPSPGCGSLAQSVCHLLNHQYEGVT
ETVYNLIRSTCPRFLLGLLDAGKMYVHRQVRPEAWLSSRPSLGSGQLLLVCHASGFYP
KPVWVTWNIRNEQEQLGTKHGDILPNADGTWYLQVILEVASEEPAGLSCRVRHSSLGGQ
DIILX~1AHTRTSCETLPPDSPIVLRTQQPRSLVOYSDAIPSTLHLNCFSFCIINIC
Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 23B.
Table 23B.
Comparison
of NOV23a
against NOV23b.
Protein SequenceNOV23a Residues/Identities/
Match ResiduesSimilarities for the Matched
Region
NOV23b 10..294 282/285 (98%)
14..298 282/285 (98%)
Further analysis of the NOV23a protein yielded the following properties shown
in Table
23C.
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Table 23C. Protein Sequence Properties NOV23a
PSort 0.4600 probability located in plasma membrane; 0.3053 probability
located in
analysis: microbody (peroxisome); 0.3000 probability located in lysosome
(membrane); 0.2800
probability located in endoplasmic reticulum (membrane}
SignalP Cleavage site between residues 16 and 17
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 23D.
Table 23D. Geneseq Results
for NOV23a
1 NOV23aId~endties/ 1
Geneseq ProteinlOrganism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
ABG13799Novel human diagnostic protein28..194 162/167 (97%)3e-95
#13790
- Homo Sapiens, 681 aa. 515..681165/167 (98%)
[WO200175067-A2, 11-OCT-2001]
ABG13799Novel human diagnostic protein28..194 162/167 (97%)3e-95
#13790
- Homo Sapiens, 681 aa. 515..681165/167 (98%)
[W0200175067-A2, 11-OCT-2001]
AAG00593Human secreted protein, I..60 56/60 (93%} 6e-27
SEQ ID NO:
4674 - Homo sapiens, 64 5..64 57/60 (94%)
aa.
[EP1033401-A2, 06-SEP-2000]
AAY94507Chicken BFIV 12 class I~MHC110..31758/209 (27%)2e-16
protein -
Gallus gallus, 355 aa. [IJS6075125-A,114..315102/209 (48%)
13-JIJN-2000]
AAP83149Probe F10-encoded protein 110..31758/209 (27%)2e-16
of 1VIHC
class I of chicken - Gallus115..316102/209 (48%)
gallus, 345 aa.
[W08809386-A, O1-DEC-1988]
In a BLAST search of public sequence datbases, the NOV23a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 23E.
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Table 23E. Public BLASTP
Results for NOV23a
Protein NOV23a Identities/
Accession Protein/Organism/Length Residues/ SimilaritiesExpect
for
Number Match the Matched Value
Residues Portion
P29017 T-cell surface glycoprotein1..329 326/329 (99%)0.0
CDlc ~
precursor (CD I c antigen)5..333 326/329 (99%)
- Homo
Sapiens (Human), 333
aa.
Q9QZY6 T-cell surface glycoprotein1..328 216/328 (65%)e-126
CDIc3
precursor (CD1-c3 antigen)5..331 253/328 (76%)
- Cavia
porcellus (Guinea pig),
332 aa.
Q9QZY8 T-cell surface glycoprotein2..328 212/327 (64%)~ e-121
CDlcl
precursor (CD 1-c 1 antigen)6..3? I 245!327 (74".)
- Cavia
porcellus (Guinea pig),
332 aa.
Q9QZY7 T-cell surface glycoprotein3 1..328 197/328 (60%)e-112
CDlc2
precursor (CDI-c2 antigen)5..331 239/328 (72%)
- Cavia
porcellus (Guinea pig),
332 aa.
P29016 T-cell surface glycoprotein2..328 197!328 (60%)e-110
CDlb
precursor (CD1 b antigen)6..332 240/328 (73%)
- Homo
sapiens (Human), 333
aa.
PFam analysis predicts that the NOV23a protein contains the domain shown in
the Table
23F.
Table 23F. Domain Analysis of NOV23a
Identities/ . I 1
Pfam Domain NOV23a Match Region Similarities Expect Value
for the Matched Region
ig 214..278 15/67 (22%) ~ 4.4e-07
48167 (72%)
Eaamule 24.
The NOV24 clone was analyzed, and the nucleotide=and encoded poIypeptide
sequences
are shown in Table 24A.
Table 24A. NOV24 Sequence Analysis
SEQ ID NO: 83 ~ 1497 by
164
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CG99608-O1 A_CATGGAGGGGAGCGCGAGCCCCCCGGAAAAGCCCCGCGCCCGCCCTGCGGCTGCCGT
DNA
Sequence GCTGTGCCGGGGCCCGGTAGAGCCGCTGGTCTTCCTGGCCAACTTTGCCTTGGTCCTG
CAGGGCCCGCTCACCACGCAGTATCTGTGGCACCGCTTCAGCGCCGACCTCGGCTACA
ATGGCACCCGCCAAAGGGGGGGCTGCAGCAACCGCAGCGCGGACCCCACCATGCAGGA
AGTGGAGACCCTTACCTCCCACTGGACCCTCTACATGAACGTGGGCGGCTTCCTGGTG
GGGCTCTTCTCGTCCACCCTGCTGGGAGCTTGGAGCGACAGTGTGGGCCGCCGCCCGC
TGCTAGTGCTGGCCTCGCTGGGCCTGCTGCTCCAGGCCCTAGTGTCCGTTTTTGTGGT
GCAGCTGCAGCTCCACGTCGGCTACTTCGTGCTGGGTCGCATCCTTTGTGCCCTCCTC
GGCGACTTCGGTGGCCTTCTGGCTGCTAGCTTTGCGTCCGTGGCAGATGTCAGCTCCA
GTCGCAGCCGCACCTTCCGGATGGCCCTGCTGGAAGCCAGCATCGGGGTGGCTGGGAT
GCTGGCAAGCCTCCTCGGGGGCCACTGGCTCCGGGCCCAGGGTTATGCCAACCCCTTC
TGGCTGGCCTTGGCCTTGCTGATAGCCATGACTCTCTATGCAGCTTTCTGCTTTGGTG
AGACCTTAAAGGAGCCAAAGTCCACCCGGCTCTTCACGTTCCGTCACCACCGATCCAT
TGTCCAGCTCTATGTGGCTCCCGCCCCAGAGAAGTCCAGGAAACATTTAGCCCTCTAC
TCACTGGCCATCTTCGTGGTGATCACTGTGCACTTTGGGGCCCAGGACATCTTAACCC
TTTATGAACTAAGCACACCCCTCTGCTGGGACTCCAAACTAATCGGCTATGGTTCTGC
AGCTCAGCATCTCCCCTACCTCACCAGCCTGCTGGCCCTGAAGCTCCTGCAGTACTGC
CTGGCCGATGCCTGGGTAGCTGAGATCGGCCTGGCCTTCAACATCCTGGGGATGGTGG
TCTTTGCCTTTGCCACTATCACGCCTCTCATGTTCACAGGATATGGGTTGCTTTTCCT
GTCATTAGTCATCACACCTGTCATCCGGGCTAAACTCTCCAAGCTGGTGAGAGAGACA
GAGCAGGGTGCTCTCTTTTCTGCTGTGGCCTGTGTGAATAGCCTGGCCATGCTGACGG
CCTCCGGCATCTTCAACTCACTCTACCCAGCCACTCTGAACTTTATGAAGGGGTTCCC
CTTCCTCCTGGGAGCTGGCCTCCTGCTCATCCCGGCTGTTCTGATTGGGATGCTGGAA
AAGGCTGATCCTCACCTCGAGTTCCAGCAGTTTCCCCAGAGCCCCTGATCTGCCTGGA
CCAGAAGACAGAGGGCAAGAGGAGCAAAGTGAACACCAAGCAACTGG
ORF Start: ATG at 61 ORF Stop:
TGA at 143 8
SEQ ID NO: 84 459 as MW at 49769.9kD
NOV24a, MEGSASPPEKPRARPAAAVLCRGPVEPLVFLANFALVLQGPLTTQYLWHRFSADLGYN
CG996O8-O1 GTRQRGGCSNRSADPTMQEVETLTSHWTLYMNVGGFLVGLFSSTLLGAWSDSVGRRPL
Protein SequenceL~SLGLLLQALVSVFWQLQLHVGYFVLGRILCALLGDFGGLLAASFASVADVSSS
RSRTFRMALLEASIGVAGMLASLLGGHWLRAQGYANPFWLALALLIAMTLYAAFCFGE
TLKEPKSTRLFTFRHHRSIVQLYVAPAPEKSRKHLALYSLAIFWITVHFGAQDILTL
YELSTPLCWDSKLIGYGSAAQHLPYLTSLLALKLLQYCLADAWVAEIGLAFNILGMW
FAFATITPLMFTGYGLLFLSLVITPVIRAKLSKLVRETEQGALFSAVACVNSLAMLTA
SGIFNSLYPATLNFMKGFPFLLGAGLLLIPAVLIGMLEKADPALEFQQFPQSP
Further analysis of the NOV24a protein yielded the following properties shown
in Table
24B.
Table 24B. Protein Sequence Properties NOV24a
PSort 0.8000 probability located in plasma membrane; 0.4000 probability
located in Golgi
analysis: 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 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 24C.
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Table 240. Geneseq Results
for NOV24a
1 NOV24a Identities/
Geneseq Protein/OrganismJLength Residues/SimilaritiesExpect
[Patent #, for
Identifier~ Date] Match the Matched Value
Residues Region
AAE04906Human transporter and ion 36..438 139/420 (33%)9e-54
channel-19
(TRICH-19) protein - Homo 1..415 212/420 (50%)
Sapiens,
445 aa. [W020014b258-A2,
28-JIJN-
200I ]
AAB41967~ Human ORFx ORF1731 polypeptide181..281 101/101 (100%)Se-52
sequence SEQ ID N0:3462 1..101 101/101 (100%)
- Homo
sapiens, 101 aa. [W0200058473-A2,
OS-
OCT-2000]
AAU14370Human novel protein #241 ~ 119..45986/365 (23%)Se-21
- Homo ~ ~
~ 1..358 156/365 (42%)
-
Sapiens, 365 aa. [WO200155437-A2,
02
AUG-2001 J
AAU 14134Human novel protein #5 - 119..459 86/365 (23%)Se-21
Homo Sapiens,
365 aa. [W0200155437-A2, 1..358 156/365 (42%)
02-AUG-
2001]
ABB59118~ Drosophila melanogaster 25..443 101/440 (22%)3e-20
polypeptide
SEQ ID NO 4146 - Drosophila403..838 1941440 (43%)
melanogaster, 856 aa. [W0200171042-
A2, 27-SEP-2001
.... ... .... ... .....
.. . _. . ......
In a BLAST search of public sequence datbases, the NOV24a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 24D.
Table 24D. Public BLASTP
Results for NOV24a
Protein NOV24a Ide~atitiQS/
AccessionProtein/Organism/Length Residues/SimilaritiesExpect
for
Number Match the Matched Value
ResiduesPortion
Q96NT5 CDNA FLJ30107 FIS, CLONE 1..459 459/459 (100%)0.0
BNGH41000198, WEAKLY SIMILAR1..459 459/459 (100%)
TO TETRACYCLINE RESISTANCE
PROTEIN, CLASS E - Homo sapiens
(Human), 459 aa.
Q96FL0 SIMILAR TO RIKEN CDNA 1..459 431/459 (93%)0.0
I 110002008 GENE - Homo SapiensI ..431 431/459 (93%)
(Human), 431 aa.
Q9D1P1 1110002C08RIK PROTEIN-Mus 1 1..459399/459 (86%)0.0
musculus (Mouse), 459 aa. ~ 1..459418/459 (90%)
Q28720 HYPOTHETICAL 31.9 KDA PROTEIN~ 181..459242/279 (86%)e-138
- Oryctolagus cuniculus (Rabbit),1..279 260/279 (92%)
293 aa.
166
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. . . ....._....._..__._. ..........._..
__._.~._._......................_......_. . . .. . .
.~,y".~,..._........_............ _.._",~,~",..........._.._.....
AAH24522 SIMILAR TO RIKEN CDNA t 264..459 ; 178/196 (90%) 3e-98
1110002008 GENE - Mus musculus ~ 1..196 186/196 (94%)
(Mouse), 196 as (fragment).
PFam analysis predicts that the NOV24a protein contains the domains shown in
the Table
24E.
Table 24E.
Domain
Analysis
of NOV24a
Identities/
Pfam DomainNOV24a Match Similarities Eacpect
Region Value
for the Matched
Region
Sugar_tr 21..459 73/S19 (14%) 0.017
26$/S 19 (S 1
%)
Eacample 25.
The NOV2S clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 25A.
Table 25A. NOV25
Sequence Analysis
SEQ ID NO: 85 564
by
NOV2Sa, TCCCTCATGTATGGCAAGAGCTCTACTCGTGCGGTGCTTCTTCTCCTTGGCATACAGC
CG99674-O1 T~~GCTCTTTGGCCTATAGCAGCTGTGGAAATTTATACCTCCCGGGTGCTGGAGGC
DNA
Sequence TGTTAATGGGACAGATGCTCGGTTTAAGGACCGGGTGTCTTGGGATGGGAATCCTGAG
CGGTACGATGCCTCCATCCTTCTCTGGAAACTGCAGTTCGACGACAATGGGACATACA
CCTGCCAGGTGAAGAACCCACCTGATGTTGATGGGGTGATAGGGGAGATCCGGCTCAG
CGTCGTGCACACTGTACGCTTCTCTGAGATCCACTTCCTGGCTCTGGCCATTGGCTCT
GCCTGTGCACTGATGATCATAATAGTAATTGTAGTGGTCCTCTTCCAGCATTACCGGA
AAAAGCGATGGGCCGAAAGAGCTCATAAAGTGGTGGAGATAAAATCAAAAGAAGAGGA
AAGGCTCAACCAAGAGAAAAAGGTCTCTGTTTATTTAGAAGACACAGACTAACAATTT
TAGATGGAAGCTGAGATGATTTCCAAGAACAAGAACCCTAGT
ORF Start: ATG at ORF Stop:
7 TAA at
514
SEQ ID NO: 86 169 as MW at 19261.11cD
NOV25a, MYGKSSTRAVLLLLGIQLTALWPIAAVEIYTSRVLEAVNGTDARFKDRVSWDGNPERY
CG99674-O1 DASILLWKLQFDDNGTYTCQVKNPPDVDGVIGEIRLSVVHTVRFSEIHFLALAIGSAC
Protein Sequence~MIIIVIVWLFQHYRKKRWAERAHKVVEIKSKEEERLNQEKKVSVYLEDTD
Further analysis of the NOV2Sa protein yielded the following properties shown
in Table
25B.
Table 25B. Protein Sequence Properties NOV25a
PSort 0.4600 probability located in plasma membrane; O.I000 probability
located in
analysis: endoplasmic reticulum (membrane); 0.1000 probability located in
endoplasmic
reticulum (lumen); 0.1000 probability located in outside
167
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SignalP ~ Cleavage site between residues 27 and 28
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 Protein/Organism/Length [PatentResidues/SimilaritiesExpect
#, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAB65275Human PRO! 192 (UNQ606) protein1..169 169/215 (78%)9e-87
sequence SEQ ID N0:389 - 1..215 169/215 (78%)
Homo
Sapiens, 215 aa. [W0200073454-A1,
07-
DEC-2000]
AAU12415Human PRO1192 polypeptide 1..169 169/215 (78%)9e-87
sequence -
Homo Sapiens, 215 aa. [W0200140466-1 ..21 169/215 (78%)
S
A2, 07-JUN-2001 ]
AAY66752Membrane-bound protein PROI I ..169 169/215 (78%)9e-87
192 -
Homo Sapiens, 215 aa. [W09963088-A2,I ..215 169/215 (78%)
09-DEC-1999]
AAB33448Human PRO1192 protein UNQ606~ 1..169169/215 (78%)9e-87
SEQ
ID NO:163 - Homo sapiens, 1..21 169/215 (78%)
215 aa. S
[W0200053758-A2, 14-SEP-2000]
AAY41673Human channel-related molecule1..169 169/215 (78%)9e-87
HCRM-
1 - Homo Sapiens, 215 aa. 1..215 169/215 (78%)
[W09943807-
A2, 02-SEP-1999]
In a BLAST search of public sequence datbases, the NOV25a protein was found to
have
homology to the proteins shown iri the BLASTP data in Table 25D.
Table 25D. Public BLASTP
Results for NOV25a
Protein NOV25a Identities/
AccessionProtein/Organism/Length Residues/Similarities Expect
for
Number Match the Matched Value
ResiduesPortion
060487 Epithelial V-like antigen I ..169 I 69/215 (78%)2e-86
1 precursor -
Homo sapiens (Human), 215 I ..215 169/215 (78%)
aa.
070255 Epithelial V-like antigen I ..169 134/215 (62%)Se-66
I precursor -
Mus musculus (Mouse), 21 I ..215 146/215 (67%)
S aa.
Q9I WI4 EPITHELIAL V-LIKE ANTIGEN I ..I 133/215 (6I 3e-65
- Mus 69 %)
musculus (Mouse), 215 aa. I ..215 1451215 (66%)
,
168
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P37301 Myelin PO protein precursor (Myelin38/94 (40%) 2e-12
45..138
protein zero) (Myelin peripheral 53/94 (SS%)
protein) 95..188
(MPP) - Gallus gallus~(Chicken),
249 aa.
91406 IPI - Salmo s , 202 aa. 45..136 36/92 (39%) 3e-12
Q ~ p ~
88..179 Sl/92 (SS%)
PFam analysis predicts that the NOV2Sa protein contains the domain shown in
the Table
25E.
Table 25E. Domain Analysis of NOV25a
Identities/
Pfam Domain NOV25a Match Region Similarities Expect Value
for the Matched Region
ig 39..79 ~ 10/42 (24%) 0.0023
34%42 (81%)
Example 26.
S 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: 87 820 by
NOV26a, A_TGGGCCACTTCCTCTCCAGTCCCTCCTGCAGCGTCTCTGCTCTGGGCCCTGCCATCT
CG99732-02 CCTGCTGTCCCTGGGCCTCGGCTTCCTGCTGCTGGTCATCATCTGTGTGGTTGGATTC
DNA
Sequence ~TTCCAAATTTCAGAGGGACCTGGTGACCCTGAGAACAGATTTTAGCAACTTCA
CCTCAAACACTGTGGCGGAGATCCAGGCACTGACTTCCCAGGGCAGCAGCTTGGAAGA
AACGATAGCATCTCTGAAAGCTGAGGTGGAGGGTTTCAAGCAGGAACGGCAGGCAGTT
CATTCTGAAATGCTCCTGCGAGTCCAGCAGCTGGTGCAAGACCTGAAGAAACTGACCT
GCCAGGTGGCTACTi TCAACAACAATGCCTCCAC'I'GAAGGGACCTvCTGCCt:iGTCAA
CTGGGTGGAGCACCAAGACAGCTGCTACTGGTTCTCTCACTCTGGGATGTCCTGGGCC
GAGGCTGAGAAGTACTGCCAGCTGAAGAACGCCCACCTGGTGGTCATCAACTCCAGGG
AGGAGCAGAATTTTGTCCAGAAATATCTAGGCTCCGCATACACCTGGATGGGCCTCAG
TGACCCTGAAGGAGCCTGGAAGTGGGTGGATGGAACAGACTATGCGACCGGCTTCCAG
AACTGGAAGCCAGACCAGCCAGACGACTGGCAGGGGCACGGGCTGGGTGGAGGCGAGG
ACTGTGCTCACTTCCATCCAGTCGGCAGGTGGAATGACGACGTCTGCCAGAGGCCCTA
CCACTGGGTCTGCGAGGCTGGCTTGGGTCAGACCAGCCAGGAGAGTCACTGAGGTACC
TTTGGTGG
ORF Start: at 3 ORF Stop:
TGA
at 804
SEQ ID NO: 88 267 as MW at 29924.3kD
NOV26a, GPLPLQSLLQRLCSGPCHLLLSLGLGFLLLVIICWGFQNSKFQRDLWLRTDFSNFT
CG99732-02 SN'~~IQALTSQGSSLEETIASLKAEVEGFKQERQAVHSEMLLRVQQLVQDLKKLTC
Protein SequenceQ~'T~'~TEGTCCPVNWVEHQDSCYWFSHSGMSWAEAEKYCQL'r~IAHLWINSRE
EQNFVQKYLGSAYTWMGLSDPEGAWKWVDGTDYATGFQNWKPDQPDDWQGHGLGGGED
CAHFHPVGRWNDDVCQRPYHWVCEAGLGQTSQESH
SEQ ID NO: 89 1072
by
NOV26b, CTCCATTTCAGCTGTGACAACCTCAGAGCCGTGTTGGCCTAAGCATGACAAGGACGTA
169
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Sequence comparison of the above protein sequences yields the following
sequence
relationships shown in Table 26B.
Table 26B. Comparison of NOV26a against NOV26b.
Protein Sequence NOV26a Residues! Identities/
Match Residues Similarities for the Matched Region
'NOV26b ~ l:.lfi'1 253%297 (85%)
23..319 253/297 (85%)
Further analysis of the NOV26a protein yielded the following properties shown
in Table
26C.
Table 26C. Protein Sequence Properties NOV26a
Psort 0.7900 probability located in plasma membrane; 0.6756 probability
located in
analysis: microbody (peroxisome); 0.3000 probability located in Golgi body;
0.2000 probability
located in endoplasmic reticulum (membrane)
SignaIP Cleavage site between residues 38 and 39
analysis:
170
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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.
Table 26D. Geneseq Results
for NOV26a
NOV26a Identities/
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAW88125 Primate DCMP2 C-lectin family1..267 263/294 (89%)e-155
gene
protein sequence - Mammalia,23..316 263/294 (89%)
316 aa.
[W09902562-A1, 21-JAN-1999]
AAW88129 Variant primate DCMP2 C-lectin1..267 246/270 (91%)e-145
family
gene protein sequence - 23..273 246/270 (91
Mammalia, 273 %)
aa. [W09902562-AI, 21-JAN-1999]
AAW 15245Asialoglycoprotein receptor1..265 161/265 (60%)1 e-98
HI - Homo
Sapiens, 291 aa. [EP773289-A2,24..287 202/265 (75%)
14-
MAY-1997]
AAW15250 Asialoglycoprotein receptor1..265 148/265 (55f)5e-88
HI
cytoplasmic+extracellular 24..270 188/265 (70%)
domains -
Chimeric Homo Sapiens, 274
aa.
[EP773289-A2, 14-MAY-1997]
AAW15249 Asialoglycoprotein receptor37..265 135/229 (58%)1e-83
Hl
extracellular domain - ChimericI ..228 173/229 (74%)
Homo
Sapiens, 232 aa. [EP773289-A2,
14-
MAY-1997]
In a BLAST search of public sequence datbases, the NOV26a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 26E.
Table 26E. Public BLASTP
Results for NOV26a .
Protein NOV26a Identities/
AccessionProtein/Organism/Length Residues/Similarities Expect
for
Number Match the Matched Value
ResiduesPortion
Q14538 MACROPHAGE LECT1N 2 - Homo1..267 263/270 (97%)e-158
.
Sapiens (Human), 292 aa. 23..292 263/270 (97%)
BAB83508 ASIALOGLYCOPROTE1N 1..265 161/265 (60%)3e-98
RECEPTOR 1 - Homo Sapiens 24..287 202/265 (75%)
(Human), 291 aa.
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P07306Asialoglycoprotein receptor 1 (Hepatic1..265161/265 3e-
lectin H1) 23..286(60%) 98
(ASGPR) (ASGP-R) - Homo sapiens (Human), 202!265
290 aa. (75%)
Q91Y84ASIALOGLYCOPROTEIN RECEPTOR MAJOR 1..263149/263 le-
(56%)
SUBUNIT (ASIALOGLYCOPROTEIN RECEPTOR 23..284197/263 91
1 ) - (74%)
Mus musculus (Mouse), 284 aa.
LNRTL Hepatic lectin - rat, 284 aa. 1..263150/263 3e-
(57%)
23..284194/263 91
(73%)
PFam analysis predicts that the NOV26a protein contains the domains shown in
the Table
26F.
Table 26F. Domain Analysis of NOV26a
Identities/
Pfam Domain NOV26a Match Region Similarities Expect v slue
for the Matched Region
lectin_c 149..257 41/127 (32%) 3e-45
94/127 (74%)
Example 27.
The NOV27 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 27A.
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Further analysis of the NOV27a protein yielded the following properties shown
in Table
27B.
Table 27B. Protein Sequence Properties NOV27a
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
SignalP Cleavage site between residues 27 and 28
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 [PatentResidues/SimilaritiesExpect
#, for
IdentifierDate) Match the Matched Value
ResiduesRegion
ABG037941 Novel human diagnostic 52..178 69/134 (S1%)4e-21
protein #3785 -
Homo Sapiens, 150 aa. [W0200175067-25..150 74/134 (54%)
A2, I1-OCT-2001]
ABG03794Novel human diagnostic protein52..178 69/134 (S1%)4e-21
#3785 -
Homo Sapiens, 150 aa. [W0200175067-25..150 74/134 (54%)
A2, 11-OCT-2001]
AAB88581Human hydrophobic domain 44..197 52/155 (33%)1e-13
containing
protein clone HP1072? #65 39..170 74/15 (47,il
-.Homo
Sapiens, 183 aa. [W0200112660-A2,
22-
FEB-2001
AAY13464Human diaphanous polypeptide83..203 32/121 (26%)0.004
(Dial) -
Homo Sapiens, 1248 aa. [W09922028-A1,605..71541/121 (33%)
06-MAY-1999]
ABG21919Novel human diagnostic protein79..203 ~ 38/125 0.006
#21910 - (30%)
Homo sapiens, 325 aa. [W0200175067-74..191 ' 44/125
(34%)
A2, I1-OCT-200I]
In a BLAST search of public sequence datbases, the NOV27a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 27D.
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Table 27D. Public BLASTP
Results for NOV27a
Protein NOV27a Identities/
AccessionProtein/Organism/Length Residues/Similarities Expect
for
Number Match the Matched Value
ResiduesPortion
Q9D702 2310043I08RIK PROTEIN 1..196 141/196 (71%)6e-76
- Mus
musculus (Mouse), 196 1..196 151/196 (76%)
aa.
Q8UW65 P8F7 - Xenopus laevis 38..16$ 67/132 (50%) 3e-31
(African
clawed frog), 229 as (fragment).59..183 87/132 (65%)
CAC33296 SEQUENCE 85 FROM PATENT 44..197 52/155 (33%) 2e-13
W00112660 - Homo sapiens 39..170 74/I55 (47%)
(Human), 183 aa.
Q96NA7 CDNA FLJ31166 FIS, CLONE 44..197 ~ 52/155 (33%)2e-13
KIDNE1000143 - Homo sapiens19..150 74/155 (47%)
(Human), 163 aa.
Q9ASK4 HYPOTHETICAL 72.7 KDA 85..203 39/135 (28%) 0.007
PROTEIN - Oryza sativa 99..228 50/135 (36%)
(Rice), 698
aa.
PFam analysis predicts that the NOV27a protein contains the domains shown in
the Table
27E.
Table 27E. Domain Analysis of NOV27a
Identities/
Pfam Domain NOV27a Match Region Similarities Expect Value
for the Matched Region
Example B: Sequencing Methodology and Identification of NOVX Clones
1. GeneCallingTM Technology: This is a proprietary method of performing
differential gene expression profiling between two or more samples developed
at GuraGen
and described by Shimkets, et al., "Gene expression analysis by transcript
profiling coupled
to a gene database query" Nature Biotechnology 17:198-803 (1999). cDNA was
derived
from various human samples representing multiple tissue types, normal and
diseased states,
physiological states, and developmental states from different donors. Samples
were
obtained as whole tissue, primary cells or tissue cultured primary cells or
cell lines. Cells
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and cell lines may have been treated with biological or chemical agents that
regulate gene
expression, for example, growth factors, chemokines or steroids. The cDNA thus
derived
was then digested with up to as many as I20 pairs of restriction enzymes and
pairs of
linker-adaptors specific for each pair of restriction enzymes were ligated to
the appropriate
end. The restriction digestion generates a mixture of unique cDNA gene
fragments.
Limited PCR amplification is performed with primers homologous to the linker
adapter
sequence where one primer is biotinylated and the other is fluorescently
labeled. The
doubly labeled material is isolated and the fluorescently labeled single
strand is resolved by
capillary gel electrophoresis. A computer algorithm compares the
electropherograms from
an experimental and control group for each of the restriction digestions. This
and additional
sequence-derived information is used to predict the identity of each
differentially expressed
gene fragment using a variety of genetic databases. i ne identity of ts~:e
gene fray en t is
confizmed by additional, gene-specific competitive PCR or by isolation and
sequencing of
the gene fragment.
IS
2. SeqCalling~ Technology: cDNA was derived from various human samples
representing multiple tissue types, normal and diseased states, physiological
states, and
developmental states from different donors. Samples were obtained as whole
tissue,
primary cells or tissue cultured primary cells or cell lines. Cells and cell
lines may have
been treated with biological or chemical agents that regulate gene expression,
for example,
growth factors, chemokines or steroids. The cDNA thus derived was then
sequenced using
CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated
manually
and edited for corrections if apprayi-iate. cDNA sequences from all ~ apples
were
assembled together, sometimes including public human sequences, using
bioinformatic
programs to produce a consensus sequence for each assembly. Each assembly
is'included in
CuraGen Corporation's database. Sequences were included as components for
assembly
when the extent of identity with another component was at least 95% over 50
bp. Each
assembly represents a gene or portion thereof and includes information on
variants, such as
splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and
other
sequence variations.
3. PathCaIIing~ Technology:
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The NOVX nucleic acid sequences are derived by laboratory screening of cDNA
library by the two-hybrid approach. cDNA fragments covering either the full
length of the
DNA sequence, or part of the sequence, or both, are sequenced. In silico
prediction was
based on sequences available in CuraGen Corporation's proprietary sequence
databases or
S in the public human sequence databases, and provided either the full length
DNA sequence,
or some portion thereof.
The laboratory screening was performed using the methods summarized below:
cDNA libraries were derived from various human samples representing multiple
tissue types, normal and diseased states, physiological states, and
developmental states
from different donors. Samples were obtained as whole tissue, primary cells or
tissue
cultured primary cells or cell lines. Cells and cell lines may have been
treated with
biological or chemical agents that regulate gene expression, for example,
growth factors,
chemokines or steroids. The cDNA thus derived was then directionally cloned
into the
appropriate two-hybrid vector (Gal4-activation domain (Gal4-AD) fusion). Such
cDNA
1 S libraries as well as commercially available cDNA libraries from Clontech
(Palo Alto, CA)
were then transferred from E.coli into a CuraGen Corporation proprietary yeast
strain
(disclosed in U. S. Patents 6,057,101 and 6,083,693, incorporated herein by
reference in
their entireties).
Gal4-binding doriiain (Gal4-BD) fusions of a CuraGen Corportion proprietary
library of human sequences was used to screen multiple Gal4-AD fusion cDNA
libraries
resulting in the selection of yeast hybrid diploids in each of :ul'uch tree
GK,'~. ~A~? fusion
contains an individual cDNA. Each sample was amplified using the polymerase
chain
reaction (PCR) using non-specific primers at the cDNA insert boundaries. Such
PCR
product was sequenced; sequence traces were evaluated manually and edited for
corrections if appropriate. cDNA sequences from all samples were assembled
together,
sometimes including public human sequences, using bioinformatic programs to
produce a
consensus sequence for each assembly. Each assembly is included in CuraGen
Corporation's database. Sequences were included as components for assembly
when the
extent of identity with another component was at least 95% over 50 bp. Each
assembly
represents a gene or portion thereof and includes information on variants,
such as splice
forms single nucleotide polymorphisms (SNPs), insertions, deletions and other
sequence
variations.
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Physical clone: the cDNA fragment derived by the screening procedure, covering
the entire open reading frame is, as a recombinant DNA, cloned into pACT2
plasmid
(CIontech) used to make the cDNA library. The recombinant plasmid is inserted
into the
host and selected by the yeast hybrid diploid generated during the screening
procedure by
the mating of both CuraGen Corporation proprietary yeast strains N106' and
YULH (U. S.
Patents 6,057,101 and 6,083,693).
4. RACE: Techniques based on the polymerase chain reaction such as rapid
amplification of cDNA ends (RACE), were used to isolate or complete the
predicted
sequence of the cDNA of the invention. Usually multiple clones were sequenced
from one
or more human samples to derive the sequences for fragments. Various human
tissue
samples from different donors were used for the RACE reaction. The sequences
derived
from these procedures were included in the SeqCalling Assembly process
described in
preceding paragraphs.
5. Exon Linking: The NOVX target sequences identified in the present invention
I S were subjected to the exon linking process to confirm the sequence. PCR
primers were
designed by starting at the most upstream sequence available, for the forward
primer, and at
the most downstream sequence available for the reverse primer. In each case,
the sequence
was examined, walking inward from the respective termini toward the coding
sequence,
until a suitable sequence that is either unique or highly selective was
encountered, or, in the
case of the reverse primer, until the stop codon was reached. Such primers
were designed
based on in silico predictions for the full length cDNA, part (one or more
exons) of the
DNA or protein sequence of the target sequence, or by t~anslatPd homology of
the
predicted exons to closely related human sequences from other species. These
primers were
then employed in PGR amplification based on the following pool of human cDNAs:
adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain -
hippocampus,
brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal
kidney, fetal liver,
fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary
gland,
placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal
cord, spleen,
stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons
were gel purified,
cloned and sequenced to high redundancy. The PCR product derived from exon
linking
was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial
clone has an
insert covering the entire open reading frame cloned into the pCR2.1 vector.
The resulting
sequences from all clones were assembled with themselves, with other fragments
in
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CuraGen Corporation's database and with public ESTs. Fragments and ESTs were
included as components for an assembly when the extent of their identity with
another
component of the assembly was at least 95% over 50 bp. In addition, sequence
traces were
evaluated manually and edited for corrections if appropriate. These procedures
provide the
sequence reported herein.
6. Physical Clone: Exons were predicted by homology and the intron/exon
boundaries were determined using standard genetic rules. Exons were further
selected and
refined by means of similarity determination using multiple BLAST (for
example, tBlastN,
BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail.
Expressed
sequences from both public and proprietary databases were also added when
available to
further define and complete the gene sequence. The DNA sequence was then
manually
corrected for apparent inconsistencies thereby obtaining the sequences
encoding the
full-length protein.
The PCR product derived by exon linking, covering the entire open reading
frame,
was cloned into the pCR2.1 vector from Invitrogen to provide clones used for
expression
and screening purposes.
Example C: Quantitative expression analysis of clones in various cells and
tissues
The 'quantitative expression of various clones was assessed using microtiter
plates
containing RNA samples from a variety of normal and pathology-derived cells,
Bell lines
and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed
on an
Applied Biosystems ABI PRISM~ 7700 or an ABI PRISM~ 7900 HT Sequence Detection
System. Various collections of samples are assembled on the plates, and
referred to as
Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing
samples
derived from tissues from normal and cancer sources), Panel 3 (containing
cancer cell
lines), Panel 4 (containing cells and cell lines from normal tissues and cells
related to
inflammatory conditions), Panel SD/SI (containing human tissues and cell lines
with an
emphasis on metabolic diseases), AI comprehensive~anel (containing normal
tissue and
samples from autoimmune diseases), Panel CNSD.O1 (containing central nervous
system
samples from normal and diseased brains) and CNS neurodegeneration~anel
(containing
samples from normal and Alzheimer's diseased brains).
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RNA integrity from all samples is controlled for quality by visual assessment
of
agarose gel electropherograms using 28S and 18S ribosomal RNA staining
intensity ratio
as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs
that would
be indicative of degradation products. Samples are controlled against genomic
DNA
contamination by RTQ PCR reactions run in the absence of reverse transcriptase
using
probe and primer sets designed to amplify across the span of a single exon.
First, the RNA samples were normalized to reference nucleic acids such as
constitutively expressed genes (for example, (3-actin and GAPDH). Normalized
RNA (5 u1)
was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix
Reagents (Applied Biosystems; Catalog No. 4309169) and gene-specific primers
according
to the manufacturer's instructions.
In other cases, non-normalized RNA samples were converted to single strand
eDNA
(sscDNA) using Superscript II (Invitrogen Corporation; Catalog No. 18064-147)
and
random hexamers according to the manufacturer's instructions. Reactions
containing up to
10 ~g of total RNA were performed in a volume of 20 ~l and incubated for 60
minutes at
42 °C. This reaction can be scaled up to 50 ~.g of total RNA in a final
volume of 100 ~1.
sscDNA samples are then normalized to reference nucleic acids as described
previously,
using 1X TaqMan~ Universal Master mix (Applied Biosystems; catalog No.
4324020),
following the manufacturer's instructions.
Probes and primers were designed for each assay according to Applied
Biosystems
Primer Express Software package (version I for Apple Computer's Ma.,intosh
Power PC) or
a similar algorithm using the target sequence as input. Default settings were
used for
reaction conditions and the following parameters were set before selecting
primers: primer
concentration = 250 nM, primer melting temperature (Tm) range = 58 °-60
°C, primer
optimal Tm = 59 °C, maximum primer difference = 2 °C, probe does
not have 5'G, probe
Tm must be 10 °C greater than primer Tm, amplicon size 75bp to 100bp.
The probes and
primers selected (see below) were synthesized by Synthegen (Houston, TX, USA).
Probes
were double purified by HPLC to remove uncoupled dye and evaluated by mass
spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3'
ends of the
probe, respectively. Their final concentrations were: forward and reverse
primers, 900nM
each, and probe, 200nM.
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PCR conditions: When working with RNA samples, normalized RNA from each
tissue and each cell line was spotted in each well of either a 96 well or a
384-well PCR
plate (Applied Biosystems). PCR cocktails included either a single gene
specific probe and
primers set, or two multiplexed probe and primers sets (a set specific for the
target clone
S and another gene-specific set multiplexed with the target probe). PCR
reactions were set up
using TaqMan~ One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No.
4313803) following manufacturer's instructions. Reverse transcription was
performed at 48
°C for 30 minutes followed by amplification/PCR cycles as follows:
9S°C I O min, then 40
cycles of 95 °C for 1 S seconds, 60 °C for 1 minute. Results
were recorded as CT values
(cycle at which a given sample crosses a threshold level of fluorescence)
using a log scale,
with the difference in RNA concentration between a given sample and the sample
with the
lowest CT value being represented as 2 to ts~e rower ref delta C'''. 'TEL
percea relWi ae
expression is then obtained by taking the reciprocal of this RNA difference
and multiplying
by 100.
1 S When working with sscDNA samples, normalized sscDNA was used as described
previously for RNA samples. PCR reactions containing one or two sets of probe
and
primers were set up as described previously, using 1X TaqMan~ Universal Master
mix
(Applied Biosystems; catalog No. 4324020), following the manufacturer's
instructions.
PCR amplification was performed as follows: 9S °C 10 min, then 40
cycles of 9S °C for 1 S
seconds, 60 °C for 1 minute. Results were analyzed and processed as
described previously.
Panels 1,1.1,1.2, and 1.3D
The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control wells (genomic
DNA
control and chemistry control) and 94 wells containing cDNA from various
samples. The
samples in these panels are broken into 2 classes: samples derived from
cultured cell lines
and samples derived from primary normal tissues. The cell lines are derived
from cancers
of the following types: lung cancer, breast cancer, melanoma, colon cancer,
prostate cancer,
CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal
cancer, gastric
cancer and pancreatic cancer. Cell lines used in these panels are widely
available through
the American Type Culture Collection (ATCC), a repository for cultured cell
lines, and
were cultured using the conditions recommended by the ATCC. The normal tissues
found
on these panels are comprised of samples derived from all major organ systems
from single
adult individuals or fetuses. These samples are derived from the following
organs: adult
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skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult
kidney, fetal kidney,
adult liver, fetal liver, adult lung, fetal lung, various regions of the
brain, the spleen, bone
marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland,
spinal cord,
thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary,
uterus, placenta,
prostate, testis and adipose.
In the results for Panels 1, 1.l, 1.2 and 1.3D, the following abbreviations
are used:
ca. = carcinoma,
* = established from metastasis,
met = metastasis,
s cell var = small cell variant,
non-s = non-sm = non-small,
squam = squamous,
p1. eff = p1 effusion = pleural effusion,
glio = glioma,
astro = astrocytoma, and
neuro = neuroblastoma.
General screening_panel v1.4
The plates for Panel 1.4 include 2 control wells (genomic DNA control and
chemistry control) and 94 wells containing cDNA from various samples. The
samples in
Panel 1.4 are broken into 2 classes: samples derived from cultured cell lines
and samples
derived from primary normal tissues. The cell lines are derived from cancers
of the
following types: lung cancer, breast cancer, melanoma, colon cancer, prostate
cancer, CNS
cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer,
gastric cancer
and pancreatic cancer. Cell lines used in Panel 1.4 are widely available
through the
American Type Culture Collection (ATCC), a repository for cultured cell lines,
and were
cultured using the conditions recommended by the ATCC. The normal tissues
found on
Panel 1.4 are comprised of pools of samples derived from all major organ
systems from 2
to 5 different adult individuals or fetuses. These samples are derived from
the following
organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal
heart, adult kidney,
fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various
regions of the brain, the
spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland,
adrenal gland,
spinal cord, thymus, stomach, small intestine, colon, bladder, trachea,
breast, ovary, uterus,
placenta, prostate, testis and adipose. Abbreviations are as described for
Panels 1, 1.I, 1.2,
and 1.3D.
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Panels 2D and 2.2
The plates for Panels 2D and 2.2 generally include 2 control wells and 94 test
samples composed of RNA or cDNA isolated from human tissue procured by
surgeons
working in close cooperation with the National Cancer Institute's Cooperative
Human
Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The
tissues
are derived from human malignancies and in cases where indicated many
malignant tissues
have "matched margins" obtained from noncancerous tissue just adjacent to the
tumor.
These are termed normal adjacent tissues and are denoted "NAT" in the results
below. The
tumor tissue and the "matched margins" are evaluated by two independent
pathologists (the
surgical pathologists and again by a pathologist at NDRI or CHTN). This
analysis provides
a gross histapathological assessment of tumor differentiation grade. Moreover,
most
samples include the original surgical pathology report that provides
information regarding
the clinical stage of the patient. These matched margins are taken from the
tissue
surrounding (i.e. immediately proximal) to the zone of surgery (designated
"NAT", for
normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were
obtained
from various human tissues derived from autopsies performed on elderly people
or sudden
death victims (accidents, etc.). These tissues were ascertained to be free of
disease and
were purchased from various commercial sources such as Clontech (Palo Alto,
CA),
Research Genetics, and Invitrogen.
Panel3D
The plates of Panel 3D are comprised of 94 cDNA samples and two control
samples. Specifically, 92 of these samples are derived from cultured human
cancer cell
lines, 2 samples of human primary cerebellar tissue and 2 controls. The human
cell lines
are generally obtained from ATCC (American Type Culture Collection), NCI or
the
German tumor cell bank and fall into the following tissue groups: Squamous
cell carcinoma
of the tongue, breast cancer, prostate cancer, melanoma, epidermaid carcinoma,
sarcomas,
bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas,
ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In
addition, there
are two independent samples of cerebellum. These cells are all cultured under
standard
recommended conditions and RNA extracted using the standard procedures. The
cell lines
in panel 3D and 1.3D are of the most common cell lines used in the scientific
literature.
Panels 4D, 4R, and 4.1D
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Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples)
composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various
human cell
lines or tissues related to inflammatory conditions. Total RNA from control
normal tissues
such as colon and lung (Stratagene, La Jolla, CA) and thymus and kidney
(Clontech) was
employed. Total RNA from liver tissue from cirrhosis patients and kidney from
lupus
patients was obtained from BioChain (Biochain Institute, Inc., Hayward, CA).
Intestinal
tissue for RNA preparation from patients diagnosed as having Crohn's disease
and
ulcerative colitis was obtained from the National Disease Research Interchange
(NDRI)
(Philadelphia, PA).
Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth
muscle
cells, small airway epithelium, bronchial epithelium, microvascular dermal
endothelial
cells, microvascular.lung endothelial cells, human pulmonary aortic
endothelial cells,
human umbilical vein endothelial cells were all purchased from Clonetics
(Walkersville,
MD) and grown in the media supplied for these cell types by Clonetics. These
primary cell
types were activated with various cytokines or combinations of cytokines for 6
and/or 12-
14 hours, as indicated. The following cytokines were used; IL-1 beta at
approximately 1-
Sng/ml, TNF alpha at approximately 5-l Ong/ml, IFN gamma at approximately 20-
SOng/ml,
IL-4 at approximately 5-lOng/ml, IL-9 at approximately 5-lOng/ml, IL-13 at
approximately
5-l Ong/ml. Endothelial cells were sometimes starved for various times by
culture in the
basal media from Clonetics with 0.1 % serum.
Mononuclear cells were prepared from blood of employees at CuraGen
Corporation, using Ficoll. LAK cells were prepared from these cells by culture
in DMEM
5% FCS (Hyclone), 100~M non essential amino acids (Gibco/Life Technologies,
Rockville, MD), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO'SM
(Gibco), and
l OmM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either
activated with
10-20ng/ml PMA and 1-2~.g/ml ionomycin, IL-12 at 5-l Ong/ml, IFN gamma at 20-
SOng/ml
and IL-18 at 5-l Ong/ml for 6 hours. In some cases, mononuclear cells were
cultured for 4-5
days in DMEM 5% FCS (Hyclone), 100p.M non essential amino acids (Gibco), 1mM
sodium pyruvate (Gibco), mercaptoethanol S.SxlO'SM (Gibco), and l OmM Hepes
(Gibco)
with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately
S~.g/ml.
Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed
lymphocyte
reaction) samples were obtained by taking blood from two donors, isolating the
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mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1
at a final
concentration of approximately 2x106cells/ml in DMEM 5% FCS (Hyclone), 100~,M
non
essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol
(S.SxlO-
SM) (Gibco), and l OmM Hepes (Gibco). The MLR was cultured and samples taken
at
various time points ranging from I- 7 days for RNA preparation.
Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve
VS selection columns and a Vario Magnet according to the manufacturer's
instructions.
Monocytes were differentiated into dendritic cells by culture in DMEM S% fetal
calf serum
(FCS) (Hyclone, Logan, UT), 100~.M non essential amino acids (Gibco), 1 mM
sodium
pyruvate (Gibco), mercaptoethanol S.SxlO-SM (Gibco), and lOmM Hepes (Gibco),
SOng/ml
GMCSF and Sng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of
monocytes for S-7 days in DMEM 5% FCS (Hyclone), 100~M non essential amino
acids
(Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM (Gibco), lOmM
Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately SOng/ml.
Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14
hours with
' lipopolysaccharide (LPS) at 1 OOng/ml. Dendritic cells were also stimulated
with anti-CD40
monoclonal antibody (Pharmingen) at 10~g/ml for 6 and 12-14 hours.
CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from
mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS
selection
columns and a Vario Magnet according to the manufacturer's instructions.
CD45RA and
CD45R0 CD4 lymphocytes were isolated by depleting mononuclear cells of CDB,
CD56,
CD14 and CD19 cells using CDB, CD56, CD14 and CD19 Miltenyi beads and positive
selection. CD45R0 beads were then used to isolate the CD45R0 CD4 lymphocytes
with
the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45R0 CD4 and
CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone),100pM non essential amino
acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol S.Sx 10'5M
(Gibco), and
l OmM Hepes (Gibco) and plated at 106cells/ml onto Falcon 6 well tissue
culture plates that
had been coated overnight with O.Spg/mI anti-CD28 (Pharmingen) and 3ug/ml anti-
CD3
(OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA
preparation. To prepare chronically activated CD8 lymphocytes, we activated
the isolated
CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then
harvested
the cells and expanded them in DMEM 5% FCS (Hyclone), 100~.M non essential
amino
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acids (Gibco), 1mM sodiumpynivate (Gibco), mercaptoethanol S.SxlO-SM (Gibco),
and
l OmM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again
with
plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was
isolated
6 and 24 hours after the second activation and after 4 days of the second
expansion culture.
The isolated NK cells were cultured in DMEM 5% FCS (Hyclone),100pM non
essential
amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM
(Gibco),
and lOmM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with
sterile dissecting scissors and then passed through a sieve. Tonsil cells were
then spun
down and resupended at 106 cells/ml in DMEM 5% FCS (Hyclone), 100~M non
essential
amino acids (Gibco), 1mM sodium pyruvate (Gibco). mercaptoethanol S.SxlO'SM
(Gibco),
and lOmM Hepes (Gibco). To activate the cells, we used PWM at Spg/ml or anti-
CD40
(Pharmingen) at approximately lOpg/ml and IL-4 at 5-lOng/ml. Cells were
harvested for
RNA preparation at 24, 48 and 72 hours.
To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon
plates
were coated overnight with l Opg/ml anti-CD28 (Pharmingen) and 2~g/ml OKT3
(ATCC),
and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic
Systems, German Town, MD) were cultured at 105-106cells/ml in DMEM 5% FCS
(Hyclone), 100~M non essential amino acids (Gibco), 1mM sodium pyruvate
(Gibco),
mercaptoethanol S.SxlO-SM (Gibco), lOmM Hepes (Gibco) and IL-2 (4ng/ml). IL-12
(Sng/ml) and anti-IL4 (1 pg/ml) were used to direct to Thl, while IL-4
(Sng/ml) and anti-
IFN gamma (1 pg/ml) were used to direct to Th2 and IL-10 at Sng/ml was used to
direct to
Trl. After 4-5 days, the activated Thl, Th2 and Trl lymphocytes were washed
once in
DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100p.M non essential
amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM
(Gibco),
lOmM Hepes (Gibco) and IL-2 (lng/ml). Following this, the activated Thl, Th2
and Trl
lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as
described above, but with the addition of anti-CD95L (lp,g/ml) to prevent
apoptosis. After
4-5 days, the Thl, Th2 and Trl lymphocytes were washed and then expanded again
with
IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this
way for a
maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2
and
Trl after 6 and 24 hours following the second and third activations with plate
bound anti-
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CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures
in
Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1,
KU-812. EOL cells were further differentiated by culture in O.lmM dbcAMP at
S Sx105cells/ml fox 8 days, changing the media every 3 days and adjusting the
cell
concentration to Sx105cells/ml. For the culture of these cells, we used DMEM
or RPMI (as
recommended by the ATCC), with the addition of S% FCS (Hyclone), 100~M non
essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol
S.SxlO-SM
(Gibco), l OmM Hepes (Gibco). RNA was either prepared from resting cells or
cells
activated with PMA at 1 Ong/ml and ionomycin at 1 ~g/ml for 6 and 14 hours.
Keratinocyte
line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained
from the
ATCC. Both were cultured in DMEM S% FCS (Hyclone), 100~.M non essential amino
acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM (Gibco),
and
IOmM Hepes (Gibco). CCDl 106 cells were activated for 6 and 14 hours with
1S approximately S ng/ml TNF alpha and lng/ml IL-1 beta, while NCI-H292 cells
were
activated for 6 and 14 hours with the following cytokines: Sng/ml IL-4, Sng/ml
IL-9,
Sng/ml IL-13 and 2Sng/ml IFN gamma.
For these cell lines and blood cells, RNA was prepared by Iysing approximately
I O~celIs/mI using TrizoI (Gibco BRL). Briefly, I/10 volume of
bromochloropropane
(Molecular Research Corporation) was added to the RNA sample, vortexed and
after 10
minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall
SS34 rotor.
The aqueous phase was removed and placed in a 1 Sml Falcon Tube. An equal
volume of
isopropanol was added and left at 20 °C overnight. The precipitated RNA
was spun down
at 9,000 rpm for 1 S min in a Sorvall SS34 rotor and washed in 70% ethanol.
The pellet was
2S redissolved in 300.1 of RNAse-free water and 35.1 buffer (Promega) 5~.1
DTT, 7p.1
RNAsin and 8~.1 DNAse were added. The tube was incubated at 37 °C for
30 minutes to
remove contaminating genomic DNA, extracted once with phenol chloroform and re-
precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of 100%
ethanol. The
RNA was spun down and placed in RNAse free water. RNA was stored at -80
°C.
AI_comprehensive panel v1.0
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The plates for AI comprehensive panel v1 .0 include two control wells and 89
test
samples comprised of cDNA isolated from surgical and postmortem human tissues
obtained from the Backus Hospital and Clinomics (Frederick, MD). Total RNA was
extracted from tissue samples from the Backus Hospital in the Facility at
CuraGen. Total
RNA from other tissues was obtained from Clinomics.
Joint tissues including synovial fluid, synovium, bone and cartilage were
obtained
from patients undergoing total knee or hip replacement surgery at the Backus
Hospital.
Tissue samples were immediately snap frozen in liquid nitrogen to ensure that
isolated
RNA was of optimal quality and not degraded. Additional samples of
osteoarthritis and
rheumatoid arthritis joint tissues were obtained from Clinomics. Normal
control tissues
were supplied by Clinomics and were obtained during autopsy of trauma victims.
Surgical specimens of psoriatic tissues and adjacent matched tissues were
provided
as total RNA by Clinomics. Two male and two female patients were selected
between the
ages of 25 and ,47. None of the patients were taking prescription drugs at the
time samples
were isolated.
Surgical specimens of diseased colon from patients with ulcerative colitis and
Crohns disease and adjacent matched tissues were obtained from Clinomics.
Bowel tissue
from three female and three male Crohn's patients between the ages of 41-69
were used.
Two patients were not on prescription medication while the others were taking
dexamethasone, phenobarbital, or tylenol. Ulcerative colitis tissue was from
three male and
four female patients. Four of tile patients were taking leb vid ~~u w~ w;.re
or~
phenobarbital.
Total RNA from post mortem lung tissue from trauma victims with no disease or
with emphysema, asthma or COPD was purchased from Clinomics. Emphysema
patients
ranged in age from 40-70 and all were smokers, this age range was chosen to
focus on
patients with cigarette-linked emphysema and to avoid those patients with
alpha-lanti-
trypsin deficiencies. Asthma patients ranged in age from 36-75, and excluded
smokers to
prevent those patients that could also have COPD. COPD patients ranged in age
from 35-
80 and included both smokers and non-smokers. Most patients were taking
corticosteroids,
and bronchodilators.
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In the labels employed to identify tissues in the AI comprehensive panel v1.0
panel, the following abbreviations are used:
AI = Autoimmunity
Syn = Synovial
Normal = No apparent disease
Rep22 /Rep20 = individual patients
RA = Rheumatoid arthritis
Backus = From Backus Hospital
OA = Osteoarthritis
(SS) (BA) (MF) = Individual patients
Adj = Adjacent tissue
Match control = adjacent tissues
-M = Male
-F = Female
COPD = Chronic obstructive pulmonary disease
Panels SD and SI
The plates for Panel SD and SI include two control wells and a variety of
cDNAs
isolated from human tissues and cell lines with an emphasis on metabolic
diseases.
Metabolic tissues were obtained from patients enrolled in the Gestational
Diabetes study.
Cells were obtained during different stages in the differentiation of
adipocytes from human
mesenchymal stem cells. Human pancreatic islets were also obtained.
In the Gestational Diabetes study subjects are young (18 - 40 years),
otherwise
healthy women with and without gestational diabetes undergoing routine
(elective)
Caesarean section. After delivery of the infant, when the surgical incisions
were being
repaired/closed, the obstetrician removed a small sample (<1 cc) of the
exposed metaboirc
tissues during the closure of each surgical level. The biopsy material was
rinsed in sterile
saline, blotted and fast frozen within 5 minutes from the time of removal. The
tissue was
then flash frozen in liquid nitrogen and stored, individually, in sterile
screw top tubes and
kept on dry ice for shipment to or to be picked up by CuraGen. The metabolic
tissues of
interest include uterine wall (smooth muscle), visceral adipose, skeletal
muscle (rectos) and
subcutaneous adipose. Patient descriptions are as follows:
Patient 2 Diabetic Hispanic, overweight,
not on insulin
Patient ?-9 Nondiabetic Caucasian and obese
(BMI>30)
Patient 10 Diabetic Hispanic, overweight,
on insulin
Patient 11 Nondiabetic African American and
overweight
Patient 12 Diabetic Hispanic on insulin
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Adipocyte differentiation was induced in donor progenitor cells obtained from
Osirus (a division of CloneticsBioWhittaker) in triplicate, except for Donor
3U which had
only two replicates. Scientists at Clonetics isolated, grew and differentiated
human
mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol
found in
Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal
Stem Cells
Science Apr 2 1999: 143-147. Clonetics provided Trizol lysates or frozen
pellets suitable
for mRNA isolation and ds cDNA production. A general description of each donor
is as
follows:
Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated Adipose
I O Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated
Donor 2 and 3 AD: Adipose, Adipose Differentiated
Human cell lines were generally obtained from ATCC (American Type Culture
Collection), NCI or the German tumor cell bank and fall into the following
tissue groups:
kidney proximal convoluted tubule, uterine smooth muscle cells, small
intestine, Liver
HepG2 cancer cells, heart primary stromal cells, and adrenal cortical adenoma
cells. These
cells are all cultured under standard recommended conditions and RNA extracted
using the
standard procedures. All samples were processed at CuraGen to produce single
stranded
cDNA.
Panel SI contains all samples previously described with the addition of
pancreatic
islets from a 58 year old female patient obtained from the Diabetes Research
Institute at the
University of Miami School of Medicine. Islet tissue was processed to total
RNA at an
outside source and delivered to Curarsen for addition to panel 5I.
In the labels employed to identify tissues in the SD and SI panels, the
following
abbreviations are used:
GO Adipose = Greater Omentum Adipose
SK = Skeletal Muscle
UT = Uterus
PL = Placenta
AD = Adipose Differentiated
AM = Adipose Midway Differentiated
U = Undifferentiated Stem Cells
Panel CNSD.Ol
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The plates for Panel CNSD.O1 include two control wells and 94 test samples
comprised of cDNA isolated from postmortem human brain tissue obtained from
the
Harvard Brain Tissue Resource Center. Brains are removed from calvaria of
donors
between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen
at -80°C in
liquid nitrogen vapor. All brains are sectioned and examined by
neuropathologists to
confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains two
brains
from each of the following diagnoses: Alzheimer's disease, Parkinson's
disease,
Huntington's disease, Progressive Supernuclear Palsy, Depression, and "Normal
controls".
Within each of these brains, the following regions are represented: cingulate
gyrus,
temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary
motor strip),
Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and
Brodman area
17 (occipital cortex). Not all brain regions are represented in all cases;
e.g., Huntington's
disease is characterized in part by neurodegeneration in the globus palladus,
thus this
region is impossible to obtain from confirmed Huntington's cases. Likewise
Parkinson's
disease is characterized by degeneration of the substantia nigra making this
region more
difficult to obtain. Normal control brains were examined for neuropathology
and found to
be free of any pathology consistent with neurodegeneration.
In the labels employed to identify tissues in the CNS panel, the following
abbreviations are
used:
PSP = Progressive supranuclear palsy
- Sub Nigra = Substantia nigra . -
Glob PaIladus= Globus palladus
Temp Pole = Temporal pole
Cing Gyr = Cingulate gyrus
BA 4 = Brodman Area 4
Panel CNS Neurodegeneration V1.0
The plates for Panel CNS Neurodegeneration V1.0 include two control wells and
47 test samples comprised of cDNA isolated from postmortem human brain tissue
obtained
from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human
Brain
and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System).
Brains are
removed from calvaria of donors between 4 and 24 hours after death, sectioned
by
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neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All
brains are sectioned and
examined by neuropathologists to confirm diagnoses with clear associated
neuropathology.
Disease diagnoses are taken from patient records. The panel contains six
brains
from Alzheimer's disease (AD) patients, and eight brains from "Normal
controls" who
showed no evidence of dementia prior to death. The eight normal control brains
are divided
into two categories: Controls with no dementia and no Alzheimer's like
pathology
(Controls) and controls with no dementia but evidence of severe Alzheimer's
like
pathology, (specifically senile plaque load rated as level 3 on a scale of 0-
3; 0 = no
evidence of plaques, 3 = severe AD senile plaque load). Within each of these
brains, the
following regions are represented: hippocampus, temporal cortex (Brodman Area
21),
parietal cortex (Brodman area 7), and occipital cortex (Brodman area 17).
These reg o_n_s
were chosen to encompass all levels of neurodegeneration in AD. The
hippocampus is a
region of early and severe neuronal Loss in AD; the temporal cortex is known
to show
neurodegeneration in AD after the hippocampus; the parietal cortex shows
moderate
neuronal death in the late stages of the disease; the occipital cortex is.
spared in AD and
therefore acts as a "control" region within AD patients. Not all brain regions
are
represented in all cases.
In the labels employed to identify tissues in the CNS Neurodegeneration V 1.0
panel, the
following abbreviations are used:
AD = Alzheimer's disease brain; patient was demented and showed AD-like
pathology upon autopsy
Control = Control brains; patient not demented, showing no neuropathology
Control (Path) = Control brains; pateint not demented but showing sever AD-
like
pathology
SupTemporal Ctx = Superior Temporal Cortex
Inf Temporal Ctx = Inferior Temporal Cortex
A. CG100104-Ol: fibronectin-malate dehydrogenase
Expression of gene CG100104-01 was assessed using the primer-probe set Ag4162,
described in Table AA. Results of the RTQ-PCR runs are shown in Tables AB and
AC.
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Table AA. Probe Name Ag4162
Start SEQ ID
PrimersSequences Length
_ P_o__sitionNo
~
Forward5~-cctcagttatgctcctgtctgt-3~ 22 _11s 93 _
Probe TES'-ttcatcttcacctcagagcggaactg-3'-26 14S 94
Reverse5~-cagttccgctcatctttgtaag-3~ 22 188 95
Table AB. General screening~anel v1.4
Rel. Exp.(!) Rel. Exp.(e)
Tissue Name Ag4162, Run Tissue Name Ag4162, Run
221000252 22100022
Adipose 0.0 Renal ca. TK-10 0.2
MeIanoma* 0.0 Bladder 0.5
~
_ .._. . ..... .. _ . . . __ _. .._.__._..
A).T .. ... . ._ .._ _. _ _ _ . .. . ..
Hs688( . . . ..... . ...... ._..... . .
.. __.. . .. .
_
Melanoma* Gastric ca. (liver
0'0 met.) 0.2
Hs688(B).T CI-N87
Melanoma* M14 ~ 0.0 Gastric ca. KATO 0.0
III
Melanoma* 0.0 Colon ca. SW-948 0.0
LOXIMVI
Melanoma* SK- 0.1 Colon ca. SW480 O.s
~
MEL-5 .. . . _ ._ . _. _ . .. ._ _ . . _ . . . _. .
_ . .. _ . ........ ... . _. . ._.
. ._. . _ . .
Squamous cell Colon ca.* (SW480
0'0 0.0
carcinoma SCC-4 met) SW620
Testis Pool 100.0 Colon ca. HT29 0.2
Prostate ca.* 0.0 Colon ca. HCT-116 0.0
(bone
met) PC-3
Prostate Pool 0.2 Colon ca. CaCo-2 0.6
Placenta 0.0 Colon cancer tissue0.0
Uterus Pool 0.0 Colon ca. SW1116 0.0
Ovarian ca. 0.4 Colon ca. Colo-20s 0.0
OVCAR-3
Ovarian ca. SK- 0.0 Colon ca. SW-48 0.0
~
OV-3 _ _ _
Ovarian ca
. 0.0 Colon Pool 0.3
OVCAR-4
Ovarian ca. p. l Small Intestine 0.0
Pool
ovcAR-s
Ovarian ca.
IGROV-1 0.0 Stomach Pool 0.0
Ovarian ca. 3.4 Bone Marrow Pool 0.0
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OVCAR-8 ..~."",.,.~.....-,...,-,_,~..,..~, ~,~. .~.",.~..~..,",W".-
..~.
Ovary 0.0 ~ Fetal Heart 0.0
Breast_ca. MCF-7 0.5 Heart Pool 0.0 _
'
Breast ca. MDA- 0.0 Lymph Node Pool 0.2
MB-231
Breast ca. BT 549 0.0 Fetal Skeletal Muscle0.1
Breast ca. T47D 0.0 Skeletal Muscle 0.7
Pool
Breast ca. MDA-N 0.0 Spleen Pool 0.0
Breast Pool 0.2 Thymus Pool 0.2
CNS cancer
Trachea 0.3 0
2
(glio/astro) U87-MG.
CNS cancer
Lung 0.2 0.3
(glio/astro) U-118-MG
CNS cancer
Fetal Lung 0.1 ~ 0.0
(_n_~prn~m__Ptl
~ $T_~ N-A C ~
~
Lung ca. NCI-N417 0.0 cancer (astro) SF- 0.0
~ 9S
'
Lung ca. LX-1 0.0 CNS cancer (astro) p.0
SNB-75
Lung ca. NCI-H146 p.7 CNS cancer (glio) 0.0
SNB-19
CNS cancer (glio)
Lung ca. SHP-77 0.5 SF 0.0
~ ~
.. _ .... _.._.__ ..._. _.._.._...295 _. .._.. .
_ . ... .. ..... ._. . _.._.._.. ..._.... .....__ ..
._ .. .. .. ._. . ....___ .... .. . .. .
._.... ... ..
... .. ...
._..._.__ . .. ..
g 0.0 , _ 0.0
Lun _ca. A549 ( ~
yg
)
Brain Am data Pool
Lung ca. NCI-H526_0.0 Brain (cerebellum) 0.0
Lung ca. NCI-H23 0.4 _ Brain (fetal) _ 0.0 _
___
Lung ca. NCI-H460 0.2 Brain (Hippocampus)03
Pool .
Lung ca. HOP-62 0.0 Cerebral Cortex 0.8
._ -_.-.. .~~ Pool __._.
....r...,. _ __..._._.___..
_..._.._._.~""~.
Brain (Substantia
Lung ca. NCI-H522 0.4 0.0
n igra) Pool
Liver 0.0 Brain (Thalamus) 0.0
Pool
Fetal Liver 0.0 Brain (whole) 0.0
Liver ca. HepG2 0.0 Spinal Cord Pool 0.0
Kidney Pool 0.4 Adrenal Gland 0.1
Fetal Kidney 0.0 Pituitary gland 0.0
Pool
Renal ca. 786-0 0.0 Salivary Gland 0.0
Renal ca. A498 0.0 Thyroid (female) 0.0
Pancreatic ca.
Renal ca. ACHN 0.2 0
4
CAPAN2 .
Renal ca. U0-31 0.0 Pancreas Pool 0.2
Table AC. Panel 4.1 D
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Rel. Exp.(%)~~ Rel. Exp.(f)
Tissue Name Ag4162, Run Tissue Name Ag4162, Run
173333854 173333854
Secondary Thl 0.0 HUVEC IL-lbeta 0.0
act
Secondary Th2 0.0 HUVEC IFN gamma 0.0
act
Secondary Trl 0.0 SEC TNF alpha + 0.0
act
IFN gamma
Secondary Thl 0.0 HUVEC TNF alpha + 0.0
rest IL4
Secondary Th2 0.0 HUVEC IL-11 0.0
rest
Lung Microvascular
Secondary Trl ~.0 EC 0.0
rest ~
. ._._ _. .. .._._.__ _.none.__..__.____...Y
. _._ __..__._ __ ._. _ _._
-_. _ _ _. _. _... _
._
Primary Thl act 0.0 Leg Microvascular _
EC 0.0
TNFalpha + IL-1 beta
Primary Th2 act 0.0 Microvascular Dermal0.0
~
EC none
Primary Trl act 0.0 Microsvasular Dermal0.0
EC
TNFalpha + IL-lbeta
Bronchial epithelium
Primary Thl rest 0.0 ~ 0.0
_ . TNFalpha + IL 1 beta_
Primary Th2 rest 0.0 small airway epithelium0.0
none
Primary Trl rest 0.0 Small airway epithelium0.0
TNFalpha + IL-1 beta
CD45RA CD4
0.0 Coronery artery SMC 0.0
rest
lymphocyte act
CD45R0 CD4 Coronery artery SMC
0'0 0
0
lymphocyte act , _ TNFalpha + IL-lbeta .
_ ~ ~. .
CD8 lymphocyte_ 0.0 Astrocytes rest 0.0
act
Secondary CD8 Astrocytes TNFalpha
0 + 0
0 ~ 0
lymphocyte rest ' ~IL-lbeta .
Secondary CD8 0,0 KU-812 (Basophil) 0.0
rest
lymphocyte act
CD4 lymphocyte 0.0 KU-812 (Basophil) 0.0
none
PMA/ionomycin
try Thl/Th2/Trl CCD1106
anti-
_ 0'0 ~(Keratinocytes) 0.0
CD95 CHl 1 __ _._. none __
_ . . .. ___ _ -. . _ __ .
~
CCD
1106
LAK cells rest 0.0 (Keratinocytes) 0.0
TNFalpha + IL-lbeta
LAK cells IL-2 0.0 Liver cirrhosis 0.5
LAK cells IL-2+IL-120.0 CI-H292 none 0.0
LAK cells IL-2+IFN
0.0 CI-H292 IL-4 0.0
gamma
LAK cells IL-2+ 0.0 CI-H292 IL-9 0.0
IL-18
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LAK cells 0.0 NCI-H292 IL-13 ~ 0
0
PMA/ionomycin .
NK Cells IL-2 rest 0.0 NCI-H292 IFN gamma 0.0
Two Way MLR 3 day _ 0.0 HPAEC none 0.0
Two Way MLR 5 day 0.0 HPAEC TNF alpha + IL- 0.0
1 beta
Two Way MLR 7 day 0.0 Lung fibroblast none 0.0
PBMC rest 0.0 Leg fibroblast TNF 0
0
alpha + IL-1 beta .
PBMC PWM 0.0 Lung fibroblast IL-4 0.0
PBMC PHA-L 0.0 Lung fibroblast IL-9 0.0
Ramos (B cell) none0.0 Lung fibroblast IL-13 0.6
Ramos (B cell) 0.6 Lung fibroblast IFN 0.0
.
ionomycin . .. gamma ..
De~rira' fi br Vblast
B lymphocytes PWM 0.0 0.0
CCD1070 rest
~
B lymphocytes CD40L Dermal fibroblast
and IL-4 0'0 CCD1070 TNF alpha 0.6
EOL-l dbcAMP 0.0 Dermal fibroblast 0
0
CCD1070IL-1 beta .
EOL-1 dbcAMP 0.0 Dermal fibroblast IFN 0
0
PMAlionomycin....__.___.._______._._.___..._..._.~ga _.... ___._...
_._..~. _ ..... _._..__._.... __._ ._ _ ~.._. _ _-.___
_.. _ _ . _.
Dendritic cells 0.0 _ _ Dermal fibroblast 0.0
none_ IL-4 _
Dendritic cells 0.0 _ __ Dermal Fibroblasts 1.1
LPS rest
Dendritic cells 0.0 Neutrophils TNFa+LPS 1.4
anti-
CD40
Monocytes rest 0.0 Neutrophils rest 1.0
Monocytes LPS 0.0 Colon 2.2
Macrophages rest 0.0 Lung _11.3
~
Macrophages LPS 0.0 Thymus 10.7~~
HUVEC none 0.0 Kidney 100.0
HUVEC starved 0.0
General screening_panel v1.4 Summary: Ag4162 Highest expression of the
CG100104-O1 gene is detected exclusively in testis (CT=28.5). Therefore,
expression of
this gene could be used to distinguish testis sample from other samples used
in this panel.
In addition, therapeutic modulation of this gene product could be useful in
treatment of
testis related disorders such fertility and hypogonadism.
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In addition, low expression of this gene is also detected in Ovarian cancer
OVCAR-8 cell
line (CT--33.4). Therefore, therapeutic modulation of this protein product may
be useful in
the treatment of ovarian cancer.
Panel 4.1D Summary: Ag4162 Highest expression of the CG100104-O1 gene is
detected
in kidney (CT=29.9). Expression of this gene is exclusively seen in normal
lung, thymus
and kidney. Thus expression of this gene could be used to distinguish these
tissue samples
from other samples in this panel. In addition, therapeutic modulation of this
gene product
could be beneficial in the treatment of inflammatory or autoimmune diseases
that affect
lung and kidney.
B. CG56785-Ol: GTP:AMP PHOSPHOTRANSFERASE
MITOCHONDRIAL
Expression of gene CG56785-O1 was assessed using the primer-probe set Ag3036,
described in Table BA. Results of the RTQ-PCR runs are shown in Tables BB, BC
and BD.
Table BA. Probe Name Ag3036
_..~,.....,......._,..,.~..,..-.~..-.-...~..
Start SEQ ID
rimersSequences Length
positionNo
Forward5'-accaatggccaagtctacaac-3' 21 427 96
PI'ObeTET-5'-attggattcaaccctcccacaactgt-3'-'
26 448 97
Reyerse5'-gtttateatcctcacgetgaat-3' 22 505 98
Table BB. CNS neurodegeneration v1.0
-_ Rel. Ezp.(%) Ag3036,~ Rel. Exln_l/..1
Tissue Name Run 211012102 Tissue Nam A o~n36.
e Run 211012102
Control (Path)
AD 1 Hippo 3.3 3 5.8
Temporal Ctx
Control (Path)
AD 2 Hippo 4.9 4 11.0
_ _
Temporal Ctx
AD 3 Hippo 0.0 AD 1 Occipital_Ctx11.9
AD 4 Hippo 2.9 AD 2 Occipital__
Ctx 0.0
(Missing)
AD 5 Hippo 100.0 AD 3 Occipital0.0
Ctx
AD 6 Hippo 6.9 AD 4 Occipital9.2
Ctx
Control 2 4.2 AD S Occipital3.3
Hippo Ctx
Control 4 3.3 AD 6 Occipital1.9
Hippo Ctx
Control (Path)7.9 Control 1 Occipital0.0
3
Hippo
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AD 1 Temporal ~ 5.0 ~~ ntrol 2~~Occipital ~...~.....-..,
i ~...~.-... g
~ ~ C g
o
C C .
~
AD 2 Temporal 18.2 Control 3 Occipital ~ 5
1
Ctx Ctx .
AD 3 Temporal 3.3 Control 4 Occipital 0
0
Ctx Ctx .
AD 4 Temporal 11.0 Control (Path) 1 Sg
6
C~_ .__... ..._......._.._....___ _ _ _ _ . .... occipital .
_ _.._ _ ~.. ... Ctx. .._. .... . .__..._._ .___.__....
_ _.~ _.. . ... .._..._ ._
.. .._
__
AD 5 Inf Temporal Control (Path) 2 '
6'5 3
3
Cbc Occipital Ctx .
AD 5 Sup Control (Path) 3
40.3 4
6
Temporal Ctx Occipital Ctx .
AD 6 Inf Temporal Control (Path) 4
10.2 4
9
C~ Occipital Ctx .
AD 6 Sup Control 1 Parietal
~ 9'3 ~ 0
0
_. C~ .
Temporal_ Ctx
Control 1 _
0.0 Control 2 Parietal 32
3
Temporal Ctx C~ ~ .
Control 2 Control 3 Parietal
3' I 8
0
Temporal Ctx C~ .
Control 3 Control (Path) 1
2'2 43
8
Temporal Ctx Parietal Ctx .
Control 3 Control (Path) 2
8'S 5
3
Temporal Ctx ._ _ _ . Parietal Ctx .
_ __...._ _. ._ _. ...... _ . . .._ .. . . _ .._.
_ _. .... .... ...... . _ . . ...._
.. . ...... ..._.
Control (Path) Control (Path) 3
1 50'7 1
7
Temporal Ctx Parietal Ctae .
Control (Path) Control (Path) 4
2 4
6
Temporal Ctx ' Parietal Ctx 14.4
Table BC. Panel I.3D
Rel. Exp.(%) Rel. Ezp.(!)
Tissue Name Ag3036, Run Tissue Name Ag3036, Run
167962459 ~ 167962459
Liver adenocarcinoma0.0 Kidney (fetal) 8.1
Pancreas 0.0 Renal ca. 786-0 3.2
Pancreatic ca. 0.0 Renal ca. A498 0
CAPAN 0
2_.. ...... _ . .
_ _ .
_ .__.__......._.......__._....... . ... ..
Adrenal gland 0.0 ... _._ . 0.0
_..._........___..._.._.. Renal ca. RXF
__. _....__... 393
__ _.._ .__.____
._. _
~
__.. _.._ _ _ _ __
Thyroid 0.0 .._ 8.2
-.Renal ca. ACHN
~
Salivary._gland 0.0 _._ _
Renal c_a. U0-310.0
~
Pituitary gland 0.0 __ Renal ca. 0.0
TK-I O
Brain (fetal) 27.9 _ Liver _ 21.9 _
Brain (whole) 22.4 Liver (fetal) 38.4
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Brain (amygdala)~._ ~ 38.7 ' Liver ca. ~ ,~,~"""~"",_0_.0
....... ...."~,~.v,........._..............._.
(hepatoblast)
HepG2
Brain (cerebellum) 38.4 Lung 17.1
Brain (hippocampus) 4.7 Lung (fetal) 3.4
Brain (substantia 4,2 LX g ca. (small 0.0
nigra) cell)
Brain (thalamus) 19.8 Leg ca. (small p.0
cell)
NCI-H69
Lung ca. (s.cell
Cerebral Cortex 13.5 var.) 0
0
_ _._ _ . . ....... SHP-77_.
. . _ .. . _._ _ _. .. .. .. ._.._
... _ ... _
Spinal cord 0.0 Leg ca. (large 0.0
cell)NCI-H460
glio/astro U87-MG 0.0 Leg ca. (non-sm. 0.0
cell) A549
glio/astro U-118-MG 0.0 Leg ca. (non-s.cell)0.0
NCI-H23
Lung ca. (non-s.cell)
astrocytoma SW1783 ~ .0
. . _. ... ._._...~_~...HOP.-62_ .._._. ......_. ._..._..._.._.
_ . . _ . ..._. ....._ _._~
.. .._._ _. __
. . _ _
.
neuro*; met SK-N-AS 0.0 Leg ca. (non-s.cl)0.0
NCI-H522
astrocytoma SF-539 0.0 Leg ca. (squam.) 0.0
~
SW 900
astrocytoma SNB-75 0.0 Leg ca. (squam.) 0.0
NCI-H596
glioma SNB-19 0.0 Mammary gland 0.0
Breast ca.* (pl.ef)
glioma U251 0.0 0.0
.. _ . _ . . .._ _. MCF-7 ' _._._..._. _
. .. _. _ _ . .. ..__ . . _
. _._ _. . .
_.
Breast ca.* (pl.efj
glioma SF-295 0.0 0.0
~
MDA-MB-231
Heart (fetal) 0.0 Breast ca.* (nl.efl~,0
T47D
Heart 0.0 Breast ca. BT-5490.0
Skeletal muscle (fetal)2.7 Breast ca. MDA 0.0
N
Skeletal muscle 4.1 Ovary 0.0
Ovarian ca.
Bone marrow 100.0 0
0
OVCAR-3 .
Ovarian ca.
Thymus 0.0 0.0
OVCAR-4 __ _ .
Ovarian ca.
Spleen 31.2 0.0
OVCAR-5
Lymph node 0.0 ' ~ 0.0
OVC~ 8
Colorectal 0.0 Ovarian ca. IGROV-0
0
.
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Stomach 0.0 Ovanan ca.* 0.0
(ascites) SK-OV-3
Small intestine 0.0 Uterus 6.2
Colon ca. SW480 0.0 Placenta 0.0
Colon ca.* e
0.0 Prostat 0.0
SW620(SW480 met)
Colon ca. HT29 0.0 Prostate ca.* (bone 0.0
met)PC-3
Colon ca. HCT-116 0.0 Testis 0.0
Colon ca. CaCo-2 0.0 Melanoma 0.0
_. _._.._ ... ..__ _ ___ Hs688(A).T
_ . _ . _ _._ _ .. ._ __... .__... _. _...._._......~.._.. _..._.
__ . ... . ... . . . _
Colon ca. Melanoma* (met)
0 0
0 0
tissue(OD03866) ' Hs688(B).T .
Colon ca. HCC-2998 0.0 6 elanoma UACC- ~ 0.0
.
Gastric ca.* (liver 0 Melanoma M14 0
met) 0 0
NCI-N87 . .
Melanoma LOX
Bladder 0.0 0
0
_. . __. . _ ICI .
_ _._. _._ . _ .._ __ _ _.
_.. _
Trachea 3.5 Melanoma* (met) 0
0
SK-MEL-5 .
Kidney 0.0 Adipose ~9.8
Table BD. Panel 4D
Rel. Exp.(%) Rel. Exp.(%)
Tissue Name Ag3036, Run Tissue Name Ag3036, Run
162427947 162427947
Secondary Thl 0.0 HUVEC IL-lbeta 0.0
act
Secondary Th2 2.5 HUVEC IFN gamma 0.0
act ~
Secondary Trl 0.0 SEC TNF alpha + 0.0
act
IFN gamma
Secondary Thl 0.0 HUVEC TNF alpha 0.0
rest + IL4
Secondary Th2 0.0 HUVEC IL-11 0.0
rest
Lung Microvascular
Secondary Trl 1.7 EC 0.0
rest .
_ . . . _..... _. . _ _ ._. none. ... __ . _
.__. _. . _ _._._. _ ._ _
_
Primary Thl act 0.0 Leg Microvascular 0.0
EC
TNFalpha+ IL-lbeta.
Primary Th2 act 0.0 Microvascular Dermal0.0
EC none
Primary Trl act 4.4 Microsvasular Dermal0,0
EC
TNFalpha + IL-lbeta
Primary Thl rest0.0 Bronchial epithelium0.0
TNFalpha + ILlbeta
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Primary Th2 rest 0.0~ Small airway~.epithelium
none
Primary Trl rest . 0.0 Small airway epithelium 0
0
.
TNFalpha + IL-1 beta
CD45RA CD4
2.1 Coronery artery SMC rest 0.0
lymphocyte act
CD45R0 CD4 Coronery artery SMC
0
0
lymphocyte act ' TNFalpha + IL-lbeta 0.0
. _. _ _ ...._.... . . . ._ . __.. _._...
. _ _ _ ...._ ...___..
... .._. . _.
_
CD8 y._ .. O-0
lymphoc _ 1.'~ . ..
a ac_,_.. ... Astr'ocytes
_ .._ . _ _.... rest
. _. _.. _. ~ . .__. __. ._ __. _.. __ _ _.
_ t . .. .._ _ .__ .__
_
.
Secondary CD8 0 _
0 Astrocytes TNFalpha +
lymphocyte rest ' IL-lbeta 0.0
Secondary CD8
0.0 KU-812 (Basophil) rest 0.0
lymphocyte act
KU-812 (Basophil)
CD4 lymphocyte 0.0 0.0
none
p~ionomycin
2 Thl/Th2/TrI CCD1106
anti-
ry
.
0.0 0 0
CD95 CH11 eratinoc es none '
._ .. ._..._... _. .___.....
~.................._._........~.........~..........._.._..._......_.._.._._.._.
_
......_ _.. .... ... _.._._..._.... . . _. . ___._. _._..._ .
..._. ..._....._ _.._.... ... . .__.
.... ... ......
CCD1106
LAK cells rest 1.9 (Keratinocytes) 0.0
TNFalpha + IL-1 beta
LAK cells IL-2 0.0 Liver cirrhosis 1 S.5
LAK cells IL-2+IL-120.0 Lupus kidney 0.0
LAK cells IL-2+IFN0.0 NCI-H292 none . 0.0
gamma
LAK cells IL-2+ 0.0 NCI-H292 IL-4 0.0
IL-18
LAK cells 0.0 CI-H292 IL-9 0
0
1'
PMA/ionomyc_in .
. _ _
.
_.
NK Cells 0.0 NCI-H292
IL-2 rest IL-13
. 0.0
.
..
Two Way MLR 3 3.5 NCI-H292 IFN
day __ __. . .._ gamma
_._____....... . __.__. . 0.0
._._.. ... .... .._._.._..... _._ _._. . ...
. .__._ . __ . ..__ ._._. .__. _.. . .__..
. _._. ___ .. _ .. - __ ....__
Two Way MLR 5 0.0 HPAEC none
day
Two Way MLR 7 2.2 HPAEC TNF alpha + IL- 0.0
day
1 beta
PBMC rest 12.3 Lung fibroblast none 0.0
PBMC PWM 0.0 Leg fibroblast TNF 0
0
.
alpha + IL-1 beta
PBMC PHA-L 3.0 Lung fibroblast IL-4 0.0
Ramos (B cell) 0.0 Lung fibroblast IL-9 0.0
none
Ramos (B cell)
0.0 Lung fibroblast IL-13 0.0
ionomycin
Lung fibroblast IFN
B lymphocytes 11.0 0.0
PWM
._ . . _. _ . ga _ _ _. .. _
. __ . . __
B lymphocytes Dermal fibroblast
CD40L
and IL-4 1$'3 CCD1070 rest 0.0
EOL-1 dbcAMP 0.0 Dermal fibroblast 1.1
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_ "~,~,-"""~",~" C_CD_107_0 TNF
alpha
EOL-1 dbcAMP Dermal fibroblast
PMAiionomycin 0'0 CCD1070IL-1 beta 0.0
Dendritic cells 0.0 g~~ fibroblast IFN p.0
none
Dendritic cells 0.0 Dermal fibroblast 0.0
LPS IL-4
Dendritic cells
anti-
0,0 IBD Colitis 2 0.0
CD40
Monocytes rest 100.0 IBD Crohn's 0.0
Monocytes LPS 14.0 Colon 0.0
Macrophages rest 0.0 Lung 10.7
Macrophages LPS 2.3 Thymus 1.1
HI1VEC none 0.0 Kidney 3.1
HUVEC starved 0.0
CNS ~eurodegeneration v1.0 Summary: Ag3036 T'nis panel does not show
differential
expression of the CG56785-Ol gene in Alzheimer's disease. However, this
expression
profile does show the presence of this gene in the brain. Therefore,
therapeutic modulation
of the expression or function of this gene may be useful in the treatment of
neurologic
disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia,
multiple
sclerosis, stroke and epilepsy.
Panel 1.3D Summary: Ag3036 Expression of the CG56785-Ol gene is exclusive to
bone
marrow (CT=34.6). This gene encodes a putative member of the adenylate kinase
family,
which has been shown to be down-regulated in various blood disorders (Walter
HD, Klin
Wochenschr 1978 May 15;56(10):483-91). Thus, expression of this gene could be
used to
differentiate between this sample and other samples on this panel and as a
marker of bone
marrow and red blood cells. Furthermore, therapeutic modulation of the
expression or
function of this gene may be useful in the treatment of blood disorders and
leukemias.
Panel 4D Summary: Ag3036 Expression of the CG56785-O1 gene is exclusive to
resting
monocytes (CT=33.3). This expression is in agreement with expression in Panel
1.3D. The
expression of this gene in resting cells of this lineage suggests that the
protein encoded by
this transcript may be involved in normal immunological processes associated
with
immune homeostasis.
C. CG56914-O1: Thrombospondin
201
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 201
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
brevets
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VOLUME
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CONTAINING PAGES 1 TO 201
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
