Note: Descriptions are shown in the official language in which they were submitted.
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CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
THERAPEUTIC POLYPEPTIDES, NUCLEIC ACIDS ENCODING
SAME, AND METHODS OF USE
FIELD OF THE INVENTION
The present invention relates to novel polypeptides, and the nucleic acids
encoding
S them, having properties related to stimulation of biochemical or
physiological responses in
a cell, a tissue, an organ or an organism. More particularly, the novel
polypeptides are
gene products of novel genes, or are specified biologically active fragments
or derivatives
thereof. Methods of use encompass diagnostic and prognostic assay procedures
as well as
methods of treating diverse pathological conditions.
CA 02486490 2004-12-03
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BACKGROUND OF THE INVENTION
Eukaryotic cells are characterized by biochemical and physiological processes,
which under normal conditions are exquisitely balanced to achieve the
preservation and
propagation of the cells. When such cells are components of multicellular
organisms such
as vertebrates or, more particularly, organisms such as mammals, the
regulation of the
biochemical and physiological processes involves intricate signaling pathways.
Frequently, such signaling pathways involve 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 pathologies the dysregulation is
manifested as
diminished or suppressed Ievel of synthesis and secretion of protein
effectors. In other
classes of pathologies the dysregulation is manifested as increased or up-
regulated level of
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synthesis and secretion of protein effectors. In a clinical setting a subject
may be
suspected of suffering from a condition brought on by altered or mis-regulated
levels of a
protein effector of interest. Therefore there is a need to assay for the level
of the protein
effector of interest in a biological sample from such a subject, and to
compare the level
with that characteristic of a nonpathological condition. There also is a need
to provide the
protein effector as a product of manufacture. Administration of the effector
to a subject in
need thereof is useful in treatment of the pathological condition.
Accordingly, there is a
need for a method of treatment of a pathological condition brought on by a
diminished or
suppressed levels of the protein effector of interest. In addition, there is a
need for a
method of treatment of a pathological condition brought on by a increased or
up-regulated
levels of the protein effector of interest.
Antibodies are multichain proteins that bind specifically to a given antigen,
and
bind poorly, or not at all, to substances deemed not to be cognate antigens.
Antibodies
are comprised of two short chains termed light chains and two long chains
termed heavy
15. chains. These chains are constituted of immunoglobulin domains, of which
generally
there are two classes: one variable domain per chain, one constant domain in
light chains,
and three or more constant domains in heavy chains. The antigen-specific
portion of the
immunoglobulin molecules resides in the variable domains; the variable domains
of one
light chain and one heavy chain associate with each other to generate the
antigen-binding
moiety. Antibodies that bind immunospecifically to a cognate or target antigen
bind with
high affinities. Accordingly, they are useful in assaying specifically for the
presence of
the antigen in a sample. In addition, they have the potential of inactivating
the activity of
the antigen.
Therefore there is a need to assay for the level of a protein effector of
interest in a
biological sample from such a subject, and to compare this level with that
characteristic of
a nonpathological condition. In particular, there is a need for such an assay
based on the
use of an antibody that binds immunospecifically to the antigen. There 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.
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S~JIVE1~IARY OF THE INVENTION
The invention is based in part upon the discovery of isolated polypeptides
including amino acid sequences selected from mature forms of the amino acid
sequences
selected from the group consisting of SEQ ID N0:2n, wherein n is an integer
between 1
and I41. The novel nucleic acids and polypeptides are referred to herein as
NOVX, or
NOVl, 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.
The invention also is based in part upon variants of a mature form of the
amino
acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is
an
integer between 1 and 141, wherein any amino acid in the mature form is
changed to a
different amino acid, provided that no more than I 5% of the amino acid
residues in the
sequence of the mature form are so changed. In another embodiment, the
invention
includes the amino acid sequences selected from the group consisting of SEQ ID
N0:2n,
wherein n is an integer between 1 and 141. In another embodiment, the
invention also
comprises variants of the amino acid sequence selected from the group
consisting of SEQ
ID N0:2n, wherein n is an integer between I and 141, 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
involves
fragments of any of the mature forms of the amino acid sequences selected from
the
group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 141,
or any
other amino acid sequence selected from this group. The invention also
comprises
fragments from these groups in which up to I S% of the residues are changed.
In another embodiment, the invention encompasses polypeptides that are
naturally
occurring allelic variants of the sequence selected from the group consisting
of SEQ ID
NO:2n, wherein n is an integer between 1 and 141. These allelic variants
include amino
acid sequences that are the translations of nucleic acid sequences differing
by a single
nucleotide from nucleic acid sequences selected from the group consisting of
SEQ ID
NOS: 2n-1, wherein n is an integer between 1 and 141. The variant polypeptide
where
any amino acid changed in the chosen sequence is changed to provide a
conservative
substitution.
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In another embodiment, the invention comprises a pharmaceutical composition
involving a polypeptide with an amino acid sequence selected from the group
consisting of
SEQ ID NO:2n, wherein n is an integer between 1 and 141, and a
pharmaceutically
acceptable carrier. In another embodiment, the invention involves a kit,
including, in one
or more containers, this pharmaceutical composition.
In another embodiment, the invention includes the use of a therapeutic in the
manufacture of a medicament for treating a syndrome associated with a human
disease,
the disease being selected from a pathology associated with a polypeptide with
an amino
acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is
an
integer between 1 and 141, wherein said therapeutic is the polypeptide
selected from this
group.
In another embodiment, the invention comprises a method for determining the
presence or amount of a polypeptide with an amino acid sequence selected from
the group
consisting of SEQ ID N0:2n, wherein n is an integer between I and 141, in a
sample, the
method involving providing the sample; introducing the sample to an antibody
that binds
immunospecifically to the polypeptide; and determining the presence or amount
of
antibody bound to the polypeptide, thereby determining the presence or amount
of
polypeptide in the sample.
In another embodiment, the invention includes a method for determining the
presence of or predisposition to a disease associated with altered levels of a
polypeptide
with an amino acid sequence selected from the group consisting of SEQ ID
NO:2n,
wherein n is an integer between 1 and 141, in a first mammalian subject, the
method
involving measuring the level of expression of the polypeptide in a sample
from the first
mammalian subject; and comparing the amount of the polypeptide in this sample
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, wherein 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 embodiment, the invention involves a method of identifying an agent
that binds to a polypeptide with an amino acid sequence selected from the
group
consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141, the
method
including introducing the polypeptide to the agent; and determining whether
the agent
binds to the polypeptide. The agent could be a cellular receptor or a
downstream effector.
CA 02486490 2004-12-03
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In another embodiment, the invention involves a method fox identifying a
potential
therapeutic agent for use in treatment of a pathology, wherein the pathology
is related to
aberrant expression or aberrant physiological interactions of a polypeptide
with an amino
acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is
an
integer between 1 and 141, the method including providing a cell expressing
the
polypeptide of the invention and having a property or function ascribable to
the
polypeptide; contacting the cell with a composition comprising a candidate
substance; and
determining whether the substance alters the property or function ascribable
to the
polypeptide; whereby, if an alteration observed in the presence of the
substance is not
observed when the cell is contacted with a composition devoid of the
substance, the
substance is identified as a potential therapeutic agent.
In another embodiment, the invention involves a method for screening for a
modulator of activity or of latency or predisposition to a pathology
associated with a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID
N0:2n, wherein n is an integer between 1 and 141, the method including
administering a
test compound to a test animal at increased risk for a pathology associated
with the
polypeptide of the invention, wherein the test animal recombinantly expresses
the
polypeptide of the invention; measuring the activity of the polypeptide in the
test animal
after administering the test compound; and comparing the activity of the
protein in the test
animal with the activity of the polypeptide in a control animal not
administered the
polypeptide, wherein a change in the activity of the polypeptide in the test
animal relative
to the control animal indicates the test compound is a modulator of latency
of, or
predisposition to, a pathology associated with the polypeptide of the
invention. The
recombinant test animal could express a test protein transgene or express the
transgene
under the control of a promoter at an increased level relative to a wild-type
test animal The
promoter may or may not b the native gene promoter of the transgene.
In another embodiment, the invention involves a method for modulating the
activity of a polypeptide with an amino acid sequence selected from the group
consisting
of SEQ ID N0:2n, wherein n is an integer between 1 and 141, the method
including
introducing a cell sample expressing the polypeptide with a compound that
binds to the
polypeptide in an amount sufficient to modulate the activity of the
polypeptide.
In another embodiment, the invention involves a method of treating or
preventing a
pathology associated with a polypeptide with an amino acid sequence selected
from the
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group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141,
the
method including administering the polypeptide to a subject in which such
treatment or
prevention is desired in an amount sufficient to treat or prevent the
pathology in the
subject. The subject could be human.
In another embodiment, the invention involves a method of treating a
pathological
state in a mammal, the method including administering to the mammal a
polypeptide in an
amount that is sufficient to alleviate the pathological state, wherein the
polypeptide is a
polypeptide having an amino acid sequence at least 95% identical to a
polypeptide having
the amino acid sequence selected from the group consisting of SEQ ID NO:2n,
wherein n
is an integer between 1 and 141, or a biologically active fragment thereof.
In another embodiment, the invention involves an isolated nucleic acid
molecule
comprising a nucleic acid sequence encoding a polypeptide having an amino acid
sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ ID NO:2n, wherein n is an integer between 1 and 141; a variant of a
mature
I S form of the amino acid sequence selected from the group consisting of SEQ
ID NO:2n,
wherein n is an integer between 1 and 141, wherein any amino acid in the
mature form of
the chosen sequence is changed to a different amino acid, provided that no
more than 15%
of the amino acid residues in the sequence of the mature form are so changed;
the amino
acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is
an
integer between Land 141, a variant of the amino acid sequence selected from
the group
consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141, in
which any
amino acid specified in the chosen sequence is changed to a different amino
acid, provided
that no more than 15% of the amino acid residues in the sequence are so
changed; a
nucleic acid fragment encoding at least a portion of a polypeptide comprising
the amino
acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is
an
integer between 1 and 141, or any variant of the polypeptide wherein any amino
acid of
the chosen sequence is changed to a different amino acid, provided that no
more than 10%
of the amino acid residues in the sequence are so changed; and the complement
of any of
the nucleic acid molecules.
In another embodiment, the invention comprises an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide comprising an amino acid
sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ ID N0:2n, wherein n is an integer between 1 and 141, wherein the
nucleic acid
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molecule comprises the nucleotide sequence of a naturally occurring allelic
nucleic acid
variant.
In another embodiment, the invention involves an isolated nucleic acid
molecule
including a nucleic acid sequence encoding a polypeptide having an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given
SEQ ID NO:2n, wherein n is an integer between 1 and 141, that encodes a
variant
polypeptide, wherein the variant polypeptide has the polypeptide sequence of a
naturally
occurring polypeptide variant.
In another embodiment, the invention comprises an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide comprising an amino acid
sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ ID NO:2n, wherein n is an integer between 1 and 141, wherein the
nucleic acid
molecule differs by a single nucleotide from a nucleic acid sequence selected
from the
group consisting of SEQ ID NOS: 2n-1, wherein n is an integer between 1 and
141.
In another embodiment, the invention includes an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide including an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given
SEQ ID N0:2n, wherein n is an integer between 1 and 141, wherein the nucleic
acid
molecule comprises a nucleotide sequence selected from the group consisting of
the
nucleotide sequence selected from the group consisting of SEQ ID NO:2n-1,
wherein n is
an integer between 1 and 141, a nucleotide sequence wherein one or more
nucleotides in
the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1,
wherein
n is an integer between 1 and 141, 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; a nucleic acid fragment of the sequence selected
from the
group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141,
and a
nucleic acid fragment wherein one or more nucleotides in the nucleotide
sequence selected
from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1
and
141, 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.
In another embodiment, the invention includes an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide including an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given
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SEQ ID N0:2n, wherein n is an integer between 1 and I41, wherein the nucleic
acid
molecule hybridizes under stringent conditions to the nucleotide sequence
selected from
the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and
14I, or a
complement of the nucleotide sequence.
In another embodiment, the invention includes an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide including an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given
SEQ ID N0:2n, wherein n is an integer between 1 and 141, wherein the nucleic
acid
molecule has a nucleotide sequence in which any nucleotide specified in the
coding
sequence of the chosen nucleotide sequence is changed from that selected from
the group
consisting of the chosen sequence to a different nucleotide provided that no
more than
1 S% of the nucleotides in the chosen coding sequence are so changed, an
isolated second
polynucleotide that is a complement of the first polynucleotide, or a fragment
of any of
them.
In another embodiment, the invention includes a vector involving the nucleic
acid
molecule having a nucleic acid sequence encoding a polypeptide including an
amino acid
sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ ID N0:2n, wherein n is an integer between 1 and 141. This vector can
have a
promoter operably linked to the nucleic acid molecule. This vector can be
located within a
cell.
In another embodiment, the invention involves a method for determining the
presence or amount of a nucleic acid molecule having a nucleic acid sequence
encoding a
polypeptide including an amino acid sequence selected from the group
consisting of a
mature form of the amino acid sequence given SEQ ID N0:2n, wherein n is an
integer
between 1 and 141, in a sample, the method including providing the sample;
introducing
the sample to a probe that binds to the nucleic acid molecule; and determining
the
presence or amount of the probe bound to the nucleic acid molecule, thereby
determining
the presence or amount of the nucleic acid molecule in the sample. The
presence or
amount of the nucleic acid molecule is used as a marker for cell or tissue
type. 'The cell
type can be cancerous.
In another embodiment, the invention involves a method for determining the
presence of or predisposition for a disease associated with altered levels of
a nucleic acid
molecule having a nucleic acid sequence encoding a polypeptide including an
amino acid
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sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ ID N0:2n, wherein n is an integer between 1 and 141, in a first
mammalian
subject, the method including measuring the amount of the nucleic acid in a
sample from
the first mammalian subject; and comparing the amount of the nucleic acid in
the sample
of step (a) to the amount of the nucleic acid present in a control sample from
a second
mammalian subject known not to have or not be predisposed to, the disease;
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.
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 compounds. The sequences are
collectively
referred to herein as "NOVX nucleic acids" or "NOVX polynucleotides" and the
corresponding encoded polypeptides are referred to as "NOVX polypeptides" or
"NOVX
proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the
novel
sequences disclosed herein. Table A provides a summary of the NOVX nucleic
acids and
their encoded polypeptides.
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TABLE A. Sequences and Corresponding SEQ ID Numbers
SEQ SEQ ID
ID
NOVX Internal NO NO gomology
AssignmentIdentification(nucleic(amino
acid) acid)
NOVla CG103945-021 2 Semaphorin sem2 (FLJ00014
protein) -
Homo Sapiens
_....._...._.... _......._..__.._ _....___...___......__.........._...___.
._.._.......___...
.. ....._.._.__..___ ....._ ____......_.
._..____..__.._.._._._......_.._._..._..
..__....__ ... ._...........__.._._._._.._.
....... _
NOVlb CG103945-O13 4 Semaphorin sem2 (FLJ00014
protein) -
Homo Sapiens
,
NOV2a CG106951-Ol5 6 Human semaphorin G-like
~ ~ ~ NHP protein
~
_ CG106951-047 _ 8 H__uman__se_maph_orin_G-like
NOV2b Y NH_P protein
OV2c 209829549 9 10 Human semaphorin G-like
NHPprotein
NOV2d _ 20982955311 12 Human semaphorin G-like
NHP protein
2e _ 209829 13_ 14 _ Human se_map_horin_G-like
NO 642 ~ -~ NHP protein
V
_ _ 15 x16 ;Human semaphorin G-like
_ 209829670 _ _ ~._. NHP protein
NOV2f~ ~ ., _ ~, =, _ ._~. __._.~..,"_..._
._,_.
NOV2g _: ~,~ , ....3 Human semaphorin
. ,. . , CG106951-02.. ' 18 ,... G-like,NHP protein,
,. .. .,.. 17. . ........
. .,
.,
.
NOV2h CG_ 106951-03_ 19 20 Human semaphorin G-like
_ -~ ~ NHP protein
n
NOV2i SNP13382456_ ~2- ~ Human semaphorin G-like
_21 NHP protein
NOV3a CG121295-01~ 24 Endothelia-1 precursor
23 (ET-1) -Homo
_ .. _. ._.. _ .. saPiens,_. .. . ...
. _ ....
.
NOV4a CG124756-O1 26 complement subcomponent
Clq chain
25 _B precursor [validated]
_
NOV4b CG124756-0227 28 complement subcomponent
Clq chain
B precursor [validated]
NOV4c SNP1338247529 30 complement subcomponent
Clq chain
_ _ - B precursor [validated]
~
OV4d SNP13382476,31 32 complement subcomponent
4 Clq chain
_ .._ .... . . _............ B. precursor. [validated]...]...
_ .. _ .... ...... . . . .
. ~~ ~
OVSa CG50353-Ol 33 34 Wnt-7a
protein precursor - Homo
Sapiens
~c
NOVSb 228753443 35 36 Wnt-7a protein precursor
~ - Homo
Sapiens
NOVSc 169475673 37 38 Wnt-7a protein precursor
- Homo
Sapiens _ _ _
~
NOVSd 228753459 39 40 Wnt-7a protein precursor
- Homo
_... ....~.........__.__._...._,._,......_..__......,.....__...~ _ ... _
Sapiens ......_ ._.._....._.._.._......._..._..,.....,__..__..._......_._..,
.._ . . . ...
..
...._...
OVSe 228753462 ~ 41 42 Wnt-7a protein precursor
- Homo
Sapiens _
NOVSf 228753446 43 44 Wnt-7a protein precursor
- Homo
Sapiens
NOVSg 228753465 45 46 Wnt-7a protein precursor
- Homo
_ _ Sapiens _ _ _
- ~V
NOVSh 228753438 47 48 Wnt-7a protein precursor
- Homo
Sapiens
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NOVSi 228753449 49 50 Wnt-7a protein precursor
- Homo
sapiens _......_._.._..._.._.__..._.._._.....
NOVSj CG50353-02 51 52 Wnt-7a protein precursor
- Homo
Sapiens
OVSk CG50353-03 53 54 Wnt-7a protein precursor
- Homo
v Sapiens
NOV51 SNP13382474 55 56 Wnt-7a protein precursor
~ - Homo
_ _ __ _ sapie_ns _ _
- v ~ ~
~
OV6a CG50709-03 57 58 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo Sapiens
NOV6b 282997951 59 60 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo Sapiens
NOV6c CG50709-OS 61 62 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo Sapiens
OV6d 277582109 63 64 Wnt-9b protein precursor
~ (Wnt-15)
_.. _. . . ........_.. . (Wnt-14b).-Homo Sapiens.
_
NOV6e 277582117 65 66 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Horno Sapiens
NOV6f CG50709-O1 67 68 Wnt-9b protein precursor
(Wnt-15)
_ ; (Wnt-14b) - Homo Sapiens
~
OV6g CG50709-02 69 70 Wnt-9b protein precursor
. (Wnt-15)
(Wnt-14b) - Homo sapiens
NOV6h CG50709-04 71 72 Wnt 9b protein precursor
(Wnt-15)
(Wnt 14b) Homo Sapiens
~
NOV6i CG50709-06 73 74 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo sapiens
NOV6j CG50709-07 75 76 Wnt-9b protein precursor
(Wnt-15)
~ . ~ (Wnt-14b) - Homo sapien_s
~~
OV6k SNP13381605 77 78 a Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo Sapiens
Y
OV61 SNP13381606 79 80 Wnt-9b protein precursor
(Wnt-15)
_ (Wnt-14b) - Homo Sapiens
~
NOV6m SNP13378337 81 82 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo sapiens
NOV6n SNP13381607 83 84 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo sapiens
NOV6o SNP13378336 85 86 Wnt-9b protein precursor
(Wnt-15)
(Wn_t-14b) -_Homo Sapiens
NOV6p SNP13378335 87 88 Wnt-9b protein precursor
(Wnt-15)
(Wnt-14b) - Homo sapiens
~
NOV7a CG53054-02 89 90 Wnt-9a protein precursor
(Wnt-14) -
Homo Sapiens
NOV7b 170251039 91 92 Wnt-9a protein precursor
(Wnt-14) -
Homo Sapiens
NOV7c 170251076 93 94 Wnt-9a protein precursor
(Wnt-14) -
Homo sapiens
NOV7d CG53054-Ol 95 96 Wnt-9a protein precursor
(Wnt-14) -
Homo sapiens
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NOV7e CG53054-03 , 97 98 Wnt-9a proteinprecursor
(Wnt-14) -
H_omo Sapiens
NOV7f CG53054-04 99 100 Wnt-9a protein precursor
(Wnt-14) -
Homo sapiens
~ F
OVBa CG53473-02 101 102 Neuromedin B-32 precursor
[Contains:
euromedin B] - Homo Sapiens
NOV8b CG53473-Ol 103 104 Neuromedin B-32 precursor
[Contains:
_ __ _ N_euromedin B] - Homo sapiens
T ~ _
NOV8c CG53473-03 105 106 euromedin B-32 precursor
~~ [Contains:
Neuromedin B] - Homo Sapiens
OV8d SNP13376396 107 108 Neuromedin B-32 precursor
[Contains:
_ Neuromedin B] - Homo Sapiens
~
NOV8e SNP13376395 109 110 Neuromedin B-32 precursor
[Contains:
Neuromedin B] - Homo Sapiens
NOV8f SNP13376394 111 112 Neuromedin B-32 precursor
[Contains:
euromedin B] - Homo Sapiens
.
NOV9a CG55184-03 113 114 Cerebellin-like glycoprotein
l precursor
- Homo Sapiens
NOV9b CG55184-O1 115 116 Cerebellin-like glycoprotein
1 precursor
- Homo Sapiens
N
NOV9c CGSSI84-02 117 118 Cerebellin-like glycoprotein
1 precursor
- Homo Sapiens
NOV9d CG55184-04 119 120 Cerebellin-like glycoprotein
1 precursor
- Homo Sapiens
y
NOV9e CG55184-OS 121 122 Cerebellin-like glycoprotein
1 precursor
- Homo Sapiens
NOVlOa CG55274-OS 123 124 Human endozepine-like ENDOS
NOVlOb CG55274-01 125 126 Human endozepine-like ENDOS
_. ... .....~_
~.._..................._..............._.........._...._................ _
......_...._..._........_...._........_..._._....._....
...... .. .....
....,....._......_.............._........._............_.__..,_...v............
.... _.... .........~..._.._
_.
NOVlOc CG55274_-02 3_127 128 Human e_ndozepi_n_e-Like
E_NDO_5
--
NOVlOd CG55274-03 '_129 ' 130 Human endozepine-like ENDOS
NOVlOe CG55274-04 131 132 Human endozepine-like ENDOS
_ _ A -
~
NOVl CG55379-04 ;133 ' 134 HDDM36 -_Hom_o s_apiens
la ~
~ i
NOVl CG55379-O1 135 _136 HDDM36 - Homo Sapiens
lb A
NOVllc 258065951 137 138 HDDM36 -Homo Sapiens
OVl_ld 209886264 139 140 _HDDM36 - Homo Sapiens
~
NOVl 209886345 141 142 HDDM36 - Homo sapiens
le
OVl 209886357 143 144 HDDM36 - Homo Sapiens
if
NOVllg C_G55379-02 145 146 HDDM36 -Homo Sapiens _
_ - y
~
NOVllh CG55379-03 147 148 HDDM36 -Homo Sapiens
'
NOVl2a CG55688-Ol 150 CYR61 protein precursor
(Cysteine-rich, angiogenic
inducer, 61)
149 (Insulin-like growth factor-binding
protein 10) (GIGl protein)
- Homo
Sapiens
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NOVl2b 254087906 152 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
151 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
_ Sapiens
NOVI2c 259278648 154 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
153 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
__ Sapiens
~
NOVl2d 259280032 CYR61 protein precursor
156
(Cysteine-rich, angiogenic inducer,
61)
155 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens
NOVl2e 254756530 158 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
I57 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens
NOVl2f 229509618 160 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
159 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens
NOVl2g 229509658 162 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
161 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens _
J
NOVl2h CG55688-02 164 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
163 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens
NOVl2i CG55688-03 166 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
165 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens
NOVl2j CG55688-04 168 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
167 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens
NOVl2k CG55688-05 170 CYR61 protein precursor
(Cysteine-rich, angiogenic inducer,
61)
169 (Insulin-like growth factor-binding
protein 10) (GIGl protein) - Homo
Sapiens
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:VOV121 CG55688-06 172 CYR61 protein precursor
~ ~~
( Cysteine-rich, angiogenic
inducer, 6I)
1 71 ( Insulin-like growth factor-binding
p rotein 10) (GIGl protein)
~ - Homo
_ _ _ _ ~_ ____._...apiens ~ _~_.~___.,~..~
__ __._.._.._~,_
.s
.___ __....___.__- _._. 174 CYR61 protein precursor
. ..__
lVOVl2m SNP13376428
( Cysteine-rich, angiogenic
inducer, 61)
173 (Insulin-like growth
factor-binding
protein 10) (GIGl protein)
- Homo
Sapiens _
NOVl3a CG56768-O1 176 Wnt-5a protein precursor-Homo
175
Sapiens
NOVlab CG56768-02 178 Wnt-Saproteinprecursor-Homo
177
__ _. . __._..__ .._.._Sa1?iens_.._..._...__.._..
......_._...._.__. ... .....___.._ ____.__. ....._...__......
.____._. ... .._._ .. . ._.......__._._._.
. ..
_..__. _ 180 Wnt-Sa protein precursor
. . ___..._CG56768-03 - Homo
___
NOV 13c
179
Sapiens
NOVl4a CG57054-03 182 rotein precursor (Wnt-12)
-
Wnt-lOb
181 p
,_._.__.. ..._._.__.._Homo sa.....lens,...._._..._._
_.__
_.._._.___..___CG57054-Ol 184 Wnt-lOb protein precursor
NOV 14b (Wnt-12) -
183 Homo Sapiens
NOV l4c CG57054-02 186 Wnt-l Ob protein precursor
(Wnt-12) -
185
__.._...______._Homo Sapiens
.._ ...........__.........
__ _._.
_.... ___
...........____. _...
_._..___
NOVlsa _ ___. ._._....._.. >
7431 03 .. (
CGS - 188 p
m-2 recursor ET-2 - Homo
Endothel-
7
18
sapiens
NOV l CG57431-OZ 190 Endothelin-2 precursor
Sb (ET-2) - Homo
189
Sapiens
J
NOVlsc CG57431-O1 192 (ET-2) - Homo
Endothelin-2 precursor
191
Sapiens
NOVlsd CG57431-04 194 Endothelin-2 precursor
(ET-2) - Homo
193
Sapiens
_......._.....__.._._._._......_._.__._......._....__.._....._........._._..._.
_..._.__.._____.._...._....._....._...._........____._......
....... .__.._..._. ............
_..__....__..._.........__
_.
_.._ ......_........._..._..._.._..___.__ ... 196 Semaphorin 6D
isofonn
NOVl6a ... 2 - Homo
CG59253-Ol
195
Sapiens
NOVl6b 194877881 198 Semaphorin 6D isoform
2 - Homo
197
Sapiens
NOVl6c CG59253-02 200 Semaphorin 6D isoform
2 - Homo
199
Sapiens
NOVl6d 191815765 202 Semaphorin 6D isoform
2 - Homo
201
_.. . _ .. .. _..... Sapiens w.._. .
_..._...._............_.........,...._..
. . . . _ .......__..._...__..._......._......_..............
.. . . . .. .
OV 16e CG59253-03 204 3 Semaphorin 6D isofonn
2 - Homo
203
'
Sapiens
OV 16f CG59253-04 206 Semaphorin 6D isoform
2 - Homo
205
_._... Sapiens _ .. . _ .. _
_ _.. .
- CG59253-OS 208 Semaphorin 6D isoform
NOV 16g 2 - Homo
207
Sapiens
NOV 16h CG59253-06 210 Semaphorin 6D isoform
2 - Homo
209
sa iens
. .. _ _. ..... __...._......_._....__..._.p
. . . . . . .. ..__..-_......_._....-...._ _.,...._.......
___. __ ..~, _.........._.....,._.._
. . ..... _.._..__~...._.
_ _.
___...
_._:..._ - 2 i i .,
NOVl6i CG59253 212 p
07 Sema horm 6D isoform
2 Homo
-
Sapiens
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NOVI6j CG59253-08 214 Semaphorin 6D isoform 2
- Homo
213
Sapiens
NOV CG59253-09 216 Semaphorin 6D isoform 2
16k - Homo
215
Sapiens
NOV161 CG59253-10 218 Semaphorin 6D isoform 2
- Homo
217
_ Sapiens
NOVl6m SNP13381547 220 Semaphorin 6D isoform 2
-Homo
219
~_. _ ____.._.__._. .._._ ___.... _Sapiens.__.._.__.._..__._...
._._ _. _..__ __ _ _. _.__..__... ..,,~,.~___.._.__._
.__ ... ... .. ._.._~
.. ._ _..._.____
_._
_
NOV SNP13378936 222 Semaphorin 6D isoform 2
16n - Homo
221
Sapiens
f
NOVl6o SNP13378935 224 Semaphorin 6D isoform 2
-Homo
223
sapiens _
~
NOVl6p SNP13381569 226 ~Semaphorin 6D isoform 2
-Homo
225
Sapiens
NOVl6q SNP13382528 228 Semaphorin 6D isoform 2
- Homo
227
Sapiens
NOV CG_95430-02 229 230 Energen-related secreted
17a protein - C2P
NOV CG95430-04 231 232 Energen-related secreted
17b protein - C2P
NOVl7c CG95430-Ol 233 234 Energen-related_secreted
protein - C2P
NOV 319194717 235 236 _ Energen-related secreted
17d protein - C2P
NOVl7e CG95430-03 237 238 Energen-related secreted
protein - C2P
NOV CG95430-05 239 240 Energen-related secreted
17f ~ protein - C2P
NOVl7g CG95430-06 241 242 Energen-related secreted
_ _ protein - C2P
~ ~ ~
y4
NOV CG95430-07 24 244_,~~sec_reted protein - C2P
17h i 3 , Energen-relate_d
NOV CG95430-08 _ _ 246 Energen-related secreted
17i , protein - C2P
245
NOVl7j CG95430-09 247 248 Energen-related_secreted
protein - C2P
~
~
NOVI7k CG95430-10 249 250 '_Ener_g_en-rela_t_ed secreted
X ~~ protein - C2P
_
NOV CG95430-11 251 252 1 Energen-related secreted
171 protein - C2P
~
OV 17m CG95430-12 253 254 E_nergen-re_lat_ed_secreted
~ ~ protein - C2P
NOVl7n RCG95430-13 255 256_~ ~Energen-related secreted
p_rot_ein - C2P
NOV SNP13379412 257 258 ~ Energen-related secreted
17o protein - C2P
OVl7p SNP13381828 259 260 Energen-related secreted
protein - C2P
NOVl7q SNP13379125 261 262 Energen-related secreted
protein - C2P
~
NOVl7r SNP13381827 263 264 Energen-related secreted
protein - C2P
OV 17s SNP13381822 265 266 Energen-related secreted
protein - C2P
NOVl7t ~SNP13381826 267 268 Energen-related secreted
~ protein - C2P
NOVlBa CG97111-Ol 269 270 Human IL-1 receptor antagonist
protein
NOVlBb CG97111-02 271 272 Human IL-1 receptor antagonist
protein
NOVl8c CG9711 I-03 273 274 Human IL-1 receptor antagonist
_.. ~., . _. _.... ......._.__.protein
_. __ ._ ....._..... _..... ._
_ . _.. . _.._.. . _._..
_ . .
...
NOVl8d SNP13382516 275 276 Human IL-1 receptor antagonist
protein
NOVl8e SNP13382517 277 278 Human IL-1 receptor antagonist
_ protein
NOVlBf SNP13382518 279 280 Human IL-1 receptor antagonist
protein
NOVl9a 10132038Ø67281 282 Domain of CG50513-05
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Table A 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 A
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 A.
Pathologies, diseases, disorders, conditions and the like that are associated
with
NOVX sequences include, but are not limited to, e.g., cardiomyopathy,
atherosclerosis,
hypertension, congenital heart defects, aortic stenosis, atrial septal defect
(ASD),
atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis,
subaortic
stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis,
scleroderma,
obesity, metabolic disturbances associated with obesity, transplantation,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer,
diabetes, metabolic
disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility,
hemophilia,
hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies,
graft versus
host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis,
treatment of
Albright Hereditary Ostoeodystrophy, infectious disease, anorexia, cancer-
associated
cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's
Disorder, immune disorders, hematopoietic disorders, and the various
dyslipidemias, the
metabolic syndrome X and wasting disorders associated with chronic diseases
and various
cancers, as well as conditions such as transplantation and fertility.
NOVA 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 A, 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.
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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 A.
The NOVX nucleic acids and polypeptides are also useful for detecting specific
cell types. Details of the expression analysis for each NOVX are presented in
Example C.
Accordingly, the NOVX nucleic acids, polypeptides, antibodies and related
compounds
according to the invention will have diagnostic and therapeutic applications
in the
detection of a variety of diseases with differential expression in normal vr.
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.
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 a research tool. These include
serving as a
specific or selective nucleic acid or protein diagnostic and/or prognostic
marker, wherein
the presence or amount of the nucleic acid or the protein are to be assessed,
as well as
potential therapeutic applications such as the following: (i) a protein
therapeutic, (ii) a
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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
ira 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
N0:2n,
wherein n is an integer between 1 and 141, (b) a variant of a mature form of
the amino
acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is
an
integer between 1 and 141, wherein any amino acid in the mature form is
changed to a
different amino acid, provided that no more than 15% of the amino acid
residues in the
sequence of the mature form are so changed; (c) an amino acid sequence
selected from the
group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141,
(d) a
variant of the amino acid sequence selected from the group consisting of SEQ
ID N0:2n,
wherein n is an integer between 1 and 141, wherein any amino acid specified in
the chosen
sequence is changed to a different amino acid, provided that no more than 15%
of the
amino acid residues in the sequence are so changed; and (e) a fragment of any
of (a)
through (d).
In another specific embodiment, the invention includes an isolated nucleic
acid
molecule comprising a nucleic acid sequence encoding a polypeptide comprising
an amino
acid sequence selected from the group consisting of (a) a mature form of the
amino acid
sequence given SEQ ID N0:2n, wherein n is an integer between 1 and 141; (b) a
variant
of a mature form of the amino acid sequence selected from the group consisting
of SEQ
ID N0:2n, wherein n is an integer between 1 and 141, wherein any amino acid in
the
mature form of the chosen sequence is changed to a different amino acid,
provided that no
more than 15% of the amino acid residues in the sequence of the mature form
are so
changed; (c) the amino acid sequence selected from the group consisting of SEQ
ID
N0:2n, wherein n is an integer between 1 and 141; (d) a variant of the amino
acid
sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an
integer
between I and 141, in which any amino acid specified in the chosen sequence is
changed
to a different amino acid, provided that no more than 15% of the amino acid
residues in
the sequence are so changed; (e) a nucleic acid fragment encoding at least a
portion of a
polypeptide comprising the amino acid sequence selected from the group
consisting of
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SEQ ID N0:2n, wherein n is an integer between 1 and 141, or any variant of
said
polypeptide wherein any amino acid of the chosen sequence is changed to a
different
amino acid, provided that no more than 10% of the amino acid residues in the
sequence
are so changed; and (f) the complement of any of said nucleic acid molecules.
In yet another specific embodiment, the invention includes an isolated nucleic
acid
molecule, wherein said nucleic acid molecule comprises a nucleotide sequence
selected
from the group consisting of-. (a) the nucleotide sequence selected from the
group
consisting of SEQ ID N0:2n-l, wherein n is an integer between 1 and 141; (b) a
nucleotide sequence wherein one or more nucleotides in the nucleotide sequence
selected
from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1
and
141, is changed from that selected from the group consisting of the chosen
sequence to a
different nucleotide provided that no more than 15% of the nucleotides are so
changed; (c)
a nucleic acid fragment of the sequence selected from the group consisting of
SEQ ID
N0:2n-I, wherein n is an integer between I and 141; and (d) a nucleic acid
fragment
wherein one or more nucleotides in the nucleotide sequence selected from.the
group
consisting of SEQ ID N0:2n-l, wherein n is an integer between 1 and 141, 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 mRNAs) and fragments for use as PCR
primers for the amplification and/or mutation of NOVX nucleic acid molecules.
As used
herein, the term "nucleic acid molecule" is intended to include DNA molecules
(e.g.,
cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA
generated using nucleotide analogs, and derivatives, fragments and homologs
thereof. The
nucleic acid molecule may be single-stranded or double-stranded, but
preferably is
comprised double-stranded DNA.
A NOVX nucleic acid can encode a mature NOVX poIypeptide. As used herein, a
"mature" form of a polypeptide or protein disclosed in the present invention
is the product
of a naturally occurring polypeptide, precursor form, or proprotein. The
naturally
CA 02486490 2004-12-03
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occurring polypeptide, precursor or proprotein includes, by way of nonlimiting
example,
the full-length gene product encoded by the corresponding gene. Alternatively,
it may be
defined as the polypeptide, precursor or proprotein encoded by an ORF
described herein.
The product "mature" form arises, by way of nonlimiting example, as a result
of one or
more naturally occurnng processing steps that may take place within the cell
(e.g., host
cell) in which the gene product arises. Examples of such processing steps
leading to a
"mature" form of a polypeptide or protein include the cleavage of the N-
terminal
methionine residue encoded by the initiation codon of an ORF or the
proteolytic cleavage
of a signal peptide or leader sequence. Thus a mature form arising from a
precursor
polypeptide or protein that has residues 1 to N, where residue 1 is the N-
terminal
methionine, would have residues 2 through N remaining after removal of the N-
terminal
methionine. Alternatively, a mature form arising from a precursor polypeptide
or protein
having residues 1 to N, in which an N-terminal signal sequence from residue 1
to residue
M is cleaved, would have the residues from residue M+1 to residue N remaining.
Further
as used herein, a "mature" form of a polypeptide or protein may arise from a
post-translational modification step 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 "probe", as utilized herein, refers to nucleic acid sequences of
variable
length, preferably between at least about 10 nucleotides (nt), about 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 used herein, is a nucleic acid
that 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
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less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about
0.5 kb, or about
0.1 kb, of nucleotide sequences which naturally flank the nucleic acid
molecule in
genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g.,
brain, heart,
liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a
cDNA
molecule, can be substantially free of other cellular material, or culture
medium, or of
chemical precursors or other chemicals.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having
the
nucleotide sequence of SEQ ID NOS: 2n-l, wherein n is an integer between 1 and
141, or
a complement of this nucleotide sequence, can be isolated using standard
molecular
biology techniques and the sequence information provided herein. Using all or
a portion
of the nucleic acid sequence of SEQ ID NOS:2n-l, wherein n is an integer
between 1 and
141, as a hybridization probe, NOVX molecules can be isolated using standard
hybridization and cloning techniques (e.g., as described in Sambrook, et al.,
(eds.),
MOLECULAR CLONING: A LABORATORY MANUAL 2"d Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 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 with 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. A short oligonucleotide sequence may be based on, or designed from,
a genomic
or cDNA sequence and is used to amplify, confirm, or reveal the presence of an
identical,
similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides
comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in
length, preferably
about 15 nt to 30 nt in length. In one embodiment of the invention, an
oligonucleotide
comprising a nucleic acid molecule less than 100 nt in length would further
comprise at
least 6 contiguous nucleotides of SEQ ID NOS:2n-l, wherein n is an integer
between 1
and 141, or a complement thereof. Oligonucleotides may be chemically
synthesized and
may also be used as probes.
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In another embodiment, an isolated nucleic acid molecule of the invention
comprises a nucleic acid molecule that is a complement of the nucleotide
sequence shown
in SEQ ID NOS:2n-l, wherein n is an integer between 1 and 141, or a portion of
this
nucleotide sequence (e.g., a fragment that can be used as a probe or primer or
a fragment
encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid
molecule
that is complementary to the nucleotide sequence of SEQ ID NOS:2n-l, wherein n
is an
integer between 1 and 141, is one that is su~ciently complementary to the
nucleotide
sequence of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 141, that
it can
hydrogen bond with few or no mismatches to a nucleotide sequence of SEQ ID
NOS:2n-1,
wherein n is an integer between 1 and 141, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base pairing between nucleotides units of a nucleic acid molecule, and the
term "binding"
means the physical or chemical interaction between two polypeptides or
compounds or
associated polypeptides or compounds or combinations thereof. Binding includes
ionic,
non-ionic, van der Waals, hydrophobic interactions, and the like. A physical
interaction
can be either direct or indirect. Indirect interactions may be through or due
to the effects
of another polypeptide or compound. Direct binding refers to interactions that
do not take
place through, or due to, the effect of another polypeptide or compound, but
instead are
without other substantial chemical intermediates.
A "fragment" provided herein is defined as a sequence 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, and is 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.
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
5' direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence
lacking an in-frame stop codon similarly encodes a truncated N-terminal
fragment of the
respective NOVX polypeptide, and requires that the corresponding full-length
cDNA
extend in the 3' direction of the disclosed sequence.
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A "derivative" is a nucleic acid sequence or amino acid sequence formed from
the
native compounds either directly, by modification or partial substitution. An
"analog" is
a nucleic acid sequence or amino acid sequence that has a structure similar
to, but not
identical to, the native compound, e.g. they differs from it in respect to
certain
components or side chains. Analogs may be synthetic or derived from a
different
evolutionary origin and may have a similar or opposite metabolic activity
compared to
wild type. A "homolog" is a nucleic acid sequence or amino acid sequence of a
particular gene that is derived from different species.
Derivatives and analogs may be full length or other than full length.
Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not
limited to,
molecules comprising regions that are substantially homologous to the nucleic
acids or
proteins of the invention, in various embodiments, by at least about 70%, 80%,
or 95%
identity (with a preferred identity of 80-95%) over a nucleic acid or amino
acid sequence
of identical size or when compared to an aligned sequence in which the
alignment is done
by a computer homology program known in the art, or whose encoding nucleic
acid is
capable of hybridizing to the complement of a sequence encoding the 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
include 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
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below) in SEQ )D NO:2n-l, wherein n is an integer between 1 and 141, as well
as a
polypeptide possessing NOVX biological activity. Various biological activities
of the
NOVX proteins are described below.
A NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could
potentially be
translated into a polypeptide. A stretch of nucleic acids comprising an ORF is
uninterrupted by a stop codon. An ORF that represents the coding sequence for
a full
protein begins with an ATG "start" codon and terminates with one of the three
"stop"
codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF
may
be any part of a coding sequence, with or without a start codon, a stop codon,
or both.
For an ORF to be considered as a good candidate for coding for a bona fide
cellular
protein, a minimum size requirement is often set, e.g., a stretch of DNA that
would
encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes
allows for the generation of probes and primers designed for use in
identifying and/or
cloning NOVX homologues in other cell types, e.g. from other tissues, as well
as NOVX
homologues from other vertebrates. The probe/primer typically comprises
substantially
purified oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide
sequence that hybridizes under stringent conditions to at least about 12, 25,
50, 100, 150,
200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ
ID
N0:2n-l, wherein n is an integer between 1 and 141; or an anti-sense strand
nucleotide
sequence of SEQ ID N0:2n-l, wherein n is an integer between 1 and 141; or of a
naturally occurring mutant of SEQ ID N0:2n-1, wherein n is an integer between
1 and
141.
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 has a detectable label attached, e.g. the label 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.
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"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 and 141, that encodes a
poIypeptide having a NOVX biological activity (the biological activities of
the NOVX
proteins are described below), expressing the encoded portion of NOVX protein
(e.g., by
recombinant expression irz 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 ID N0:2rz-l, wherein n is an integer between 1 and
141,
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-l, wherein n is an integer
between 1 and 141. 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 and 141.
In addition to the human NOVX nucleotide sequences of SEQ ID N0:2n-1,
wherein n is an integer between 1 and I41, it will be appreciated by those
skilled in the
art that DNA sequence polymorphisms that lead to changes in the amino acid
sequences
of the NOVX polypeptides may exist within a population (e.g., the human
population).
Such genetic polymorphism in the NOVX genes may exist among individuals within
a
population due to natural allelic variation. As used herein, the terms "gene"
and
"recombinant gene" refer to nucleic acid molecules comprising an open reading
frame
(ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such
natural
allelic variations can typically result in 1-5°!° variance in
the nucleotide sequence ofthe
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.
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Moreover, nucleic acid molecules encoding NOVX proteins from other species,
and thus that have a nucleotide sequence that differs from a human SEQ ID
N0:2n-l,
wherein n is an integer between 1 and 141, are intended to be within the scope
of the
invention. Nucleic acid molecules corresponding to natural allelic variants
and
homologues of the NOVX cDNAs of the invention can be isolated based on their
homology to the human NOVX nucleic acids disclosed herein using the human
cDNAs,
or a portion thereof, as a hybridization probe according to standard
hybridization
techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 6 nucleotides in length and hybridizes under stringent
conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2n-
1,
wherein n is an integer between 1 and 141. 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
1 S 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 about 65% homologous to each other typically remain
hybridized to
each other.
Homologs (i. e., nucleic acids encoding NOVX proteins derived from species
other
than human) or other related sequences (e.g., paraIogs) can be obtained by
low, moderate
or high stringency hybridization with all or a portion of the particular human
sequence as
a probe using methods well known in the art for nucleic acid hybridization and
cloning.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but
to no other sequences. Stringent conditions are sequence-dependent and will be
different
in different circumstances. Longer sequences hybridize specifically at higher
temperatures than shorter sequences. Generally, stringent conditions are
selected to be
about 5 °C lower than the thermal melting point (Tm) for the specific
sequence at a
defined ionic strength and pH. The Tm is the temperature (under defined ionic
strength,
pH and nucleic acid concentration) at which 50% of the probes complementary to
the
target sequence hybridize to the target sequence at equilibrium. Since the
target
sequences are generally present at eaccess, at Tm, 50% of the probes are
occupied at
equilibrium. Typically, stringent conditions will be those in which the salt
concentration
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is less than about I .0 M sodium ion, typically about O.OI 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 oligonucIeotides. Stringent conditions may also be achieved with
the
addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that
sequences at least
about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other
typically remain hybridized to each other. A non-limiting example of stringent
hybridization conditions are hybridization in a high salt buffer comprising 6X
SSC, 50
mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500
mg/ml denatured salmon sperm DNA at 65°C, followed by one or more
washes in 0.2X
SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the
invention that
hybridizes under stringent conditions to a sequence of SEQ ID N0:2n-l, wherein
n is an
integer between 1 and 141, corresponds to a naturally-occurring nucleic acid
molecule.
As used herein, a "naturally-occurnng" nucleic acid molecule refers to an RNA
or DNA
molecule having a nucleotide sequence that occurs in nature (e.g., encodes a
natural
protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of SEQ ID N0:2n-l, wherein n
is an
integer between 1 and 141, 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 and 100 mg/ml denatured salmon sperm DNA at 55 °C, followed by one
or more
washes in 1X SSC, 0.1% SDS at 37 °C. Other conditions of moderate
stringency that
may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.),
1993,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger,
1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule comprising the nucleotide sequences of SEQ ID N0:2ra-l, wherein n is
an
integer between l and 141, or fragments, analogs or derivatives thereof, under
conditions
of low stringency, is provided. A non-limiting example of low stringency
hybridization
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WO 2004/000997 PCT/US2003/017512
conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCI (pH
7.5), 5
mM EDTA, 0.02% PVP, 0.02°!° Ficoll, 0.2% BSA, 100 mg/ml
denatured salmon sperm
DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more
washes in 2X SSC,
25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50 °C. Other
conditions of
low stringency that may be used are well known in the art (e.g., as employed
for
cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993,
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990,
GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo
and Weinberg, 1951. Proc Natl Acad Sci USA 78: 6759-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may
exist in the population, the skilled artisan will further appreciate that
changes can be
introduced by mutation into the nucleotide sequences of SEQ ID N0:2n-1,
wherein n is
an integer between 1 and 141, thereby leading to changes in the amino acid
sequences of
1 S the encoded NOVX protein, without altering the functional ability of that
NOVX protein.
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 and 141. A "non-essential" amino acid
residue is a
residue that can be altered from the wild-type sequences of the NOVX proteins
without
altering their biological activity, whereas an "essential" amino acid residue
is required for
such biological activity. For example, amino acid residues that are conserved
among the
NOVX proteins of the invention are predicted to be particularly non-amenable
to
alteration. Amino acids for which conservative substitutions can be made are
well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding
NOVX proteins that contain changes in amino acid residues that are not
essential for
activity. Such NOVX proteins differ in amino acid sequence from SEQ ID N0:2n-
1,
wherein n is an integer between 1 and 141, yet retain biological activity. In
one
embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence
encoding a protein, wherein the protein comprises an amino acid sequence at
least about
40% homologous to the amino acid sequences of SEQ ID N0:2n, wherein n is an
integer
between 1 and 141. Preferably, the protein encoded by the nucleic acid
molecule is at
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least about 60% homologous to SEQ ID N0:2n, wherein n is an integer between 1
and
141; more preferably at least about 70% homologous to SEQ ID N0:2n, wherein n
is an
integer between 1 and 141; still more preferably at least about 80% homologous
to SEQ
ID N0:2n,.wherein n is an integer between 1 and I41; even more preferably at
least
about 90% homologous to SEQ ID N0:2n, wherein n is an integer between 1 and
141;
and most preferably at least about 95% homologous to SEQ ID N0:2n, wherein n
is an
integer between 1 and 14I .
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 and 141, 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 and
141,
such that one or more amino acid substitutions, additions or deletions are
introduced into
the encoded protein.
Mutations can be introduced any one of SEQ ID NO:2n-1, wherein n is an integer
between 1 and 141, by standard techniques, such as site-directed mutagenesis
and
PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions
are made
at one or more predicted, non-essential amino acid residues. A "conservative
amino acid
substitution" is one in which the amino acid residue is replaced with an amino
acid
residue having a similar side chain. Families of amino acid residues having
similar side
chains have been defined within the art. These families include amino acids
with basic
side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains
(e.g:, threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino
acid residue
in the NOVX protein is replaced with another amino acid residue from the same
side
chain family. Alternatively, in another embodiment, mutations can be
introduced
randomly along all or part of a NOVX coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for NOVX biological
activity to
identify mutants that retain activity. Following mutagenesis of a nucleic acid
of SEQ ID
N0:2n-I, wherein n is an integer between 1 and 14I, the encoded protein can be
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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).
Interfering RNA
In one aspect of the invention, NOVX gene expression can be attenuated by RNA
interference. One approach well-known in the art is short interfering RNA
(siRNA)
mediated gene silencing where expression products of a NOVX gene are targeted
by
specific double stranded NOVX derived siRNA nucleotide sequences that are
complementary to at least a 19-25 nt long segment of the NOVX gene transcript,
including
the 5' untranslated (UT) region, the ORF, or the 3' UT region. See, e.g., PCT
applications
WO00/44895, WO99/32619, WO01/75164, WO01/92513, WO01/29058, WO01/89304,
W002/16620, and W002/29858, each incorporated by reference herein in their
entirety.
Targeted genes can be a NOVX gene, or an upstream or downstream modulator of
the
NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX
gene include, e.g., a transcription factor that binds the NOVX gene promoter,
a kinase or
phosphatase that interacts with a NOVX polypeptide, and polypeptides involved
in a
NOVX regulatory pathway.
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According to the methods of the present invention, NOVX gene expression is
silenced using short interfering RNA. A NOVX polynucleotide according to the
invention
includes a siRNA polynucleotide. Such a NOVX siRNA can be obtained using a
NOVX
polynucleotide sequence, for example, by processing the NOVX
ribopolynucleotide
sequence in a cell-free system, such as but not limited to a Drosophila
extract, or by
transcription of recombinant double stranded NOVX RNA or by chemical synthesis
of
nucleotide sequences homologous to a NOVX sequence. See, e.g., Tuschl, Zamore,
Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated
herein by
reference in its entirety. When synthesized, a typical 0.2 micromolar-scale
RNA synthesis
provides about 1 milligram of siRNA, which is sufficient for 1000 transfection
experiments using a 24-well tissue culture plate format.
The most efficient silencing is generally observed with siRNA duplexes
composed
of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to
have a 2-nt
3' overhang. The sequence of the 2-nt 3' overhang makes an additional small
contribution
to the specificity of siRNA target recognition. The contribution to
specificity is localized
to the unpaired nucleotide adjacent to the first paired bases. In one
embodiment, the
nucleotides in the 3' overhang are ribonucIeotides. In an alternative
embodiment, the
nucleotides in the 3' overhang are deoxyribonucleotides. Using 2'-
deoxyribonucleotides.
in the 3' overhangs is as efficient as using ribonucleotides, but
deoxyribonucleotides are
often cheaper to synthesize and are most likely more nuclease resistant.
A contemplated recombinant expression vector of the invention comprises a
NOVX DNA molecule cloned into an expression vector comprising operatively-
linked
regulatory sequences flanking the NOVX sequence in a manner that allows for
expression
(by transcription of the DNA molecule) of both strands. An RNA molecule that
is
antisense to NOVX mRNA is transcribed by a first promoter (e.g., a promoter
sequence 3'
of the cloned DNA) and an RNA molecule that is the sense strand for the NOVX
mRNA
is transcribed by a second promoter (e.g., a promoter sequence 5' of the
cloned DNA).
The sense and antisense strands may hybridize in vivo to generate siRNA
constructs for
silencing of the NOVX gene. Alternatively, two constructs can be utilized to
create the
sense and anti-sense strands of a siRNA construct. Finally, cloned DNA can
encode a
construct having secondary structure, wherein a single transcript has both the
sense and
complementary antisense sequences from the target gene or genes. In an example
of this
embodiment, a hairpin RNAi product is homologous to all or a portion of the
target gene.
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In another example, a hairpin RNAi product is a siRNA. The regulatory
sequences
flanking the NOVX sequence may be identical or may be different, such that
their
expression may be modulated independently, or in a temporal or spatial manner.
In a specific embodiment, siRNAs are transcribed intracellularly by cloning
the
NOVX gene templates into a vector containing, e.g., a RNA pol III
transcription unit from
the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA H I . One example
of a
vector system is the GeneSuppressorTM RNA Interference kit (commercially
available
from Imgenex). The U6 and Hl promoters are members of the type III class of
Pol III
promoters. The +I nucleotide of the U6-like promoters is always guanosine,
whereas the
I O +1 for H1 promoters is adenosine. The termination signal for these
promoters is defined by
five consecutive thyrnidines. The transcript is typically cleaved after the
second uridine.
Cleavage at this position generates a 3' UU overhang in the expressed siRNA,
which is
similar to the 3' overhangs of synthetic siRNAs. Any sequence less than 400
nucleotides in
length can be transcribed by these promoter, therefore they are ideally suited
for the
expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-
nucleotide RNA
stem-loop transcript.
A siRNA vector appears to have an advantage over synthetic siRNAs where long
term knock-down of expression is desired. Cells transfected with a siRNA
expression
vector would experience steady, long-term mRNA inhibition. In contrast, cells
transfected
with exogenous synthetic siRNAs typically recover from mRNA suppression within
seven
days or ten rounds of cell division. The long-term gene silencing ability of
siRNA
expression vectors may provide for applications in gene therapy.
In general, siRNAs are chopped from longer dsRNA by an ATP-dependent
ribonuclease called DICER. DICER is a member of the RNase III family of
double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular
proteins
into an endonuclease complex. In vitro studies in Drosophila suggest that the
siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex,
called
an RNA-induced silencing complex (RISC), which contains an endoribonuclease
that is
distinct from DICER. RISC uses the sequence encoded by the antisense siRNA
strand to
find and destroy mRNAs of complementary sequence. The siRNA thus acts as a
guide,
restricting the ribonuclease to cleave only mRNAs complementary to one of the
two
siRNA strands.
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A NOVX mRNA region to be targeted by siRNA is generally selected from a
desired NOVX sequence beginning 50 to 100 nt downstream of the start codon.
Alternatively, S' or 3' UTRs and regions nearby the start codon can be used
but are
generally avoided, as these may be richer in regulatory protein binding sites.
UTR binding
proteins and/or translation initiation complexes may interfere with binding of
the siRNP or
RISC endonuclease complex. An initial BLAST homology search for the selected
siRNA
sequence is done against an available nucleotide sequence library to ensure
that only one
gene is targeted. Specificity of target recognition by siRNA duplexes indicate
that a single
point mutation located in the paired region of an siRNA duplex is sufficient
to abolish
target mRNA degradation. See, Elbashir et al. 2001 EMBO J. 20(23):6877-88.
Hence,
consideration should be taken to accommodate SNPs, polymorphisms, allelic
variants or
species-specific variations when targeting a desired gene.
In one embodiment, a complete NOVX siRNA experiment includes the proper
negative control. A negative control siRNA generally has the same nucleotide
composition as the NOVX siRNA but lack significant sequence homology to the
genome.
Typically, one would scramble the nucleotide sequence of the NOVX siRNA and do
a
homology search to make sure it lacks homology to any other gene.
Two independent NOVX siRNA duplexes can be used to knock-down a target
NOVX gene. This helps to control for specificity of the silencing effect. In
addition,
expression of two independent genes can be simultaneously knocked down by
using equal
concentrations of different NOVX siRNA duplexes, e.g., a NOVX siRNA and an
siRNA
for a regulator of a NOVX gene or polypeptide. Availability of siRNA-
associating
proteins is believed to be more limiting than target mRNA accessibility.
A targeted NOVX region is typically a sequence of two adenines (AA) and two
thymidines (TT) divided by a spacer region of nineteen (N19) residues (e.g.,
AA(N19)TT). A desirable spacer region has a CI/C-content of approximately 30%
to 70%,
and more preferably of about 50%. If the sequence AA(N19)TT is not present in
the
target sequence, an alternative target region would be AA(N21). The sequence
of the
NOVX sense siRNA corresponds to (Nl9)TT or N21, respectively. In the latter
case,
conversion of the 3' end of the sense siRNA to TT can be performed if such a
sequence
does not naturally occur in the NOVX polynucleotide. The rationale for this
sequence
conversion is to generate a symmetric duplex with respect to the sequence
composition of
the sense and antisense 3' overhangs. Symmetric 3' overhangs may help to
ensure that the
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siRNPs are formed with approximately equal ratios of sense and antisense
target
RNA-cleaving siRNPs. See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes &
Dev.
15: 188-200, incorporated by reference herein in its entirely. The
modification of the
overhang of the sense sequence of the siRNA duplex is not expected to affect
targeted
mRNA recognition, as the antisense siRNA strand guides target recognition.
Alternatively, if the NOVX target mRNA does not contain a suitable AA(N21)
sequence, one may search for the sequence NA(N21). Further, the sequence of
the sense
strand and antisense strand may still be synthesized as 5' (Nl9)TT, as it is
believed that the
sequence of the 3'-most nucleotide of the antisense siRNA does not contribute
to
specificity. Unlike antisense or ribozyme technology, the secondary structure
of the target
mRNA does not appear to have a strong effect on silencing. See, Harborth, et
al. (2001) J.
Cell Science 114: 4557-4565, incorporated by reference in its entirety.
Transfection of NOVX siRNA duplexes can be achieved using standard nucleic
acid transfection methods, for example, OLIGOFECTAMINE Reagent (commercially
available from Invitrogen). An assay for NOVX gene silencing is generally
performed
approximately 2 days after transfection. No NOVX gene silencing has been
observed in
the absence of transfection reagent, allowing for a comparative analysis of
the wild-type
and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-
well
plate, approximately 0.84 ~g of the siRNA duplex is generally sufficient.
Cells are
typically seeded the previous day, and are transfected at about 50%
confluence. The
choice of cell culture media and conditions are routine to those of skill in
the art, and will
vary with the choice of cell type. The efficiency of transfection may depend
on the cell
type, but also on the passage number and the confluency of the cells. The time
and the
manner of formation of siRNA-liposome complexes (e.g. inversion versus
vortexing) are
also critical. Low transfection efficiencies are the most frequent cause of
unsuccessful
NOVX silencing. The efficiency of transfection needs to be carefully examined
for each
new cell line to be used. Preferred cell are derived from a mammal, more
preferably from
a rodent such as a rat or mouse, and most preferably from a human. Where used
for
therapeutic treatment, the cells are preferentially autologous, although non-
autologous cell
sources are also contemplated as within the scope of the present invention.
For a control experiment, transfection of 0.84 ~g single-stranded sense NOVX
siRNA will have no effect on NOVX silencing, and 0.84 ltg antisense siRNA has
a weak
silencing effect when compared to 0.84 ~g of duplex siRNAs. Control
experiments again
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allow for a comparative analysis of the wild-type and silenced NOVX
phenotypes. To
control for transfection efficiency, targeting of common proteins is typically
performed,
for example targeting of lamin A/C or transfection of a CMV-driven EGFP-
expression
plasmid (e.g. commercially available from Clontech). In the above example, a
determination of the fraction of lamin A/C knockdown in cells is determined
the next day
by such techniques as immunofluorescence, Western blot, Northern blot or other
similar
assays for protein expression or gene expression. Lamin A/C monoclonal
antibodies may
be obtained from Santa Cruz Biotechnology.
Depending on the abundance and the half life (or turnover) of the targeted
NOVX
polynucleotide in a cell, a knock-down phenotype may become apparent after 1
to 3 days,
or even later. In cases where no NOVX knock-down phenotype is observed,
depletion of
the NOVX polynucleotide may be observed by immunofluorescence or Western
blotting.
If the NOVX polynucleotide is still abundant after 3 days, cells need to be
split and
transferred to a fresh 24-well plate for re-transfection. If no knock-down of
the targeted
protein is observed, it may be desirable to analyze whether the target mRNA
(NOVX or a
NOVX upstream or downstream gene) was effectively destroyed by the transfected
siRNA
duplex. Two days after transfection, total RNA is prepared, reverse
transcribed using a
target-specific primer, and PCR-amplified with a primer pair covering at least
one
exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR
of a
non-targeted mRNA is also needed as control. Effective depletion of the mRNA
yet
undetectable reduction of target protein may indicate that a large reservoir
of stable
NOVX protein may exist in the cell. Multiple transfection in sufficiently long
intervals
may be necessary until the target protein is finally depleted to a point where
a phenotype
may become apparent. If multiple transfection steps are required, cells are
split 2 to 3
days after transfection. The cells may be transfected immediately after
splitting.
An inventive therapeutic method of the invention contemplates administering a
NOVX siRNA construct as therapy to compensate for increased or aberrant NOVX
expression or activity. The NOVX ribopolynucleotide is obtained and processed
into
siRNA fragments, or a NOVX siRNA is synthesized, as described above. The NOVX
siRNA is administered to cells or tissues using known nucleic acid
transfection techniques,
as described above. A NOVX siRNA specific for a NOVX gene will decrease or
knockdown NOVX transcription products, which will lead to reduced NOVX
polypeptide
production, resulting in reduced NOVX polypeptide activity in the cells or
tissues.
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The present invention also encompasses a method of treating a disease or
condition
associated with the presence of a NOVX protein in an individual comprising
administering
to the individual an IRNAi construct that targets the mRNA of the protein (the
mRNA that
encodes the protein) for degradation. A specific RNAi construct includes a
siRNA or a
double stranded gene transcript that is processed into siRNAs. Upon treatment,
the target
protein is not produced or is not produced to the extent it would be in the
absence of the
treatment.
Where the NOVX gene function is not correlated with a known phenotype, a
control sample of cells or tissues from healthy individuals provides a
reference standard
for determining NOVX expression levels. Expression levels are detected using
the assays
described, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the
like. A
subject sample of cells or tissues is taken from a mammal, preferably a human
subject,
suffering from a disease state. The NOVX ribopolynucleotide is used to produce
siRNA
constructs, that are specific for the NOVX gene product. These cells or
tissues are treated
by administering NOVX siItNA's to the cells or tissues by methods described
for the
transfection of nucleic acids into a cell or tissue, and a change in NOVX
polypeptide or
polynucleotide expression is observed in the subject sample relative to the
control sample,
using the assays described. This NOVX gene knockdown approach provides a rapid
method for determination of a NOVX minus (NOVX-) phenotype in the treated
subject
sample. The NOVX-phenotype observed in the treated subject sample thus serves
as a
marker for monitoring the course of a disease state during treatment.
In specific embodiments, a NOVX siRNA is used in therapy. Methods for the
generation and use of a NOVX siRNA are known to those skilled in the art.
Example
techniques are provided below.
Production of RNAs
Sense RNA (ssRNA) and antisense RNA (asRNA) of NOVX are produced using
known methods such as transcription in RNA expression vectors. In the initial
experiments, the sense and antisense RNA are about 500 bases in length each.
The
produced ssRNA and asRNA (0.5 ~.M) in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCI
were heated to 95° C for 1 min then cooled and annealed at room
temperature for 12 to 16
h. The RNAs are precipitated and resuspended in lysis buffer (below). To
monitor
annealing, RNAs are electrophoresed in a 2% agarose gel in TBE buffer and
stained with
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ethidium bromide. See, e.g., Sambrook et aL, Molecular Cloning. Cold Spring
Harbor
Laboratory Press, Plainview, N.Y. (1989).
Lysate Preparation
Untreated rabbit reticulocyte lysate (Ambion) are assembled according to the
manufacturer's directions. dsRNA is incubated in the lysate at 30° C
for 10 min prior to the
addition of mRNAs. Then NOVX mRNAs are added and the incubation continued for
an
additional 60 rnin. The molar ratio of double stranded RNA and mRNA is about
200:1.
The NOVX mRNA is radiolabeled (using known techniques) and its stability is
monitored
by gel electrophoresis.
In a parallel experiment made with the same conditions, the double stranded
RNA
is internally radiolabeled with a 3aP-ATP. Reactions are stopped by the
addition of 2 X
proteinase K buffer and deproteinized as described previously (Tuschl et al.,
Genes Dev.,
13:3191-3197 (1999)). Products are analyzed by electrophoresis in 15% or 18%
polyacrylamide sequencing gels using appropriate RNA standards. By monitoring
the gels
1 S for radioactivity, the natural production of 10 to 25 nt RNAs from the
double stranded
RNA can be determined.
The band of double stranded RNA, about 21-23 bps, is eluded. The efficacy of
these 21-23 mers for suppressing NOVX transcription is assayed in vitro using
the same
rabbit reticulocyte assay described above using 50 nanomolar of double
stranded 21-23
mer for each assay. The sequence of these 21-23 mers is then determined using
standard
nucleic acid sequencing techniques.
RNA Preparation
21 nt RNAs, based on the sequence determined above, are chemically synthesized
using Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo,
Germany). Synthetic oligonucleotides are deprotected and gel-purred (Elbashir,
Lendeckel, & Tuschl, Genes & Dev. 15, 188-200 (2001)), followed by Sep-Pak C18
cartridge (Waters, Milford, Mass., USA) purification (Tuschl, et al.,
Biochemistry,
32:11658-11668 (1993)).
These RNAs (20 pM) single strands are incubated in annealing buffer (100 mM
potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1
min at
90° C followed by 1 h at 37° C.
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Cell Culture
A cell culture known in the art to regularly express NOVX is propagated using
standard conditions. 24 hours before transfection, at approx. ~0% confluency,
the cells are
trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3 X 105
cellslml) and
transferred to 24-well plates (500 mllwell). Transfection is performed using a
commercially available lipofection kit and NOVX expression is monitored using
standard
techniques with positive and negative control. A positive control is cells
that naturally
express NOVX while a negative control is cells that do not express NOVX. Base-
paired
21 and 22 nt siRNAs with overhanging 3' ends mediate efficient sequence-
specific mRNA
degradation in lysates and in cell culture. Different concentrations of siRNAs
are used.
An efficient concentration for suppression in vitro in mammalian culture is
between 25
nM to 100 nM final concentration. This indicates that siRNAs are effective at
concentrations that are several orders of magnitude below the concentrations
applied in
conventional antisense or ribozyme gene targeting experiments.
The above method provides a way both for the deduction of NOVX siRNA
sequence and the use of such siRNA for in vitro suppression. In vivo
suppression may be
performed using the same siRNA using well known in vivo transfection or gene
therapy
transfection techniques.
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 N0:2ra-l, wherein n is an integer
between
1 and 141, or fragments, analogs or derivatives thereof. An "antisense"
nucleic acid
comprises a nucleotide sequence that is complementary to a "sense" nucleic
acid
encoding a protein (e.g., complementary to the coding strand of a double-
stranded cDNA
molecule or complementary to an mRNA sequence). In specific aspects, antisense
nucleic acid molecules are provided that comprise a sequence complementary to
at least
about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand,
or to
only a portion thereof. Nucleic acid molecules encoding fragments, homologs,
derivatives and analogs of a NOVX protein of SEQ ID N0:2ra, wherein n is an
integer
between 1 and 141, or antisense nucleic acids complementary to a NOVX nucleic
acid
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sequence of SEQ ID NO:2n-1, wherein n is an integer between 1 and I41, 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
modified
nucleotides designed to increase the biological stability of the molecules or
to increase
the physical stability of the duplex formed between the antisense and sense
nucleic acids
(e.g., phosphorothioate derivatives and acridine substituted nucleotides can
be used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-
iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-
thiouridine,
5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil,
dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil,
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4-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, S-methyluracil,
uracil-S-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 andlor translation). The
hybridization can be by
conventional nucleotide complementarity to form a stable duplex, or, for
example, in the
case of an antisense nucleic acid molecule that binds to DNA duplexes, through
specific
interactions in the major groove of the double helix. An example of a route of
administration of antisense nucleic acid molecules of the invention includes
direct
injection at a tissue site. Alternatively, antisense nucleic acid molecules
can be modified
to target selected cells and then administered systemically. For example, for
systemic
administration, antisense molecules can be modified such that they
specifically bind to
receptors or antigens expressed on a selected cell surface (e.g., by linking
the antisense
nucleic acid molecules to peptides or antibodies that bind to cell surface
receptors or
antigens). The antisense nucleic acid molecules can also be delivered to cells
using the
vectors described herein. To achieve sufficient nucleic acid molecules, vector
constructs
in which the antisense nucleic acid molecule is placed under the control of a
strong pol II
or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is
an 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. Nucl. Acids Res. 15:
6131-6148)
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or a chimeric RNA DNA analogue (See, e.g., moue, et al., 1987. FEBSLett. 215:
327-330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified
bases, and nucleic acids whose sugar phosphate backbones are modified or
derivatized.
These modifications are carried out at least in part to enhance the chemical
stability of
the modified nucleic acid, such that they 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., SEQ ID N0:2n-1,
wherein
n is an integer between 1 and 141). For example, a derivative of a
Tetrahyrnena L-19
IVS RNA can be constructed in which the nucleotide sequence of the active site
is
complementary to the nucleotide sequence to be cleaved in a NOVX-encoding
mRNA.
See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to
Cech, et al.
NOVX mRNA can also be used to select a catalytic RNA having a specific
ribonuclease
activity from a pool.of RNA molecules. See, e.g., Bartel et al., (1993)
Science
261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the NOVX nucleic acid
(e.g., the
NOVX promoter and/or enhancers) to form triple helical structures that prevent
transcription of the NOVX gene in target cells. See, e.g., Helene, 1991.
Anticancer Drug
Des. 6: 569-84; Helene, et al. 1992. Anra. 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
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CA 02486490 2004-12-03
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nucleic acids can be modified to generate peptide nucleic acids. See, e.g.,
Hyrup, et al.,
1996. Bioorg Med Claem 4: 5-23. As used herein, the terms "peptide nucleic
acids" or
"PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in which the
deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only the four
natural
nucleotide bases are retained. The neutral backbone of PNAs has been shown to
allow
for specific hybridization to DNA and RNA under conditions of low ionic
strength. The
synthesis of PNA oligomer can be performed using standard solid phase peptide
synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe,
et al.,
1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For
example, PNAs can be used as antisense or antigene agents for sequence-
specific
modulation of gene expression by, e.g., inducing transcription or translation
arrest or
inhibiting replication. PNAs of NOVX can also be~used, for example, in the
analysis of
single base pair mutations in a gene (e.g., PNA directed PCR clamping; as
artificial
restriction enzymes when used in combination with other enzymes, e.g., S1
nucleases
(See, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and
hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996.
supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of NOVX can be
generated
that may combine the advantageous properties of PNA and DNA. Such chimeras
allow
DNA recognition enzymes (e.g., RNase H and DNA polyrnerases) to interact with
the
DNA portion while the PNA portion would provide high binding affinity and
specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths selected
in terms
of base stacking, number of bonds between the nucleotide bases, and
orientation (see,
Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras can be
performed as
described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res
24:
3357-3363. For example, a DNA chain can be synthesized on a solid support
using
standard phosphoramidite coupling chemistry, and modified nucleoside analogs,
e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used
between the
PNA and the 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
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with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra.
Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and
a 3'
PNA segment. See, e.g:, Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
S such as peptides (e.g., for targeting host cell receptors izz vivo), or
agents facilitating
transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc.
Natl. Acad.
~'ci. 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 (see, e.g., Krol, et al., 1988. BioTeclzniques 6:958-
976) or
intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this
end, the
oligonucleotide may be conjugated to another molecule, e.g., a peptide, a
hybridization
triggered cross-linking agent, a transport agent, a hybridization-triggered
cleavage agent,
and the like.
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 N0:2zz, wherein n is an integer between 1 and 141. 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 NO:2n, wherein n is an
integer
between 1 and 141, while 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 further include the possibility of inserting an
additional residue or
residues between two residues of the parent protein as well as the possibility
of deleting
one or more residues from the parent sequence. Any amino acid substitution,
insertion,
or deletion is encompassed by the invention. In favorable circumstances, the
substitution
is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and
biologically-active portions thereof, or derivatives, fragments, analogs or
homologs
thereof. Also provided are polypeptide fragments suitable for use as
immunogens to
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raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be
isolated
from cells or tissue sources by an appropriate purification scheme using
standard protein
purification techniques. In another embodiment, NOVX proteins are produced by
recombinant DNA techniques. Alternative to recombinant expression, a NOVX
protein
or polypeptide can be synthesized chemically using standard peptide synthesis
techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active
portion
thereof is substantially free of cellular material or other contaminating
proteins from the
cell or tissue source from which the NOVX protein is derived, or substantially
free from
chemical precursors or other chemicals when chemically synthesized. The
language
"substantially free of cellular material" includes preparations of NOVX
proteins in which
the protein is separated 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 preparafiions of NOVX proteins having less than about 30%
(by dry
weight) of non-NOVX proteins (also referred to herein as a "contaminating
protein"),
more preferably less than about 20% of non-NOVX proteins, still more
preferably less
than about 10% of non-NOVX proteins, and most preferably less than about 5% of
non-NOVX proteins. When the NOVX protein or biologically-active portion
thereof is
recombinantly-produced, it is also preferably substantially free of culture
medium, i.e.,
culture medium represents less than about 20%, more preferably less than about
10%,
and most preferably less than about 5% of the volume of the NOVX protein
preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from
chemical
precursors or other chemicals that are involved in the synthesis of the
protein. In one
embodiment, the language "substantially free of chemical precursors or other
chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry
weight) of
chemical precursors or non-NOVX chemicals, more preferably less than about 20%
chemical precursors or non-NOVX chemicals, still more~preferably less than
about 10%
chemical precursors or non-NOVX chemicals, and most preferably less than about
5%
chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequences of
the NOVX proteins (e.g., the amino acid sequence of SEQ ID N0:2ra, wherein n
is an
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integer between 1 and 141) that include fewer amino acids than the full-length
NOVX
proteins, and exhibit at least one activity of a NOVX protein. Typically,
biologically-active portions comprise a domain or motif with at least one
activity of the
NOVX protein. A biologically-active portion of a NOVX protein can be a
polypeptide
which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the
protein
are deleted, can be prepared by recombinant techniques and evaluated for one
or more of
the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID
NO:2n, wherein n is an integer between 1 and 141. In other embodiments, the
NOVX
protein is substantially homologous to SEQ ID N0:2n, wherein n is an integer
between 1
and 141, and retains the functional activity of the protein of SEQ ID NO:2n,
wherein n is
an integer between 1 and 141, yet differs in amino acid sequence due to
natu~'al 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 and 141, and retains the functional activity of the NOVX
proteins of
SEQ ID N0:2rz, wherein n is an integer between 1 and 141.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be
introduced in the sequence of a first amino acid or nucleic acid sequence for
optimal
alignment with a second amino or nucleic acid sequence). The amino acid
residues or
nucleotides at corresponding amino acid positions or nucleotide positions are
then
compared. When a position in the first sequence is occupied by the same amino
acid
residue or nucleotide as the corresponding position in the second sequence,
then the
molecules are homologous at that position (i.e., as used herein amino acid or
nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known in the art, such as GAP software provided in the GCG program package.
See,
Needleman and Wunsch, 1970. ,7Mol Biol 48: 443-453. Using GCG GAP software
with
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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 N0:2n-1, wherein n is an integer between 1 and 141.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region
of comparison. The term "percentage of sequence identity" is calculated by
comparing
two optimally aligned sequences over that region of comparison, determining
the number
of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or
I, in the case
of nucleic acids) occurs in both sequences to yield the number of matched
positions,
dividing the number of matched positions by the total number of positions in
the region
of comparison (i.e., the window size), and multiplying the result by 100 to
yield the
percentage of sequence identity. The term "substantial identity" as used
herein denotes a
1 S characteristic of a polynucleotide sequence, wherein the polynucleotide
comprises a
sequence that has at least 80 percent sequence identity, preferably at least
85 percent
identity and often 90 to 95 percent sequence identity, more usually at least
99 percent
sequence identity as compared to a reference sequence over a comparison
region.
Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein,
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 and 141, whereas a "non-NOVX
polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a
protein that is
not substantially homologous to the NOVX protein, e.g., a protein that is
different from
the NOVX protein and that is derived from the same or a different organism.
Within a
NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of
a
NOVX protein. In one embodiment, a NOVX fusion protein comprises at least one
biologically-active portion of a NOVX protein. In another embodiment, a NOVX
fusion
protein comprises at least two biologically-active portions of a NOVX protein.
In yet
another embodiment, a NOVX fusion protein comprises at least three
biologically-active
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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
heteroIogous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian
host cells), expression and/or secretion of NOVX can be increased through use
of a
heterologous signal sequence.
In yet another embodiment, the fusion protein is a NOVX-imriiunoglobulin
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 irz
vivo. The
NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability
of a
NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be
useful
therapeutically for both the treatment of proliferative and differentiative
disorders, as
well as modulating (e.g: promoting or inhibiting) cell survival. Moreover, the
NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens
to
produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in
screening
assays to identify molecules that inhibit the interaction of NOVX with a NOVX
ligand.
A NOVX chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
Iigation,
restriction enzyme digestion to provide for appropriate termini, filling-in of
cohesive
ends as appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and
enzymatic ligation. In another embodiment, the fusion gene can be synthesized
by
conventional techniques including automated DNA synthesizers. Alternatively,
PCR
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amplification of gene fragments can be carried out using anchor primers that
give rise to
complementary overhangs between two consecutive gene fragments that can
subsequently be annealed and reamplified to generate a chimeric gene sequence
(see,
e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Tohn
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 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.
1 S 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 occurnng 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., forphage
display) containing the set of NOVX sequences therein. There are a variety of
methods
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which can be used to produce libraries of potential NOVX variants from a
degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can
be
performed in an automatic DNA synthesizer, and the synthetic gene then ligated
into an
appropriate expression vector. Use of a degenerate set of genes allows for the
provision,
in one mixture, of all of the sequences encoding the desired set of potential
NOVX
sequences. Methods for synthesizing degenerate oligonucleotides are well-known
within
the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984.
Annu. Rev.
Bioclaern. 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 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 SI 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
CA 02486490 2004-12-03
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combination with the screening assays to identify NOVX variants. See, e.g.,
Arkin and
Yourvan, 1992. Proc. IVatl. 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
imrnunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that
contain an antigen binding site that specifically binds (immunoreacts with) an
antigen.
Such antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, single
chain, Fab, Fab' and F~ab~z fragments, and an Fab expression library. In
general, antibody
molecules obtained from humans relates to any of the classes IgG, IgM, IgA,
IgE and
IgD, which differ from one another by the nature of the heavy chain present in
the
molecule. Certain classes have subclasses as well, such as IgGI, IgG2, and
others.
Furthermore, in humans, the light chain may be a kappa chain or a lambda
chain.
Reference herein to antibodies includes a reference to all such classes,
subclasses and
types of human antibody species.
An isolated protein of the invention intended to serve as an antigen, or a
portion or
fragment thereof, can be used as an immunogen to generate antibodies that
immunospecifically bind the antigen, using standard techniques for polyclonal
and
monoclonal antibody preparation. The full-length protein can be used or,
alternatively,
the invention provides antigenic peptide fragments of the antigen for use as
immunogens.
An 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 and I4I, and encompasses an epitope thereof
such that
an antibody raised against the peptide forms a specific immune complex with
the full
length protein or with any fragment that contains the epitope. Preferably, the
antigenic
peptide comprises at least 10 amino acid residues, or at least 15 amino acid
residues, or at
least 20 amino acid residues, or at least 30 amino acid residues. Preferred
epitopes
encompassed by the antigenic peptide are regions of the protein that are
located on its
surface; commonly these are hydrophilic regions.
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
SI
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indicate which regions of a NOVX polypeptide are particularly hydrophilic and,
therefore, are likely to encode surface residues useful for targeting antibody
production.
As a means for targeting antibody production, hydropathy plots showing regions
of
hydrophilicity and hydrophobicity may be generated by any method well known in
the
art, including, for example, the Kyte Doolittle or the Hopp Woods methods,
either with
or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Pros. 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 characteristics. A NOVX polypeptide or a fragment thereof
comprises at
least one antigenic epitope. An anti-NOVA antibody of the present invention is
said to
specifically bind to antigen NOVX when the equilibrium binding constant (KD)
is __<1
~.M, preferably < 100 nM, more preferably <_ 10 nM, and most preferably < 100
pM to
about 1 pM, as measured by assays such as radioligand binding assays or
similar assays
known to those skilled in the art.
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
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
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the native protein, a synthetic variant thereof, or a derivative of the
foregoing. An
appropriate immunogenic preparation can contain, for example, the naturally
occurring
immunogenic protein, a chemically synthesized polypeptide representing the
immunogenic protein, or a recombinantly expressed immunogenic protein.
Furthermore,
the protein may be conjugated to a second protein known to be immunogenic in
the
mammal being immunized. Examples of such immunogenic proteins include but are
not
limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and
soybean
trypsin inhibitor. The preparation can further include an adjuvant. Various
adjuvants used
to increase the immunological response include, but are not limited to,
Freund's (complete
and incomplete), mineral gels (e.g., aluminum hydroxide), surface active
substances (e.g.,
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium
parwm, or similar immunostimulatory agents. Additional examples of adjuvants
which
can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic
trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be isolated from the mammal (e.g., from the blood) and further purified by
well known
techniques, such as amity 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.
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Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma
method, a
mouse, hamster, or other appropriate host animal, is typically immunized with
an
immunizing agent to elicit lymphocytes that produce or are capable of
producing
antibodies that will specifically bind to the immunizing agent. Alternatively,
the
lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof or a fusion protein thereof. Generally, either peripheral blood
lymphocytes are
used if cells of human origin axe desired, or spleen cells or lymph node cells
are used if
non-human mammalian sources are desired. The lymphocytes are then fused with
an
immortalized cell line using a suitable fusing agent, such as polyethylene
glycol, to form a
hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,
Academic
Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed
mammalian
cells, particularly myeloma cells of rodent, bovine and human origin. Usually,
rat or
mouse myeloma cell lines are employed. The hybridoma cells can be cultured in
a
suitable culture medium that preferably contains one or more substances that
inhibit the
growth or survival of the unfused, immortalized cells. For example, if the
parental cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the
culture medium for the hybridomas typically will include hypoxanthine,
aminopterin, and
thymidine ("HAT medium"), which substances prevent the growth of HGPRT-
deficient
cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to
.a medium such as HAT medium. More preferred immortalized cell lines are
murine
myeloma lines, which can be obtained, fox instance, from the Salk Institute
Cell
Distribution Center, San Diego, California and the American Type Culture
Collection,
Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines
also
have been described for the production of human monoclonal antibodies (Kozbor,
J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Technigues
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
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determined by immunoprecipitation or by an in vitro binding assay, such as
radioimrnunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such
techniques and assays are known in the art. The binding affinity of the
monoclonal
antibody can, for example, be determined by the Scatchard analysis of Munson
and
Pollard, Anal. Biochem., 107:220 (1980). It is an objective, especially
important in
therapeutic applications of monoclonal antibodies, to identify antibodies
having a high
degree of specificity and a high binding affinity for the target antigen.
After the desired hybridoma cells are identified, the clones can be subcloned
by
limiting dilution procedures and grown by standard methods (Goding,1986).
Suitable
culture media for this purpose include, for example, Dulbecco's Modified
Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo
as
ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified
from the culture medium or ascites fluid by conventional immunoglobulin
purification
procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography,
gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such
as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of the invention can be readily isolated and sequenced using
conventional
procedures (e.g., by using oligonucleotide probes that are capable of binding
specifically
to genes encoding the heavy and light chains of murine antibodies). 'The
hybridoma cells
of the invention serve as a preferred source of such DNA. Once isolated, the
DNA can be
placed into expression vectors, which are then transfected into host cells
such as simian
COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not
otherwise
produce immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the
recombinant host cells. The DNA also can be modified, for example, by
substituting the
coding sequence for human heavy and light chain constant domains in place of
the
homologous murine sequences (U.S. 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.
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Humanized Antibodies
The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against
the administered immunoglobulin. Humanized forms of antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
F(ab')2 or other antigen-binding subsequences of antibodies) that are
principally comprised
of the sequence of a human immunoglobulin, and contain minimal sequence
derived from
a non-human immunoglobulin. Humanization can be performed following the method
of
I O 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
I S residues. Humanized antibodies can also comprise residues which are found
neither in the
recipient antibody nor in the imported CDR or framework sequences. In general,
the
humanized antibody will comprise substantially all of at least one, and
typically two,
variable domains, in which all or substantially all of the CDR regions
correspond to those
of a non-human irnmunoglobulin and all or substantially all of the framework
regions are
20 those of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise at least a portion of an immunoglobulin constant
region (Fc),
typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et
al., 1988; and
Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Human Antibodies
25 Fully human antibodies essentially relate to antibody molecules in which
the entire
sequence of both the light chain and the heavy chain, including the CDRs,
arise from
human genes. Such antibodies are termed "human antibodies", or "fully human
antibodies" herein. Human monoclonal antibodies can be prepared by the trioma
technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983
Immunol
30 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
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present invention and may be produced by using human hybridomas (see Cote? et
al.,
1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells
with
Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES
AND
CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in 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 BiotechnoloQV 4 845-51 (1996));
Neuberger
(Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol.
13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals which are modified so as to produce fully human antibodies rather than
the
animal's endogenous antibodies in response to challenge by an antigen. (See
PCT
publication W094/02602). The endogenous genes encoding the heavy and light
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 96134096.
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.
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Additionally, the genes encoding the immunoglobulins with human variable
regions can
be recovered and expressed to obtain the antibodies directly, or can be
further modified to
obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S.
Patent No. 5,939,598. It can be obtained by a method including deleting the J
segment
genes from at least one endogenous heavy chain locus in an embryonic stem cell
to
prevent rearrangement of the locus and to prevent formation of a transcript of
a rearranged
immunoglobulin heavy chain locus, the deletion being effected by a targeting
vector
containing a gene encoding a selectable marker; and producing from the
embryonic stem
cell a transgenic mouse whose somatic and germ cells contain the gene encoding
the
selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression
vector that
contains a nucleotide sequence encoding a heavy chain into one mammalian host
cell in
culture, introducing an expression vector containing a nucleotide sequence
encoding a
light chain into another mammalian host cell, and fusing the two cells to form
a hybrid
cell. The hybrid cell expresses an antibody containing the heavy chain and the
light chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an antigenic protein of the invention (see
e.g., U.S.
Patent No. 4,946,778). In addition, methods can be adapted for the
construction of Fab
expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to
allow rapid
and effective identification of monoclonal Fab fragments with the desired
specificity for a
protein or derivatives, fragments, analogs or homologs thereof. Antibody
fragments that
contain the idiotypes to a protein antigen may be produced by techniques known
in the art
including, but not limited to: (i) an F~ab')2 fragment produced by pepsin
digestion of an
antibody molecule; (ii) an Fab fragment generated by reducing the disulfide
bridges of an
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F~~b72 fragment; (iii) an Fab fragment generated by the treatment of the
antibody molecule
with papain and a reducing agent and (iv) F" fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies
that have binding specificities for at least two different antigens. In the
present case, one
of the binding specificities is for an antigenic protein of the invention. The
second binding
target is any other antigen, and advantageously is a cell-surface protein or
receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispeciBc 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
1 S correct bispecific structure. The purification of the correct molecule is
usually
accomplished by affinity chromatography steps. Similar procedures are
disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., I0:3655-
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 brst
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 Enzymolo~y,
121:210
(1986).
According to another approach described in WO 96/2701 l, the interface between
a
pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
which are recovered from recombinant cell culture. The preferred interface
comprises at
least a part of the CH3 region of an antibody constant domain. In this method,
one or
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more small amino acid side chains from the interface of the first antibody
molecule are
replaced with larger side chains (e.g_ tyrosine or tryptophan). Compensatory
"cavities" of
identical or similar size to the large side chains) are created on the
interface of the second
antibody molecule by replacing large amino acid side chains with smaller ones
(e.g.
alanine or threonine). This provides a mechanism for increasing the yield of
the
heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating
bispecific
antibodies from antibody fragments have been described in the literature. For
example,
bispecific antibodies can be prepared using chemical linkage. Brennan et al.,
Science
229:81 (1985) describe a procedure wherein intact antibodies are
proteolytically cleaved to
generate F(ab')2 fragments. These fragments are reduced in the presence of the
dithiol
complexing agent sodium arsenite to stabilize vicinal 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.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., 3. 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
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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 Fv (sFv) dimers has
also been
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60
(1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of
which originates in the protein antigen of the invention. Alternatively, an
anti-antigenic
arm of an immunoglobulin molecule can be combined with an arm which binds to a
triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.
CD2, CD3,
CD28, or B7), or Fc receptors for IgG (FcYR), such as FcyRI (CD64), FcyRII
(CD32) and
FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell
expressing the
particular antigen. Bispecific antibodies can also be used to direct cytotoxic
agents to cells
which express a particular antigen. These antibodies possess an antigen-
binding arm and
an arm which binds a cytotoxic agent or a radionuclide chelator, such as
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 (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared
in vitro
using known methods in synthetic protein chemistry, including those involving
crosslinking agents. For example, immunotoxins can be constructed using a
disulfide
exchange reaction or by forming a thioether bond. Examples of suitable
reagents for this
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purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for
example, in IT.S. Patent No. 4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. 'For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing
interchain disulfide bond formation in this region. The homodimeric antibody
thus
generated can have improved internalization capability and/or increased
complement-mediated cell killing and antibody-dependent cellular cytotoxicity
(ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol.,
148:
2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can
also be
prepared using heterobifunctional cross-linkers as described in Wolff et al.
Cancer
Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered
that has
dual Fc regions and can thereby have enhanced complement lysis and ADCC
capabilities.
See Stevenson et al., Anti-Cancer Drug Desi~,n, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g.,
an
enzymatically active toxin of bacterial, fungal, plant, or animal origin, or
fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above. 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 2~ZBi, i3il, l3iln, 9oY, and
I$6Re.
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
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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
W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate
is administered to the patient, followed by removal of unbound conjugate from
the
circulation using a clearing agent and then administration of a "ligand"
(e.g., avidin) that is
I S in turn conjugated to a cytotoxic agent.
Immunoliposomes
The antibodies disclosed herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art,
such as
described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985);
Hwang et al.,
Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and
4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation
method with a lipid composition comprising phosphatidylcholine, cholesterol,
and
PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded
through
filters of defined pore size to yield liposomes with the desired diameter.
Fab' fragments of
the antibody of the present invention can be conjugated to the 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
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use in measuring levels of the protein within appropriate physiological
samples, for use in
diagnostic methods, for use in imaging the protein, and the like). In a given
embodiment,
antibodies against the proteins, or derivatives, fragments, analogs or
homologs thereof,
that contain the antigen binding domain, are utilized as pharmacologically-
active
compounds (see below).
An antibody specific for a protein of the invention can be used to isolate the
protein by standard techniques, such as immunoaffinity chromatography or
immunoprecipitation. Such an antibody can facilitate the purification of the
natural
protein antigen from cells and of recombinantly produced antigen expressed in
host cells.
Moreover, such an antibody can be used to detect the antigenic protein (e.g.,
in a cellular
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; examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material
includes luminol; examples of bioluminescent materials include luciferase,
luciferin, and
aequorin, and examples of suitable radioactive material include ~ZSI, lsih ssS
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 subject. An antibody
preparation,
preferably one having high specificity and high affinity for its target
antigen, is
administered to the subject and will generally have an effect due to its
binding with the
target. Such an effect may be one of two kinds, depending on the specific
nature of the
interaction between the given antibody molecule and the target antigen in
question. In the
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first instance, administration of the antibody may abrogate or inhibit the
binding of the
target with an endogenous ligand to which it naturally binds. In this case,
the antibody
binds to the target and masks a binding site of the naturally occurring
ligand, wherein the
ligand serves as an effector molecule. Thus the receptor mediates a signal
transduction
pathway for which ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a
physiological
result by virtue of binding to an effector binding site on the target
molecule. In this case
the target, a receptor having an endogenous Iigand which may be absent or
defective in the
disease or pathology, binds the antibody as a surrogate effector ligand,
initiating a
receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates
generally
to the amount needed to achieve a therapeutic objective. As noted above, this
may be a
binding interaction between the antibody and its target antigen that, in
certain cases,
interferes with the functioning of the target, and in other cases, promotes a
physiological
response. The amount required to be administered will furthermore depend on
the binding
affinity of the antibody for its specific antigen, and will also depend on the
rate at which
an administered antibody is depleted from the free volume other subject to
which it is
administered. Common ranges for therapeutically effective dosing of an
antibody or
antibody fragment of the invention may be, by way of nonlimiting example, from
about
0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies
may
range, for example, from twice daily to once a week.
Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a protein of the invention, as well as other
molecules identified by the screening assays disclosed herein, can be
administered for the
treatment of various disorders in the form of pharmaceutical compositions.
Principles and
considerations involved in preparing such compositions, as well as guidance in
the choice
of components are provided, for example, in Remington : The Science And
Practice Of
Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton,
Pa. : 1995;
Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And
Trends,
Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein
Drug
Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
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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,
albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in
macroemulsions.
The formulations to be used for in vivo administration must be sterile. This
is
readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of solid
hydrophobic
polymers containing the antibody, which matrices are in the form of shaped
articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices include
polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)),
polylactides (LT.S. 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 TM (injectable microspheres composed
of
lactic acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl
acetate and
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lactic acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels
release proteins for shorter time periods.
ELISA Assay
An agent for detecting an analyte protein is an antibody capable of binding to
an
analyte protein, preferably an antibody with a detectable label. Antibodies
can be
polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment
thereof
(e.g., Fab or F(ab)2) can be used. The term "labeled", with regard to the
probe or antibody,
is intended to encompass direct labeling of the probe or antibody by coupling
(i.e.,
physically linking) a detectable substance to the probe or antibody, as well
as indirect
labeling of the probe or antibody by reactivity with another reagent that is
directly labeled.
Examples of indirect labeling include detection of a primary antibody using a
fluorescently-labeled secondary antibody and end-labeling of a DNA probe with
biotin
such that it can be detected with fluorescently-labeled streptavidin. The term
"biological
sample" is intended to include tissues, cells and biological fluids isolated
from a subject,
as well as tissues, cells and fluids present within a subject. Included within
the usage of
the term "biological sample", therefore, is blood and a fraction or component
of blood
including blood serum, blood plasma, or lymph. That is, the detection method
of the
invention can be used to detect an analyte mRNA, protein, or genomic DNA in a
biological sample in vitro as well as in vivo. For example, ira vitro
techniques for detection
of an analyte mRNA include Northern hybridizations and in situ hybridizations.
In vitro
techniques for detection of an analyte protein include enzyme linked
immunosorbent
assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
In vitro
techniques for detection of an analyte genomic DNA include Southern
hybridizations.
Procedures for conducting immunoassays are described, for example in "ELISA:
Theory
and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.)
Human Press,
Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic
Press,
Inc., San Diego, CA, 1996; and "Practice and Theory 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.
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NOVX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding a NOVX protein, or derivatives, fragments,
analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule
capable of transporting another nucleic acid to which it has been linked. One
type of
vector is a "plasmid", which refers to a circular double stranded DNA loop
into which
additional DNA segments can be ligated. Another type of vector is a viral
vector, wherein
additional DNA segments can be ligated into the viral genome. Certain vectors
are
capable of autonomous replication in a host cell into which they are
introduced (e.g.,
bacterial vectors having a bacterial origin of replication and episomal
mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the
genome of a host cell upon introduction into the host cell, and thereby are
replicated along
with the host genome. Moreover, certain vectors are capable of directing the
expression of
genes to which they are operatively-linked. Such vectors are referred to
herein as
"expression vectors". In general, useful expression vectors in recombinant DNA
techniques are often in the form of plasmids. In the present specification,
"plasmid" and
"vector" can be used interchangeably as the plasmid is the most commonly used
form of
vector. However, the invention is intended to include such other forms of
expression
vectors, such as viral vectors (e.g., replication defective retroviruses,
adenoviruses and
adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means
that the recombinant expression vectors include one or more regulatory
sequences,
selected on the basis of the host cells to be used for expression, that is
operatively-Linked
to the nucleic acid sequence to be expressed. Within a recombinant expression
vector,
"operably-linked" is intended to mean that the nucleotide sequence of interest
is linked to
the regulatory sequences) in a manner that allows for expression of the
nucleotide
sequence (e.g., in an in vitro transcription/translation system or in a host
cell when the
vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers
and
other expression control elements (e.g., polyadenylation signals). Such
regulatory
sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:
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METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory
sequences include those that direct constitutive expression of a nucleotide
sequence in
many types of host cell and those that direct expression of the nucleotide
sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It will be
appreciated by
those skilled in the art that the design of the expression vector can depend
on such factors
as the choice of the host cell to be transformed, the level of expression of
protein desired,
etc. The expression vectors of the invention can be introduced into host cells
to thereby
produce proteins or peptides, including fusion proteins or peptides, encoded
by nucleic
acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins,
fusion
proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of NOVX proteins in prokaryotic or eukaryotic cells. For example,
NOVX
proteins can be expressed in bacterial cells such as 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 izz vitro, for
example
using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia
coli
with vectors containing constitutive or inducible promoters directing the
expression of
either fusion or non-fusion proteins. Fusion vectors add a number of amino
acids to a
protein encoded therein, usually to the amino terminus of the recombinant
protein. Such
fusion vectors typically serve three purposes: (i) to increase expression of
recombinant
protein; (ii) to increase the solubility of the recombinant protein; and (iii)
to aid in the
purification of the recombinant protein by acting as a ligand in affinity
purification.
Often, in fusion expression vectors, a proteolytic cleavage site is introduced
at the junction
of the fusion moiety and the recombinant protein to enable separation of the
recombinant
protein from the fusion moiety subsequent to purification of the fusion
protein. Such
enzymes, and their cognate recognition sequences, include Factor Xa, thrombin
and
enterokinase. Typical fusion expression vectors include pGEX (Pharmacia
Biotech Inc;
Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,
Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-
transferase
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(GST), maltose E binding protein, or protein A, respectively, to the target
recombinant
protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET 11 d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990)
60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express
the protein in a host bacteria with an impaired capacity to proteolytically
cleave the
recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another
strategy
is to alter the nucleic acid sequence of the nucleic acid to be inserted into
an expression
vector so that the individual codons for each amino acid are those
preferentially utilized in
E. c~li (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such
alteration of
nucleic acid sequences of the invention can be carned out by standard DNA
synthesis
1 S techniques.
In another embodiment, the NOVX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Sacclzaromyces cerivisae include
pYepSec 1
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell
30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2
(Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,
Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells
(e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell.
Biol. 3: 2156-2165)
and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDMB (Seed, 1987. Nature 329: 840) and pMT2PC
(Kaufinan, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells,
the
expression vector's control functions are often provided by viral regulatory
elements. For
example, commonly used promoters are derived from polyoma, adenovirus 2,
cytomegalovirus, and simian virus 40. For other suitable expression systems
for both
prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et
al.,
CA 02486490 2004-12-03
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MOLECULAR CLONING: A LABORATORY MAt~ItlAZ.. 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 axe used to express the nucleic acid).
Tissue-specific
regulatory elements are known in the art. Non-limiting examples of suitable
tissue-specif c promoters include the albumin promoter (liver-specific;
Pinkert, et al.,
1987. Genes Dev. 1: 268-277), lymphoid-speciEc promoters (Calame and Eaton,
1988.
Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors
(Winoto and
Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al.,
1983. Cell
33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific
promoters
(e.g., the neuroflament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad.
Sci. USA 86:
5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230:
9I2-916), and
mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,3I6
I S and European Application Publication No. 264,166). Developmentally-
regulated
promoters are also encompassed, e.g., the murine hox promoters (Kessel and
Gruss, 1990.
Science 249: 374-379) and the oc-fetoprotein promoter (Campes and Tilghman,
1989.
Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation.
That is, the DNA molecule is operatively-linked to a regulatory sequence in a
manner that
allows for expression (by transcription of the DNA molecule) of an RNA
molecule that is
antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic
acid
cloned in the antisense orientation can be chosen that direct the continuous
expression of
the antisense RNA molecule in a variety of cell types, for instance viral
promoters and/or
enhancers, or regulatory sequences can be chosen that direct constitutive,
tissue specific or
cell type specific expression of antisense RNA. The antisense expression
vector can be in
the form of a recombinant plasmid, phagemid or attenuated virus in which
antisense
nucleic acids are produced under the control of a high efficiency regulatory
region, the
activity of which can be determined by the cell type into which the vector is
introduced.
For a discussion of the regulation of gene expression using antisense genes
see, e.g.,
Weintraub, et al., "Antisense RNA as a molecular tool for genetic analysis,"
Reviews-Trends in Genetics, Vol. 1(I) 1986.
7I
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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
transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory
manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Various selectable
markers include those that confer resistance to drugs, such as 6418,
hygromycin and
methotrexate. Nucleic acid encoding a selectable marker can be introduced into
a host cell
on the same vector as that encoding NOVX or can be introduced on a separate
vector.
Cells stably transfected with the introduced nucleic acid can be identified by
drug
selection (e.g., cells that have incorporated the selectable marker gene will
survive, while
the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture,
can be used to produce (i. e., express) NOVX protein. Accordingly, the
invention further
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provides methods for producing NOVX protein using the host cells of the
invention. In
one embodiment, the method comprises culturing the host cell of invention
(into which a
recombinant expression vector encoding NOVX protein has been introduced) in a
suitable
medium such that NOVX protein is produced. In another embodiment, the method
further
comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized
oocyte or an embryonic stem cell into which NOVX protein-coding sequences have
been
introduced. Such host cells can then be used to create non-human transgenic
animals in
which exogenous NOVX sequences have been introduced into their genome or
homologous recombinant animals in which endogenous NOVX sequences have been
altered. Such animals are useful for studying the function and/or activity of
NOVX
protein and for identifying and/or evaluating modulators of NOVX protein
activity. As
used herein, a "transgenic animal" is a non-human animal, preferably a mammal,
more
preferably a rodent such as a rat or mouse, in which one or more of the cells
of the animal
includes a transgene. Other examples of transgenic animals include non-human
primates,
sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous
DNA that
is integrated into the genome of a cell from which a transgenic animal
develops and that
remains in the genome of the mature animal, thereby directing the expression
of an
encoded gene product in one or more cell types or tissues of the transgenic
animal. As
used herein, a "homologous recombinant animal" is a non-human animal,
preferably a
r
mammal, more preferably a mouse, in which an endogenous NOVX gene has been
altered
by homologous recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic cell of the
animal, prior
to development of the animal.
A transgenic animal of the invention can be created by introducing
NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte
(e.g., by
microinjection, retroviral infection) and allowing the oocyte to develop in a
pseudopregnant female foster animal. The human NOVX cDNA sequences, i.e., any
one
of SEQ ID NOS:2n-l, wherein n is an integer between 1 and I41, can be
introduced as a
transgene into the genome of a non-human animal. Alternatively, a non-human
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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 andlor
expression of NOVX mRNA in tissues or cells of the animals. A transgenic
founder
animal can then be used to breed additional animals carrying the transgene.
Moreover,
transgenic animals carrying a transgene-encoding NOVX protein can further be
bred to
other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at least a portion of a NOVX gene into which a deletion, addition or
substitution has been
introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The
NOVX gene
can be a human gene (e.g., the cDNA of any one of SEQ ID NOS:2n-1, wherein n
is an
integer between I and 141), but more preferably, is a non-human homologue of a
human
NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID
NOS:2n-1, wherein n is an integer between 1 and 141, can be used to construct
a
homologous recombination vector suitable for altering an endogenous NOVX gene
in the
mouse genome. In one embodiment, the vector is designed such that, upon
homologous
recombination, the endogenous NOVX gene is functionally disrupted (i.e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination, the endogenous NOVX gene is mutated or otherwise altered but
still
encodes functional protein (e.g., the upstream regulatory region can be
altered to thereby
alter the expression of the endogenous NOVX protein). In the homologous
recombination
vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-
termini by
additional nucleic acid of the NOVX gene to allow for homologous recombination
to
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occur between the exogenous NOVX gene carried by the vector and an endogenous
NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid
is of
sufficient length for successful homologous recombination with the endogenous
gene.
Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini)
are included
in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description
of homologous
recombination vectors. The vector is ten introduced into an embryonic stem
cell line (e.g.,
by electroporation) and cells in which the introduced NOVX gene has
homologously-recombined with the endogenous NOVX gene are selected. See, e.g.,
Li, et
al., 1992. Cell 69: 915.
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 ANn
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. Opirz. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO
90/I 1354;
WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain selected systems that allow for regulated expression of the transgene.
One
example of such a system is the cre/IoxP recombinase system of bacteriophage
P1. For a
description of the cre/loxP recombinase system, See, e.g., Lakso, et al.,
1992. Proc. Natl.
Acad. Sci. ZISA 89: 6232-6236. Another example of a recombinase system is the
FLP
recombinase system of Sacclzarornyces cerevisiae. See, O'Gorman, et al., 1991.
Science
251:1351-1355. If a cre/loxP recombinase system is used to regulate expression
of the
transgene, animals containing transgenes encoding both the Cre recombinase and
a
selected protein are required. Such animals can be provided through the
construction of
"double" transgenic animals, e.g., by mating two transgenic animals, one
containing a
transgene encoding a selected protein and the other containing a transgene
encoding a
recombinase.
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Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilmut, et al., 1997. Nature 3 ~5: ~ 10-
~ 13. 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 carrier" is intended to include any
and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents, and the like, compatible with pharmaceutical
administration.
Suitable carriers are described in the most recent edition of Remington's
Pharmaceutical
Sciences, a standard reference text in the field, which is incorporated herein
by reference.
Preferred examples of such carriers or diluents include, but are not limited
to, water,
saline, finger's solutions, dextrose solution, and 5% human serum albumin.
Liposomes
and non-aqueous vehicles such as fixed oils may also be used. The use of such
media and
agents for pharmaceutically active substances is well known in the art. Except
insofar as
any conventional media or agent is incompatible with the active compound, use
thereof in
the compositions is contemplated. Supplementary active compounds can also be
incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with
its intended route of administration. Examples~of routes of administration
include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation), transdermaI
(i.e., topical), transmucosal, and rectal administration. Solutions or
suspensions used for
parenteral, intradermal, or subcutaneous application can include the following
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components: a sterile diluent such as water for injection, saline solution,
fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial
agents such as benzyl alcohol or methyl parabens; antioxidants such as
ascorbic acid or
sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid
(EDTA);
buffers such as acetates, citrates or phosphates, and agents for the
adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with acids or
bases, such as
hydrochloric acid or sodium hydroxide. The parenteral preparation can be
enclosed in
ampoules, disposable syringes or multiple dose vials made of glass or plastic.
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 Garners 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 Garner can be a solvent or dispersion medium containing, for example,
water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be maintained,
for example,
by the use of a coating such as lecithin, by the maintenance of the required
particle size in
the case of dispersion and by the use of surfactants. Prevention of the action
of
microorganisms can be achieved by various antibacterial and antifungal agents,
for
example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like. In many
cases, it will be preferable to include isotonic agents, for example, sugars,
polyalcohols
such as manitol, sorbitol, sodium chloride in the composition. Prolonged
absorption of the
injectable compositions can be brought about by including in the composition
an agent
which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound
(e.g., 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
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preparation of sterile injectabIe solutions, methods of preparation are vacuum
drying and
freeze-drying that yields a powder of the active ingredient plus any
additional desired
ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can
be enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral
therapeutic administration, the active compound can be incorporated with
excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also
be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid Garner is
applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible
binding agents, and/or adjuvant materials can be included as part of the
composition. °The
tablets, pills, capsules, troches and the 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
barner to be
permeated are used in the formulation. Such penetrants are generally known in
the art,
and include, for example, for transmucosal administration, detergents, bile
salts, and
fusidic acid derivatives. Transmucosal administration can be accomplished
through the
use of nasal sprays or suppositories. For transdermal administration, the
active
compounds are formulated into ointments, salves, gels, or creams as generally
known in
the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention
enemas for rectal delivery.
In one embodiment, the active compounds-are prepared with Garners that will
protect the compound against rapid elimination from the body, such as a
controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
7~
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biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation
of such formulations will be apparent to those skilled in the art. The
materials can also be
obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal
antibodies to
viral antigens) can also be used as pharmaceutically acceptable Garners. 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
Garner. The specification for the dosage unit forms of the invention are
dictated by and
1 S directly dependent on the unique characteristics of the active compound
and the particular
therapeutic effect to be achieved, and the limitations inherent in the art of
compounding
such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and
used
as gene therapy vectors. Gene therapy vectors can be delivered to a subject
by, for
example, intravenous.injection, local administration (see, e.g., U.S. Patent
No. 5,328,470)
or by stereotactic injection (see, e.g:, Chen, et al., 1994. Proc. Natl.
A,cad. Sci. USA 91:
3054-3057). The pharmaceutical preparation of the gene therapy vector can
include the
gene therapy vector in an acceptable diluent, or can comprise a slow release
matrix in
which the gene delivery vehicle is imbedded. Alternatively, where the complete
gene
delivery vector can be produced intact from recombinant cells, e.g.,
retroviral vectors, the
pharmaceutical preparation can include one or more cells that produce the gene
delivery
system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy
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applications), to detect NOVVX mRNA (e.g., in a biological sample) or a
genetic lesion in a
NOVX gene, and to modulate NOVX activity, as described further, below. In
addition,
the NOVX proteins can be used to screen drugs or compounds that modulate the
NOVX
protein activity or expression as well as to treat disorders characterized by
insufficient or
excessive production of NOVX protein or production of NOVX protein forms that
have
decreased or aberrant activity compared to NOVX wild-type protein (e.g.;
diabetes
(regulates insulin release); obesity (binds and transport lipids); metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting
disorders associated with chronic diseases and various cancers, and infectious
disease(possesses anti-microbial activity) and the various dyslipidemias. In
addition, the
anti-NOVX antibodies of the invention can be used to detect and isolate NOVX
proteins
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 thereof. The test
compounds
of the invention can be obtained using any of the numerous approaches in
combinatorial
library methods known in the art, including: biological libraries; spatially
addressable
parallel solid phase or solution phase libraries; synthetic library methods
requiring
deconvolution; the "one-bead one-compound" library method; and synthetic
library
methods using affinity chromatography selection. The biological library
approach is
limited to peptide libraries, while the other four approaches are applicable
to peptide,
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non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam,
1997.
Anticarzcer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about 5 kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules. Libraries of
chemical
and/or biological mixtures, such as fungal, bacterial, or algal extracts, are
known in the art
and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in
the
art, for example in: DeWitt, et al., 1993. 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. Chezn.
37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew.
Clzem. Irzt. 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.
Bioteclzzziques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on
chips
(Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No.
5,223,409), spores
(Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl.
Acad. Sci. USA
89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390;
Devlin, 1990.
Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:
6378-6382;
Felici, 1991. J. Mol. Biol_ 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses
a membrane-bound form of NOVX protein, or a biologically-active portion
thereof, on the
cell surface is contacted with a test compound and the ability of the test
compound to bind
to a NOVX protein determined. The cell, for example, can of mammalian origin
or a yeast
cell. Determining the ability of the test compound to bind to the NOVX protein
can be
accomplished, for example, by coupling the test compound with a radioisotope
or
enzymatic label such that binding of the test compound to the NOVX protein or
biologically-active portion thereof can be determined by detecting the labeled
compound
in a complex. For example, test compounds can be labeled with ~ZSI, 3sS, iaC,
or 3H, either
directly or indirectly, and the radioisotope detected by direct counting of
radioemission or
by scintillation counting. Alternatively, test compounds can be enzymatically-
labeled
with, for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the
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enzymatic label detected by determination of conversion of an appropriate
substrate to
product. In one embodiment, the assay comprises contacting a cell which
expresses a
membrane bound form of NOVX protein, or a biologically-active portion thereof,
on the
cell surface with a known compound which binds NOVX to form an assay mixture,
contacting the assay mixture with a test compound, and determining the ability
of the test
compound to interact with a NOVX protein, wherein determining the ability of
the test
compound to interact with a NOVX protein comprises determining the ability of
the test
compound to preferentially bind to NOVX protein or a biologically-active
portion thereof
as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane bound form of NOVX protein, or a biologically-active
portion
thereof, on the cell surface with a test compound and determining the ability
of the test
compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX
protein or
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
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molecule can be determined by detecting induction of a cellular second
messenger of the
target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting
catalyticlenzymatic
activity of the target an appropriate substrate, detecting the induction of a
reporter gene
(comprising a NOVX-responsive regulatory element operatively linked to a
nucleic acid
encoding a detectable marker, e.g., luciferase), or detecting a cellular
response, for
example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay
comprising contacting a NOVX protein or biologically-active portion thereof
with a test
compound and determining the ability of the test compound to bind to the NOVX
protein
or biologically-active portion thereof. Binding of the test compound to the
NOVX protein
can be determined either directly or indirectly as described above. In one
such
embodiment, the assay comprises contacting the NOVX protein or biologically-
active
portion thereof with a known compound which binds NOVX to form an assay
mixture,
contacting the assay mixture with a test compound, and determining the ability
of the test
compound to interact with a NOVX protein, wherein determining the ability of
the test
compound to interact with a NOVX protein comprises determining the ability of
the test
compound to preferentially bind to NOVX or biologically-active portion thereof
as
compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising
contacting
NOVX protein or biologically-active portion thereof with a test compound and
determining the ability of the test compound to modulate (e.g. stimulate or
inhibit) the
activity of the NOVX protein or biologically-active portion thereof.
Determining the
ability of the test compound to modulate the activity of NOVX can be
accomplished, for
example, by determining the ability of the NOVX protein to bind to a NOVX
target
molecule by one of the methods described above for determining direct binding.
In an
alternative embodiment, determining the ability of the test compound to
modulate the
activity of NOVX protein can be accomplished by determining the ability of the
NOVX
protein further modulate a NOVX target molecule. For example, the
catalytic%nzy~natic
activity of the target molecule on an appropriate substrate can be determined
as described,
supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically-active portion thereof with a known compound which
binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test
compound, and
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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, n-dodecylglucoside, n-dodecylinaltoside, octanoyl-N-
methylglucamide,
decanoyl-N-methylglucamide, Triton~ X-I00, Triton~ X-114, Thesit~,
Isotridecypoly(ethylene glycol ether)n, N-dodecyl--N,N-dimethyl-3-ammonio-1-
propane
sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it
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 be dissociated from the matrix, and the level of NOVX
protein binding
or activity determined using standard techniques.
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Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example, either the NOVX protein or its
target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g.,
biotinylation
kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated
96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX
protein
or target molecules, but which do not interfere with binding of the NOVX
protein to its
target molecule, can be deriyatized to the wells of the plate, and unbound
target or NOVX
protein trapped in the wells by antibody conjugation. Methods for detecting
such
complexes, in addition to those described above for the GST-immobilized
complexes,
include immunodetection of complexes using antibodies reactive with the NOVX
protein
or target molecule, as well as enzyme-linked assays that rely on detecting an
enzymatic
activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in
a method wherein a cell is contacted with a candidate compound and the
expression of
NOVX mRNA or protein in the cell is determined. The level of expression of
NOVX
mRNA or protein in the presence of the candidate compound is compared to the
level of
expression of NOVX mRNA or protein in the absence of the candidate compound.
The
candidate compound can then be identified as a modulator of NOVX mRNA or
protein
expression based upon this comparison. For example, when expression of NOVX
mRNA
or protein is greater (i.e., statistically significantly greater) in the
presence of the candidate
compound than in its absence, the candidate compound is identified as a
stimulator of
NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA
or
protein is less (statistically significantly less) in the presence of the
candidate compound
than in its absence, the candidate compound is identified as an inhibitor
ofNOVX mRNA
or protein expression. The level of NOVX mRNA or protein expression in the
cells can be
determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait
proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent
No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Claem.
268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et
al., 1993.
Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that
bind to
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or interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX
activity. Such NOVX-binding proteins are also likely to be involved in the
propagation of
signals by the NOVX proteins as, for example, upstream or downstream elements
of the
NOVX pathway.
The two-hybrid system is based on the modular nature of most transcription
factors, Which consist of separable DNA-binding and activation domains.
Briefly, the
assay utilizes two different DNA constructs. In one construct, the gene that
codes for
NOVX is fused to a gene encoding the DNA binding domain of a known
transcription
factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library
of DNA
sequences, that encodes an unidentified protein ("prey" or "sample") is fused
to a gene that
codes for the activation domain of the known transcription factor. If the
"bait" and the
"prey" proteins are able to interact, in vivo, forming 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 a NOVX
sequence,
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i. e., of SEQ ID NOS:2n-1, wherein n is an integer between 1 and 141, or
fragments or
derivatives thereof, can be used to map the location of the NOVX genes,
respectively, on a
chromosome. The mapping of the NOVX sequences to chromosomes is an important
first
step in correlating these sequences with genes associated, with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the NOVX sequences. Computer analysis of
the
NOVX, sequences can be used to rapidly select primers that do not span more
than one
exon in the genomic DNA, thus complicating the amplification process. These
primers
can then be used for PCR screening of somatic cell hybrids containing
individual human
chromosomes. Only those hybrids containing the human gene corresponding to the
NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different
mammals
(e.g., human and mouse cells). As hybrids of human and mouse cells grow and
divide,
they gradually lose human chromosomes in random order, but retain the mouse
chromosomes. By using media in which mouse cells cannot grow, because they
lack a
particular enzyme, but in which human cells can, the one human chromosome that
contains the gene encoding the needed enzyme will be retained. By using
various media,
panels of hybrid cell lines can be established. Each cell line in a panel
contains either a
single human chromosome or a small number of human chromosomes, and a full set
of
mouse chromosomes, allowing easy mapping of individual genes to specific human
chromosomes. See, e.g., D'Eustachio, et al., 193. Seience 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
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of light and dark bands develops on each chromosome, so that the chromosomes
can be
identified individually. The FISH technique can be used with a DNA sequence as
short as
500 or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of
binding to a unique chromosomal location with sufficient signal intensity for
simple
detection. Preferably 1,000 bases, and more preferably 2,000 bases, will
suffice to get
good results at a reasonable amount of time. For a review of this technique,
see, Verma, et
al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New
York 1988).
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding regions of the genes actually are preferred for mapping purposes.
Coding
sequences are more likely to be conserved within gene families, thus
increasing the chance
of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data.
Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN,
available
on-line through Johns Hopkins University Welch Medical Library). The
relationship
between genes and disease, mapped to the same chromosomal region, can then be
identified through linkage analysis (co-inheritance of physically adjacent
genes), described
in, e.g., Egeland, et al., 1987. Nature, 325: 783-787.
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.
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Tissue Typing
The NOVX sequences of the invention can also be used to identify individuals
from minute biological samples. In this technique, an individual's genomic DNA
is
digested with one or more restriction enzymes, and probed on a Southern blot
to yield
unique bands for identification. The sequences of the invention are useful as
additional
DNA markers for RFLP ("restriction fragment length polymorphisms," described
in U.S.
Patent No. 5,272,057).
Furthermore, the sequences of the invention can be used to provide an
alternative
technique that determines the actual base-by-base DNA sequence of selected
portions of
an individual's genome. Thus, the NOVX sequences described herein can be used
to
prepare two PCR primers from the 5'- and 3'-termini of the sequences. These
primers can
then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner, can provide unique individual identifications, as each individual will
have a
unique set of such DNA sequences due to allelic differences. The sequences of
the
invention can be used to obtain such identification sequences from individuals
and from
tissue. The NOVX sequences of the invention uniquely represent portions of the
human
genome. Allelic variation occurs to some degree in the coding regions of these
sequences,
and to a greater degree in the noncoding regions. It is estimated that allelic
variation
between individual humans occurs with a frequency of about once per each 500
bases.
Much of the allelic variation is due to single nucleotide polymorphisms
(SNPs), which
include restriction fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes.
Because greater numbers of polymorphisms occur in the noncoding regions, fewer
sequences are necessary to differentiate individuals. The noncoding sequences
can
comfortably provide positive individual identification with a panel of perhaps
10 to 1,000
primers that each yield a noncoding amplified sequence of 100 bases. If coding
sequences, such as those of SEQ ID NOS:2n-l, wherein n is an integer between 1
and 141,
are used, a more appropriate number of primers for positive individual
identification
would be 500-2,000.
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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 andlor nucleic acid expression as well as NOVX activity, in the
context of
a biological sample (e.g., blood, serum, cells, tissue) to thereby determine
whether an
individual is afflicted with a disease or disorder, or is at risk of
developing a disorder,
associated with aberrant NOVX expression or activity. The disorders include
metabolic
disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated
cachexia,
cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's
Disorder, immune
disorders, and hematopoietic disorders, and the various dyslipidemias,
metabolic
disturbances associated with obesity, the metabolic syndrome X and wasting
disorders
associated with chronic diseases and various cancers. The invention also
provides for
prognostic (or predictive) assays for determining whether an individual is at
risk of
developing a disorder associated with NOVX protein, nucleic acid expression or
activity.
For example, mutations in a NOVX gene can be assayed in a biological sample.
Such
assays can be used for prognostic or predictive purpose to thereby
prophylactically treat an
individual prior to the onset of a disorder characterized by or associated
with NOVX
protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein,
nucleic acid expression or activity in an individual to thereby select
appropriate
therapeutic or prophylactic agents for that individual (referred to herein as
"pharmacogenomics"). Pharmacogenomics allows 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.
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Diagnostic Assays
An exemplary method for detecting the presence or absence of NOVX in a
biological sample involves obtaining a biological sample from a test subject
and
contacting the biological sample with a compound or an agent capable of
detecting NOVX
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein
such that
the presence of NOVX is detected in the biological sample. An agent for
detecting NOVX
mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to
NOVX
mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length
NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS:2n-1, wherein n is
an integer
between 1 and 141, or a portion thereof, such as an oligonucleotide of at
least 15, 30, 50,
100, 250 or 500 nucleotides in length and sufficient to specifically hybridize
under
stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for
use in
the diagnostic assays of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be
polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,
Fab or
F(ab')2) can be used. The term "labeled", with regard to the probe or
antibody, is intended
to encompass direct labeling of the probe or antibody by coupling (i.e.,
physically linking)
a detectable substance to the probe or antibody, as well as indirect labeling
of the probe or
antibody by reactivity with another reagent that is directly labeled. Examples
of indirect
labeling include detection of a primary antibody using a fluorescently-labeled
secondary
antibody and end-labeling of a DNA probe with biotin such that it can be
detected with
fluorescently-labeled streptavidin. The term "biological sample" is intended
to include
tissues, cells and biological fluids isolated from a subject, as well as
tissues, cells and
fluids present within a subject.. That is, the detection method of the
invention can be used
to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro
as well
as in vivo. For example, in vitro techniques for detection of NOVX mRNA
include
Northern hybridizations and izz 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
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antibody. For example, the antibody can be labeled with a radioactive marker
whose
presence and location in a subject can be detected by standard imaging
techniques.
In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test
subject or genomic DNA molecules from the test subject. A preferred biological
sample is
a peripheral blood leukocyte sample isolated by conventional means from a
subject.
In another embodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
capable of detecting NOVX protein, mRNA, or genomic DNA, such that the
presence of
NOVX protein, mRNA or genomic DNA is detected in the biological sample, and
comparing the presence of NOVX protein, mRNA or genomic DNA in the control
sample
with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a
biological sample. For example, the kit can comprise: a labeled compound or
agent
capable of detecting NOVX protein or mRNA in a biological sample; means for
determining the amount of NOVX in the sample; and means for comparing the
amount of
NOVX in the sample with a standard. The compound or agent can be packaged in a
suitable container. The kit can further comprise instructions for using the
kit to detect
NOVX protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant
NOVX expression or activity. For example, the assays described herein, such as
the
preceding diagnostic assays or the following assays, can be utilized to
identify a subject
having or at risk of developing a disorder associated with NOVX protein,
nucleic acid
expression or activity. Alternatively, the prognostic assays can be utilized
to identify a
subject having or at risk for developing a disease or disorder. Thus, the
invention provides
a method for identifying a disease or disorder associated with aberrant NOVX
expression
or activity in which a test sample is obtained from a subject and NOVX protein
or nucleic
acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX
protein or
nucleic acid is diagnostic for a subject having or at risk of developing a
disease or disorder
associated with aberrant NOVX expression or activity. As used herein, a "test
sample"
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refers to a biological sample obtained from a subject of interest. For
example, a test
sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein,~peptide, nucleic acid, small molecule, or other drug
candidate) to
treat a disease or disorder associated with aberrant NOVX expression or
activity. For
example, such methods can be used to determine whether a subject can be
effectively
treated with an agent for a disorder. Thus, the invention provides methods for
determining
whether a subject can be effectively treated with an agent for a disorder
associated with
aberrant NOVX expression or activity in which a test sample is obtained and
NOVX
protein or nucleic acid is detected (e.g., wherein the presence of NOVX
protein or nucleic
acid is diagnostic for a subject that can be administered the agent to treat a
disorder
associated with aberrant NOVX expression or activity).
The methods of the invention can also be used to detect genetic lesions in a
NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a
disorder
characterized by aberrant cell proliferation and/or differentiation. In
various
embodiments, the methods include detecting, in a sample of cells from the
subject, the
presence or absence of a genetic lesion characterized by at least one of an
alteration
affecting the integrity of a gene encoding a NOVX-protein, or the
misexpression of the
NOVX gene. For example, such genetic lesions can be detected by ascertaining
the
existence of at least one of (i) a deletion of one or more nucleotides from a
NOVX gene;
(ii) an addition of one or more nucleotides to a NOVX gene; (iii) a
substitution of one or
more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX
gene;
(v) an alteration in the level of a messenger RNA transcript of a NOVX gene,
(vi) aberrant
modification of a NOVX gene, such as of the methylation pattern of the genomic
DNA,
(vii) the presence of a non-wild-type splicing pattern of a messenger RNA
transcript of a
NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss
of a NOVX
gene, and (x) inappropriate post-translational modification of a NOVX protein.
As
described herein, there are a large number of assay techniques known in the
art which can
be used for detecting lesions in a NOVX gene. A preferred biological sample is
a
peripheral blood leukocyte sample isolated by conventional means from a
subject.
However, any biological sample containing nucleated cells may be used,
including, for
example, buccal mucosal cells.
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In certain embodiments, detection of the lesion involves the use of a
probe/primer
in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195
and
4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation
chain
reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080;
and
Nakazawa, et al., 1994. Pr~c. Natl. Acad. Sei. USA 91: 360-364), the latter of
which can
be particularly useful for detecting point mutations in the NOVX-gene (see,
Abravaya, et
al., 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of
collecting a
sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or
both) from
the cells of the sample, contacting the nucleic acid sample with one or more
primers that
specifically hybridize to a NOVX gene under conditions such that hybridization
and
amplification of the NOVX gene (if present) occurs, and detecting the presence
or absence
of an amplification product, or detecting the size of the amplification
product and
comparing the length to a control sample. It is anticipated that PCR and/or
LCR may be
desirable to use as a preliminary amplification step in conjunction with any
of the
techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication
(see,
Guatelli, et al., 1990. Proc. Natl. Aead. Sci. USA 87: 1874-1878),
transcriptional
amplification system (see, Kwoh, et al., 1989. Prac. Natl. Acad. Sci. USA 86:
1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTechn~logy 6: 1197), or any other
nucleic acid
amplification method, followed by the detection of the amplified molecules
using
techniques well known to those of skill in the art. These detection schemes
are especially
useful for the detection of nucleic acid molecules if such molecules are
present in very low
numbers.
In an alternative embodiment, mutations in a NOVX gene from a sample cell can
be identified by alterations in restriction enzyme cleavage patterns. For
example, sample
and control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and
compared. Differences in fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the
presence of
specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by
hybridizing sample and control nucleic acids, e.g., DNA or RNA to high-density
arrays
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containing hundreds or thousands of oligonucleotide 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. Aead: Sci. LISA 74: 560 or Sanger, 1977. Proc.
Natl. Acad.
Sci. TISA 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. Adu
Chromatography
36: 127-162; and Grin, 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
RNAlRNA
or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Scierzce 230: 1242.
In
general, the art technique of "mismatch cleavage" starts by providing
heteroduplexes of
formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX
sequence
with potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded
duplexes are treated with an agent that cleaves single-stranded regions of the
duplex such
as which will exist due to basepair mismatches between the control and sample
strands.
For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with SI nuclease to enzymatically digesting the mismatched regions. In
other
embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with
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hydroxylamine or osmium tetroxide and with piperidine in order to digest
mismatched
regions. After digestion of the mismatched regions, the resulting material is
then
separated by size on denaturing polyacrylamide gels to determine the site of
mutation.
See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba,
et al., 1992.
Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be
labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point
mutations
in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of
E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa
cells
cleaves T at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15:
1657-1662.
According to an exemplary embodiment, a probe based on a NOVX sequence, e.g.,
a
wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a
test
cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the
cleavage
products, if any, can be detected from electrophoresis protocols or the like.
See, e.g., U.S.
Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify mutations in NOVX genes. For example, single strand conformation
polymorphism (SSCP) may be used to detect differences in electrophoretic
mobility
between mutant and wild type nucleic acids. See, e.g., Orita, et al., 1989.
Proc. Natl.
Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi,
1992. Geraet.
Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and
control NOVX
nucleic acids will be denatured and allowed to renature. The secondary
structure of
single-stranded nucleic acids varies according to sequence, the resulting
alteration in
electrophoretic mobility enables the detection of even a single base change.
The DNA
fragments may be labeled or detected with labeled probes. The sensitivity of
the assay
may be enhanced by using RNA (rather than DNA), in which the secondary
structure is
more sensitive to a change in sequence. In one embodiment, the subject method
utilizes
heteroduplex analysis to separate double stranded heteroduplex molecules on
the basis of
changes in electrophoretic mobility. See, e.g., Keen, et al.; 1991. Trends
Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing
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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. Bioplzys. 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. Acad. Sci. USA 86: 6230.
Such allele
specific oligonucleotides are hybridized to PCR amplified target DNA or a
number of
different mutations when the oligonucleotides are attached to the hybridizing
membrane
and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on
selective
PCR amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the
mutation of
interest in the center of the molecule (so that amplification depends on
differential
hybridization; see, e.g., Gibbs, et al., 1989. Nuel. Acids Res. 17: 2437-2448)
or at the
extreme 3'-terminus of one primer where, under appropriate conditions,
mismatch can
prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech.
11: 238). In
addition it may be desirable to introduce a novel restriction site in the
region of the
mutation to create cleavage-based detection. See, e.g., Gasparini, et al.,
1992. Mol. Cell
Probes 6: 1. It is anticipated that in certain embodiments amplification may
also be
performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc.
Natl. Acad.
Sci. USA 88: 189. In such cases, ligation will occur only if there is a
perfect match at the
3'-terminus of the 5' sequence, making it possible to detect the presence of a
known
mutation at a specific site by looking for the presence or absence of
amplification.
The methods described herein may be performed, for example, by utilizing
pre-packaged diagnostic kits comprising at least one probe nucleic acid or
antibody
reagent described herein, which may be conveniently used, e.g., in clinical
settings to
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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 NOVA is expressed may be utilized in the prognostic assays described
herein.
However, any biological sample containing nucleated cells may be used,
including, for
example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity (e.g., NOVX gene expression), as identified by a screening assay
described herein
can be administered to individuals to treat (prophylactically or
therapeutically) disorders
(The disorders include metabolic disorders, diabetes, obesity, infectious
disease, anorexia,
cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's
Disease,
Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the
various
dyslipidemias, metabolic disturbances associated with obesity, the metabolic
syndrome X
and wasting disorders associated with chronic diseases and various cancers.)
In
conjunction with such treatment, the pharmacogenomics (i.e., the study of the
relationship
between an individual's genotype and that individual's response to a foreign
compound or
drug) of the individual may be considered. Differences in metabolism of
therapeutics can
lead to severe toxicity or therapeutic failure by altering the relation
between dose and
blood concentration of the pharmacologically active drug. Thus, the
pharmacogenomics
of the individual permits the selection of effective agents (e.g., drugs) for
prophylactic or
therapeutic treatments based on a consideration of the individual's genotype.
Such
pharmacogenomics can further be used to determine appropriate dosages and
therapeutic
regimens. Accordingly, the activity of NOVX protein, expression of NOVX
nucleic acid,
or mutation content of NOVX genes in an individual can be determined to
thereby select
appropriate agents) for therapeutic or prophylactic treatment of the
individual.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected persons.
See e.g., Eichelbaum, 1996. Clirt. Exp. Pharrraacol. 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
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altering the way the body acts on drugs (altered drug metabolism). These
pharmacogenetic conditions can occur either as rare defects or as
polymorphisms. For
example, glucose-6-phosphate dehydrogenase (G6PD) deftciency is a common
inherited
enzymopathy in which the main clinical complication is hemolysis after
ingestion of
oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of
fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major determinant of both the intensity and duration of drug action. The
discovery of
genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase
2 (NAT
2) and cytochrome Pregnancy Zone Protein Precursor enzymes CYP2D6 and CYP2C19)
.
has provided an explanation as to why some patients do not obtain the expected
drug
effects or show exaggerated drug response and serious toxicity after taking
the standard
and safe dose of a drug. These polymorphisms are expressed in two phenotypes
in the
population, the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence
of PM is different among different populations. For example, the gene coding
for
CYP2D6 is highly polymorphic and several mutations have been identified in PM,
which
all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and side effects
when
they receive standard doses. If a metabolite is the active therapeutic moiety,
PM show no
therapeutic response, as demonstrated for the analgesic effect of codeine
mediated by its
CYP2D6-formed metabolite morphine. At the other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or
mutation content of NOVX genes in an individual can be determined to thereby
select
appropriate agents) for therapeutic or prophylactic treatment of the
individual. In
addition, pharmacogenetic studies can be used to apply genotyping of
polymorphic alleles
encoding drug-metabolizing enzymes to the identification of an individual's
drug
responsiveness phenotype. This knowledge, when applied to dosing or drug
selection, can
avoid adverse reactions or therapeutic failure and thus enhance therapeutic or
prophylactic
efficiency when treating a subject with a NOVX modulator, such as a modulator
identified
by one of the exemplary screening assays described herein.
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Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of NOVX (e.g., the ability to modulate aberrant cell proliferation
and/or
differentiation) can be applied not only in basic drug screening, but also in
clinical trials.
For example, the effectiveness of an agent determined by a screening assay as
described
herein to increase NOVX gene expression, protein levels, or upregulate NOVX
activity,
can be monitored in clinical trails of subjects exhibiting decreased NOVX gene
expression, protein levels, or downregulated NOVX activity. Alternatively, the
effectiveness of an agent determined by a screening assay to decrease NOVX
gene
expression, protein levels, or downregulate NOVX activity, can be monitored in
clinical
trails of subjects exhibiting increased NOVX gene expression, protein levels,
or
upregulated NOVX activity. In such clinical trials, the expression or activity
of NOVX
and, preferably, other genes that have been implicated in, for example, a
cellular
proliferation or immune disorder can be used as a "read out" or markers of the
immune
responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule)
that modulates NOVX activity (e.g., identified in a screening assay as
described herein)
can be identified. Thus, to study the effect of agents on cellular
proliferation disorders, for
example, in a clinical trial, cells can be isolated and RNA prepared and
analyzed for the
levels of expression of NOVX and other genes implicated in the disorder. The
levels of
gene expression (i.e., a gene expression pattern) can be quantified by
Northern blot
analysis or RT-PCR, as described herein, or alternatively by measuring the
amount of
protein produced, by one of the methods as described herein, or by measuring
the levels of
activity of NOVX or other genes. In this manner, the gene expression pattern
can serve as
a marker, indicative of the physiological response of the cells to the agent.
Accordingly,
this response state may be determined before, and at various points during,
treatment of
the individual with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness of treatment of a subject with an agent (e.g., an agonist,
antagonist, protein,
peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified
by the 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, mltNA, 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, mIRNA, 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, hypercoaguIation, 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.
Diseases 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; (ii) antibodies to an
aforementioned peptide;
(iii) nucleic acids encoding an aforementioned peptide; (iv) administration of
antisense
nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a
heterologous insertion
within the coding sequences of coding sequences to an aforementioned peptide)
that are
utilized to "knockout" endogenous function of an aforementioned peptide by
homologous
recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v)
modulators
i.e., inhibitors, agonists and antagonists, including additional peptide
mimetic of the
invention or antibodies specific to a peptide of the invention) that alter the
interaction
between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that increase (i. e., are agonists to) activity. Therapeutics
that upregulate
activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that
may be utilized include, but are not limited to, an aforementioned peptide, or
analogs,
derivatives, fragments or homologs thereof; or an agonist that increases
bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide
and/or
RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and
assaying it in
vitro for RNA or peptide levels, structure and/or activity of the expressed
peptides (or
mRNAs of an aforementioned peptide). Methods that are well-known within the
art
include, but are not limited to, immunoassays (e.g., by Western blot analysis,
immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide
gel
electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to
detect
expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization,
and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a
disease or condition associated with an aberrant NOVX expression or activity,
by
administering to the subject an agent that modulates NOVX expression or at
least one
NOVX activity. Subjects at risk for a disease that is caused or contributed to
by aberrant
NOVX expression or activity can be identified by, for example, any or a
combination of
diagnostic or prognostic assays as described herein. Administration of a
prophylactic
agent can occur prior to the manifestation of symptoms characteristic of the
NOVX
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aberrancy, such that a disease or disorder is prevented or, alternatively,
delayed in its
progression. Depending upon the type of NOVX aberrancy, for example, a NOVX
agonist or NOVX antagonist agent can be used for treating the subject. The
appropriate
i
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-occurnng cognate ligand of a NOVX protein, a peptide, a NOVX
peptidomimetic, or other small molecule. In one embodiment, the agent
stimulates one or
more NOVX protein activity. Examples of such stimulatory agents include active
NOVX
protein and a nucleic acid molecule encoding NOVX that has been introduced
into the
cell. In another embodiment, the agent inhibits one or more NOVX protein
activity.
Examples of such inhibitory agents include antisense NOVX nucleic acid
molecules and
anti-NOVX antibodies. These modulatory methods can be performed in vitro
(e.g., by
culturing the cell with the agent) or, alternatively, in vivo (e.g., by
administering the agent
to a subject). As such, the invention provides methods of treating an
individual afflicted
with a disease or disorder characterized by aberrant expression or activity of
a NOVX
protein or nucleic acid molecule. In one embodiment, the method involves
administering
an agent (e.g., an agent identified by a screening assay described herein), or
combination
of agents that modulates (e.g., up-regulates or down-regulates) NOVX
expression or
activity. In another embodiment, the method involves administering a NOVX
protein or
nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX
expression
or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is
abnormally downregulated and/or in which increased NOVX activity is likely to
have a
beneficial effect. One example of such a situation is where a subject has a
disorder
characterized by aberrant cell proliferation andlor 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 irz vitro or irz 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 compositions of the
invention will
have efficacy for treatment of patients suffering from: metabolic disorders,
diabetes,
obesity, infectious disease, anorexia, cancer-associated cachexia, cancer,
neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune
disorders, hematopoietic disorders, and the various dyslipidemias.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of
the invention, or fragments thereof, may also be useful in diagnostic
applications, wherein
the presence or amount of the nucleic acid or the protein are to be assessed.
A further use
could be as an anti-bacterial molecule (i.e., some peptides have been found to
possess
anti-bacterial properties). These materials are further useful in the
generation of
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antibodies, which immunospecifically-bind to the novel substances of the
invention for
use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do
not
limit the scope of the invention described in the claims.
S E~~AMPLES
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 lA.
lA. NOV1
.a, CG103945-02 SEQ ID NO: 1 2414 by
Sequence ORF Start: ATG at l~. ~Y ' ORF Stop: TAG at 2401
AGGGGGCCTCCTGCTCCATGGGGGTAGCTCTGGCCCCA
TACCGAGGAGCCGTGGTCCGAAAGCCTTCCAGCACCAT
CAGGCCATGTACCTAGATGAGTACCGAGACCGCCTCTTTCTGGGTGGCCTGGACGCCCTCTACT
CACCTTCATAGACGGGGAGCTGTACACGGGTCTCACTGCTGACTTCCTGGGGCGAGAGGCCATG
GATGGCCGCCCGGATCCCTGAGAACTCTGACCAGGACAATGACAAGGTGTACTTCTTCTTCTCGGAGAC
CCCTCGCCCGATGGTGGCTCGAACCATGTCACTGTCAGCCGCGTGGGCCGCGTCTGCGTGAATGATGCT
CTGGTGGTGCCGAGACCCACTTTGACCAGCTAGAGGATGTGTTCCTGCTGTGGCCCAAGGCCGGGAAGAGCCT
GACATCTGGGAGGTTTTCAACGGGCCCTTTGCCCACCGAGATGGGCCTCAGCACCAGTGGGGGCCCTATGGGG
GCAAGGTGCCCTTCCCTCGCCCTGGCGTGTGCCCCAGCAAGATGACCGCACAGCCAGGACGGCCTTTTGGCAG
CACCAAGGACTACCCAGATGAGGTGCTGCAGTTTGCCCGAGCCCACCCCCTCATGTTCTGGCCTGTGCGGCCT
CGACATGGCCGCCCTGTCCTTGTCAAGACCCACCTGGCCCAGCAGCTACACCAGATCGTGGTGGACCGCGTGG
AGGCAGAGGATGGGACCTACGATGTCATTTTCCTGGGGACTGACTCAGGGTCTGTGCTCAAAGTCATCGCTCT
CCAGGCAGGGGGCTCAGCTGAACCTGAGGAAGTGGTTCTGGAGGAGCTCCAGGTGTTTAAGGTGCCAACACCT
ATCACCGAAP.TGGAGATCTCTGTCAAAAGGCAAATGCTATACGTGGGCTCTCGGCTGGGTGTGGCCCAGCTGC
ACTGTGCCTG
CTTGCAGAGGCCAGGGGATGAGGGGCCTGACCAGGTGAAGACGGACGAGCGAGTCTTGCACACGGAGCGGGGG
TCCTGCAG
CACGGAAT
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CG103945-02 ~SEQ ID NO: 2 X800 as BMW at 88800.3kD
MAPSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYRGAVVRKPSSTMWMETFSRYLLSANRSAIFLGPQGS
LNLQAMYLDEYRDRLFLGGLDALYSLRLDQAWPDPREVLWPPQPGQREECVRKGRDPLTECANFVRVLQPHNR
THLLACGTGAFQPTCALITVGHRGEHVLHLEPGSVESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAM
IFRSGGPRPALRSDSDQSLLHDPRFVMAARIPENSDQDNDKVYFFFSETVPSPDGGSNHVTVSRVGRVCVNDA
GGQRVLVNKWSTFLKARLVCSVPGPGGAETHFDQLEDVFLLWPKAGKSLEVYALFSTVSAVFQGFAVCVYHMA
DIWEVFNGPFAHRDGPQHQWGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPDEVLQFARAHPLMFWPVRP
RHGRPVLVKTHLAQQLHQIVVDRVEAEDGTYDVIFLGTDSGSVLKVIALQAGGSAEPEEVVLEELQVFKVPTP
ITEMEISVKRQMLYVGSRLGVAQLRLHQCETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRH
GNPALQCLGQSQEEEAVGLVAATMVYGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERG
LLFRRLSRFDAGTYTCTTLEHGFSQTVVRLALVVIVASQLDNLFPPEPKPEEPPARGGLASTPPKAWYKDILQ
LIGFANLPRVDEYCERVWCRGTTECSGCFRSRSRGKQARGKSWAGLELGKKMKSRVHAEHNRTPREVEAT
dOVlb, CG103945-Ol SEQ ID NO 3 _ 4700 by _ _ _
>NA Sequence ' ORF Start ATG at 1 ~~Y ORF Sto-:-TAG at 2347
_.___ _~__.___.._._~.__. __~__-_~~re___.~~ ,r.:~~:.-.,~:~.~~_. _.
.TGGCCCCCTCGGCCTGGGCCATTTGCTGGCTGCTAGGGGGCCTCCTGCTCCATGGGGGTAGCTCTGGCCCCA
~CCCCGGCCCCAGTGTGCCCCGCCTGCGGCTCTCCTACCGAGACCTCCTGTCTGCCAACCGCTCTGCCATCTT
'CTGGGCCCCCAGGGCTCCCTGAACCTCCAGGCCATGTACCTAGATGAGTACCGAGACCGCCTCTTTCTGGGT
TCACAACCGGACCCACCTGCTAGCCTGTGGCACTGGGGCCTTCCAGCCCACCTGTGCCCTCATC
CACCGTGGGGAGCATGTGCTCCACCTGGAGCCTGGCAGTGTGGAAAGTGGCCGGGGGCGGTGCC
CCAGCCGTCCCTTTGCCAGCACCTTCATAGACGGGGAGCTGTACACGGGTCTCACTGCTGACTT
AGAGGCCATGATCTTCCGAAGTGGAGGTCCTCGGCCAGCTCTGCGTTCCGACTCTGACCAGAGT
.TGCTGGGGGCCAGCGGGTGCTGGTGAACAAATGGAGCACTTTCCTCAAGGCCAGGCTG
GTGTACGCGCTGTTCAGCACCGTCAGTGCCGTGTTCCAGGGCTTCGCCGT
TCTGGGAGGTTTTCAACGGGCCCTTTGCCCACCGAGATGGGCCTCAGCAC
GCGGCCTCGACATGGCCGCCCTGTCCTTGTCAAGACCCACCTGGCCCAGCAGCTACACCAG
CGCGTGGAGGCAGAGGATGGGACCTACGATGTCATTTTCCTGGGGACTGACTCAGGGTCTG
TCGCTCTCCAGGCAGGGGGCTCAGCTGAACCTGAGGAAGTGGTTCTGGAGGAGCTCCAGGT
AACACCTATCACCGAAATGGAGATCTCTGTCAAAAGGCAAATGCTATACGTGGGCTCTCGG
TGGTGCCTCCTGTACCCACTACCGCCCCAGCCTTGGCAAGCGCCGGTTCCG
TGGTCTACGGCACGGAGCACAATAGCACCTTCCTGGAGTGCCTGCCCAAGTCTCCCC
GCTCTTGCAGAGGCCAGGGGATGAGGGGCCTGACCAGGTGAAGACGGACGAGCGAGT
GGGCTGCTGTTCCGCAGGCTTAGCCGTTTCGATGCGGGCACCTACACCTGCACCACT
AGCAGCCCCCAGCAT
ACACCGCTACTGGGGTCTAATGGAGGGGCTGGGTTCTTGAAGCCTGTTCCCTGCCCTTCTCTGTGCTCTTAGA
CCCAGCTGGAGCCAGCACCCTCTGGCTGCTGGCAGCCCCAAGGGATCTGCCATTTGTTCTCAGAGATGGCCTG
(GCTTCCGCAACACATTTCCGGGTGTGCCCAGAGGCAAGAGGGTTGGGTGGTTCTTTCCCAGCCTACAGAACAA
~TGGCCATTCTGAGTGACCCTCAGAGTGGGTGTGTGGGTGCGTCTAGGGGGTATCCCGGTAGGGGGCCTGCAGG
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~ACACAGAGGTGTTGGGAAGGTGGAGCAACAATGCACCTCCCCTCCTGTCGCGCCGTGATATCTTGGTGGCTCC
CTGCCACTGCCCACCGCCTCTTCTCCATCTGAGAATCACGGAGAGGTGTAGATAATCTAGAGGCATAGACTGC
TGCCG
AGAGGGCAGGAGACCCTTAGGAT
AAGGGGGAAACAAGGTAGAGAAAAGGACGAAGAAGTGTAAGTCCCGCTGATTCTCGGGGGTAAGGCTCGGATG
~GCAAGGACGCGTTCTGCCTGGGCATGTAGGGGAGGTGTTTTTGCCATCACCAGTTTCTCAGGCTGGGGAGCAC
AGAGGGGAGGAGGAGGACTAAATGAAAAGTTGTTCCCAGCCTGCACATGAACACATTCATGACACACAAAACT
CAGAGGGGATTAAAGAGGGGAGGAGAGAGTGCAGAGCTCCAGGAAAGGGTATCAGAGCTGCAGCCAGCTCTGC
CCTCTACCCTAGGGAGGCCAGAAAGACACAAACAGCCCTCCGGGCCTTTACGCTGGACTCTGGCTTGGCAGGC
TCCAGGCAGGGTCCTCTGGGAAGTTACTCTAGAAAACGAAGGGAGGAGGAGCACAAGATCCTCAGCAACGAAC
ACCTGCACTTAGAAAAAGTGGACAGCTTCTGCCAACCACACCCTACCCATGGTACTGTATGCTATTAACTCCT
~GAGCACATTTCTTGTAATTACTATTGTTATTTTTATTGTCATGACTGCCCCTGAGCTCTGGTGAGAAAAGCTG
AATTTACAAGGAAAGGGATGAAGTTAATATTTGCATCACATAATTATATCATTACTGTGTATCTGTGTATTGT
ACTAAATGGACTGATGCTGCGCACATGAGCTGAAAATGAAGAGCCCTCCCATCC
b, CG103945-Ol ~SEQ ID NO: 4 782 as 1MW at 86699.9kD
iSequence
APSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYRDLLSANRSAIFLGPQGSLNLQAMYLDEYRDRLFLG
LDALYSLRLDQAWPDPREVLWPPQPGQREECVRKGRDPLTECANFVRVLQPHNRTHLLACGTGAFQPTCALI
VGHRGEHVLHLEPGSVESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAMIFRSGGPRPALRSDSDQS
LHDPRFVMAARIPENSDQDNDKWFFFSETVPSPDGGSNHWVSRVGRVCVNDAGGQRVLVNKWSTFLKARL
CSVPGPGGAETHFDQLEDVFLLWPKAGKSLEWALFSTVSAVFQGFAVCWHMADIWEVFNGPFAHRDGPQH
WGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPDEVLQFARAHPLMFWPVRPRHGRPVLVKTHLAQQLHQ
VVDRVEAEDGTYDVIFLGTDSGSVLKVIALQAGGSAEPEEVVLEELQVFKVPTPITEMEISVKRQMLYVGSR
GVAQLRLHQCETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRHGNPALQCLGQSQEEEAVG
VAATMVYGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERGLLFRRLSRFDAGTYTCTT
EHGFSQTVVRLALWIVASQLDNLFPPEPKPEEPPARGGLASTPPKAWYKDILQLIGFANLPRVDEYCERVW
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 1B.
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Table 1B. Comparison of the NOVl protein sequences.
NOVla MAPSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYRGAVVRKPSSTMWMETFSRYLLS
NOVlb MAPSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYR-------------D-----LLS
NOVla ANRSAIFLGPQGSLNLQAMYLDEYRDRLFLGGLDALYSLRLDQAWPDPREVLWPPQPGQR
NOVlb ANRSAIFLGPQGSLNLQAMYLDEYRDRLFLGGLDALYSLRLDQAWPDPREVLWPPQPGQR
~!NOVla EECVRKGRDPLTECANFVRV.LQPHNRTHLLACGTGAFQPTCALITVGHRGEHVLHLEPGS
',NOVlb EECVRKGRDPLTECANFVRVLQPHNRTHLLACGTGAFQPTCALITVGHRGEHV.LHLEPGS
',NOVla VESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAMIFRSGGPRPALRSDSDQSLLH
'NOVlb VESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAMIFRSGGPRPALRSDSDQSLLH
NOVla DPRFVMAARIPENSDQDNDKWFFFSETVPSPDGGSNHVTVSRVGRVCVNDAGGQRVLVN
NOVlb DPRFVMAARIPENSDQDNDKWFFFSETVPSPDGGSNHVTVSRVGRVCVNDAGGQRVLVN
NOVla KWSTFLKARLVCSVPGPGGAETHFDQLEDVFLLWPKAGKSLEWALFSTVSAVFQGFAVC
NOVlb KWSTFLKARLVCSVPGPGGAETHFDQLEDVFLLWPKAGKSLEWALFSTVSAVFQGFAVC
NOVIa VYHMADIWEVFNGPFAHRDGPQHQWGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPD
NOVlb VYHMADIWEVFNGPFAHRDGPQHQWGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPD
NOVla EVLQFARAHPLMFWPVRPRHGRPVLVKTHLAQQLHQIVVDRVEAEDGTYDVIFLGTDSGS
NOVlb EVLQFARAHPLMFWPVRPRHGRPVLVKTHLAQQLHQIVVDRVEAEDGTYDVIFLGTDSGS
NOVla VLKVIALQAGGSAEPEEVVLEELQVFKVPTPITEMEISVKRQMLWGSRLGVAQLRLHQC
NOVlb VLKVIALQAGGSAEPEEVVLEELQVFKVPTPITEMEISVKRQMLWGSRLGVAQLRLHQC
NOVla ETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRHGNPALQCLGQSQEEEA
NOVlb, ETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRHGNPALQCLGQSQEEEA
NOVla VGLVAATMVYGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERGLLF
NOVlb VGLVAATMWGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERGLLF
NOVla RRLSRFDAGTYTCTTLEHGFSQTWRLALWIVASQLDNLFPPEPKPEEPPARGGLASTP
NOVlb RRLSRFDAGTYTCTTLEHGFSQTVVRLALWIVASQLDNLFPPEPKPEEPPARGGLASTP
NOVla PKAWYKDILQLIGFANLPRVDEYCERVWCRGTTECSGCFRSRSRGKQARGKSWAGLELGK
NOVlb PKAWKDILQLIGFANLPRWEYCERVWCRGTTECSGCFRSRSRGKQARGKSWAGLELGK
NOVla KMKSRVHAEHNRTPREVEAT
NOVlb KMKSRVHAEHNRTPREVEAT
NOVla (SEQ ID NO: 2)
NOVlb (SEQ ID NO: 4)
Further analysis of the NOV1 a protein yielded the following properties shown
in Table
1 C.
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Table 1C. Protein Sequence Properties NOVla
SignalP analysis: Cleavage site between residues 23 and 24
PSORT II analysis: '
PSG: a new signal peptide prediction method
N-region: length 0; pos.chg 0; neg.chg 0
H-region: length 31; peak value 9.35
PSG score: 4.95
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 1.50
possible cleavage site: between 22 and 23
» > Seems to have a cleavable signal peptide (1 to 22)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 23
Tentative number of TMS(s) for the threshold 0.5: 3
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood = -2.02 Transmembrane 345 - 361
PERIPHERAL Likelihood = 2.86 (at 150)
ALOM score: -2.02 (number of TMSs: 1)
'MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 11
Charge difference: 0.5 C( 1.5) - N( 1.0)
C > N: C-terminal side will be inside
» >Caution: Inconsistent mtop result with signal peptide
» > membrane topology: type 1a (cytoplasmic tail 362 to 800)
MITDISC: discrimination of mitochondrial targeting seq
R content: 4 Hyd Moment(75): 2.13
Hyd Moment(95): 2.46 G content: 7
D/E content: 1 S/T content: 9
Score: -2.94
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 73 NRS~AI
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: PSLGKRR (3) at 570
bipartite: none
content of basic residues: 11.4&
NLS Score: -0.22
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif.
type 1: none
type 2: none
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NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: too long tail
'Dileucine motif in the tail: found
LL at 633
~i LL at 658
checking 63 PROSITE DNA binding motifs: none
checking 71 PR05ITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 89
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
44.4 %: endoplasmic reticulum
22.2 %: Golgi
22_2 %: extracellular, including cell wall
11_1 %: plasma membrane
» prediction for CG103945-02 is end (k=9)
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 1D.
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Table
1D.
Geneseq
Results
for
NOVla
NOVla Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the MatchedValue
ResiduesRegion
AAG65620Novel human protein (NHP)1..800 781/800 0.0
sequence (97%)
- Homo Sapiens, 782 aa. 1..782 781/800
(97%)
[W0200170806-A2, 27-SEP-2001]
AAG65619Novel human protein (NHP)1..800 781/800 0.0
sequence (97%)
- Homo Sapiens, 875 aa. 94..875 781/800
(97%)
[W0200170806-A2, 27-SEP-2001]
AAB23609Human secreted protein L.800 781/800 0.0
SEQ ID NO: (97%)
18 - Homo sapiens, 782 1..782 781/800
aa. (97%)
[W0200049134-Al, 24-AUG-2000]
AAB23636Human secreted protein 1..800 7811803 0.0
SEQ >D NO: (97%)
92 - Homo Sapiens, 785 2..785 781/803
aa. (97%)
[WO200049134-Al, 24-AUG-2000]
AAG78481Human ZSMF-16 - Homo 1..800 778/800 0.0
sapiens, (97%)
779 aa. [US2001049432-Al,1..779 779/800
(97!0)
06-DEC-2001]
In a BLAST search of public sequence databases, the NOV 1 a protein was found
to have
homology to the proteins shown in the BLASTP data in Table lE.
S
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Table
lE. Public
BLASTP
Results
for NOVla
NOVla Identities/
Protein Residues/SimilaritiesExpect
for
AccessionProtein/Organism/Length Match the Matched Value
Number ResiduesPortion
Q9NS98 Semaphorin sem2 (FLJ000141..800 781/800 (97%)0.0
protein) - Homo Sapiens 1..782 781/800 (97%)
(Human),
782 aa.
CAC42673 Sequence 1 from Patent 1..800 778/800 (97%)0.0
W00140278
~ 1..779 779/800 (97%)
- Homo Sapiens (human),
779 aa.
Q9QX23 Semaphorin M-SemaK - 1..795 399/805 (49%)0.0
Mus
musculus (Mouse), 775 1..770 525/805 (64%)
aa.
P70275 Semaphorin 3E precursor 1..795 398/805 (49%)0.0
(Semaphorin H) (Sema 1..?70 524/805 (64%)
H) - Mus
musculus (Mouse), 775
aa.
042237 Semaphorin 3E precursor 1..797 398/806 (49%)0.0
(Collapsin-5) (COLL-5) 5..782 519/806 (64%)
- Gallus
gallus (Chicken), 785
aa.
PFam analysis predicts that the NOV 1 a protein contains the domains shown in
the Table
1F.
Table 1F. Domain Analysis
of NOVla
Identities/
Pfam Domain NOVla Match RegionSimilarities Expect Value
for the Matched
Region
Sema 76..521 217/497 (44%) 1.3e-176
360/497 (72%)
PSI 539..622 14/101 (14%) 0.76
56/101 (SS%)
ig 614..675 15/66 (23%) 0.0061
45/66 (68%)
Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 2A.
112
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2A. NOV2
CG106951-O1 ~SEO mNO: S
Sequence ORF Start: ATG at 1400 ORF Stop: TGA at 5456
TGGTCCAGAGCTCATTGTCCTTGTTGATAAAATGATAGATTTGGACTCAATATCC
CCTTTCTTTTCTTCTCTGCATCTCTGCCTTTGTGTCCAGAGCGTGTTTTCCCTTTGCAAGTTTCTCTCCATTC
TGCACATTATGAGTTTCAGCATTTCTGTTGCCCTAGAAAGTCTATCTTTGAGATCTTGCACTGTTTCTCTTTT
TACAGTGTCTCATAAACTCCCTTCTTGGATTCAGAACCACCCTTTCTTTCCCATTATCCTGTCAAACTGCTTC
TTGCCATGGTCCAGGGGTAGGAGGATGGCAGGCAGGAGGTGCTTCTCTGGGGCTCTTAGTGTCTCAATTCTTC
ATTTTTATTTCATAGT
CTTAGGTCACCTTTTTTTACATTTTCAAATATATTTTTTGTTCAGCAGAGGGCTCCCTTCCCATCCCTCTTGC
AGCCCGGGCAGCTAGGATTTGAAGCTTGCCCCTTGAATCTTTCTCTCCCGCCTTCTAGCCATCAGAAACACTA
GATCACTTAAACTTGTAAACAATTCGGCCTCGCTCCTTGTGATTGCGCTAAACCTTCCGTCCTCAGCTGAGAA
CGCTCCACCACCTCCCCGGATCGCTCATCTCTTGGCTGCCCTCCCACTGTTCCTGATGTTATTTTACTCCCCG
TATCCCCTACTCGTTCTTCACAATTCTGTAGGGTGCGTATTACTAACCCCAGTTTACAGCTGAGGAAACTGAG
GCTTGGAGAGGTTCGCTCGGTATCGTACAGTTTGCAAGGTTAACCCTAATCCGGCCAGTTCTGGCTTTCCAGC
CCAGCCCAGCAGCCTAGCCTCCCTCTCTGCCGCTGCAGGTTATAACGGCTCTCCCCCGTTTTACACGAGGTCC
CTTCCCCTTCAAATCCACAGGCAGGAAGATCGTTCCGAACTGACGGGGCTGGGGAATGTGGGAGTCCGGAGTG
~CATTCGAGATGGGGTGACCGAGAACGGCAAGGCGGGATGTGGCAAACGGCGGCAAGTGCTCGGAGTCCTAGGT
CTTGCCGCCGGAATGCCGGCCGGGGAAGGGGCTTCGGCCCACCGGGCTGGTCACCACACTCGGCAGGCCCGGG
CAGCGCATCCAGCGGCTGCTGGGAGCCCGAGCGCAGCGGGCGCGGGCCCGGGTGGGGACTGCACCGGAGCG
GAGAGCTGGAGGCCGTTCCTGCGCGGCCGCCCCATTCCCAGACCGGCCGCCAGCCCATCTGGTTAGCTCCC
CGCTCCGCGCCGCCCGGGAGTCGGGAGCCGCGGGGAACCGGGCACCTGCACCCGCCTCTGGGAGTGAGTGG
TGGACAGTAGGGGGCTGGCTTCTCTCAC
CAGAGGGGCCTATCATGGTGCTTGCAGG
TGTC
ACCTCTTCAGACTCAGCCTTGCCAATGTCTCTCTTCTTCAGGCCACA
TCGTCGCCGGCCGGAAGGTGTTCATGTGTGGAACCAATGCCTTTTCCCCCATGTGCAC
GTGGGCCACCGCTTCGCACTGCCCAATATAACTCCAAGTGGCT
TATTGGGCTGTTTGCATACTTCTTCCTGCGGGAGAACGCAGTG
CGCGTGGCCCGCGTGTGCAAGAATGACGTGGGGGGCCGATTCC
ATAACGAGCTGCAGAGTGCCTTCCACTTGCCAGAGCAGGACCTCATCTATGGAGTTTTCACAACCAACGTA
CAGCATCGCGGCTTCTGCTGTCTGCGCCTTCAACCTCAGTGCTATCTCCCAGGCTTTCAATGGCCCATTTC
GCCTGAGACCGGTCCCAACGAGAACCTGACGGAGCGCAGCCTGCAGGACGCGCAGCGCCTCTTCCTGATGAGC
CTGGGACGGGAAGCAGCAACGTTGCAGCACACTCGAGGACAGCTCCAACATGAGCCTCTGGACCCAGAACATC
113
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TCGGCTTCCAGGTCCGCCAGCGAAGTTGCAGCAACCCTGCTC
TCTGTCTCGGGCTGCACACGGAGGAGGCACTATGTGCCACACAGGCC
TTCCCGTCATCCTGCCAGCCTCCAGCATGGAGGAGGCCACCGGCTGTGCAGGGTTCAATCTCATCCAC
GGCCACGGGCATCTCCTGCTTCTTGGGCTCTGGGCTCCTGACCCTAGCAGTGTACCTGTCTTGCCAGC
CAGCGTCAGTCCCAGGAGTCCACACTGGTCCATCCTGCCACCCCCAACCATTTGCACTACAAGGGCGG
CCCCGAAGAATGAAAAGTACACACCCATGGAATTCAAGACCCTGAACAAGAATAACTTGATCCCTGAT
AGCCAACTTCTACCCATTGCAGCAGACCAAZ'GTGTACACGACTACTTACTACCCAAGCCCCCTGAACA
CCCT
~TTATCTTCCAACCCACTGTCACGCTGACACTATGCTCCCATGCCTGGGCTGTGGACCTACTGGGCATTTC,AC,r
CACA
AGCTGAGTCTGGCCTTCTCCAGCTGGCCCCAAAAAAGGCCTTTGCTACATCCTGATTATCTCTGAAAGTAATC
AATCAAGTGGCTCCAGTAGCTCTGGATTTTCTGCCAGGGCTGGGCCATTGTGGTGCTGCCCCAGTATGACATG
~GGACCAAGGCCAGCGCAGGTTATCCACCTCTGCCTGGAAGTCTATACTCTACCCAGGGCATCCCTCTGGTCAG
GCCTTGCCCTCAATGCACGAAAGGTGGCCCAGGAGAGAGGATCAATGCCACAGGAGGCAGAAGTCTGGCCTCT
~GTGCCTCTATGGAGACTATCTTCCAGTTGCTGCTCAACAGAGTTGTTGGCTGAGACCTGCTTGGGAGTCTCTG
AATTAAAGATGATATCCAGTCTCC
2a, CG106951-O1 SEQ ID NO: 6 1352 as MW at 145674.1kD
MPAGEGASAHRAGHHTRQARGGSRPSSRGMQGAPSRSSARLEAGGCSARRGRSAPAPSSFSLPLPSFSPFACN
SSPTAPSLLLLPRSPPPCSLRAPGRELVGARGLVPEPSSAEPGGSAAHPAAAGSPSAAGAGPGGDCTGALRAG
GRSCAAAPFPDRPPAHLVSSRRSAPPGSREPRGTGHLHPPLGVSGSSWCLACVSWMPCGFSPSPVAHHLVPGP
PDTPAQQLRCGWTVGGWLLSLVRGLLPCLPPGARTAEGPTMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEP
SSEQQLCALSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQATEWAS
SEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRTTEKINGVARCPYDPRHNS
TAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLFAYFFLRENAVEHDC
GRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIA
ASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQ
PVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHVLPPGRREPLR
SLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQNITACP
VRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWAL
CSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRA
CDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVG
QACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEG
TDDGAQSRSRHCEELLPGSSACAGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVAT
LTLAVYLSCQHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLTPDDRAN
TTTYYPSPLNKHSFRPEASPGQRCFPNS
114
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?b, CG1069S1-04 SEQ ID NO: 7 3631 by
Sequence__ ORF Start: ATG at 1S4 ORF Stop: TGA at 3544
TGGACAGTAGGGGGCTGGCTTCTCTCACTGGTCAGGGGTCTTCTCCCCTGTC
CAGAGGGGCCTATCATGGTGCTTGCAGGCCCCCTGGCTGTCTCGCTGTTGCT
AGCAAGCACCCCACCGTGGCCTTTGAAGACCTGCAGCCGTGGGTCTCTAACTTCACCT
GGTGTTCATGTGTGGAACCAATGCCTTTTCCCCCATGTGCACCAGCAGACAGGTGGGGAACCTCAG
ACTGAGAAGATCAATGGTGTGGCCCGCTGCCCCTATGACCCACGCCACAACTCCACAGCTGTCATC
AGGGGGAGCTCTATGCAGCCACGGTCATCGACTTCTCAGGTCGGGACCCTGCCATCTACCGCAGCC
ATAACGAGCTGCAGAGTGCCTT
'rCr~u't'UC'1'A'1'C'sCCCAGGCTTTCAATGGCCCATTTCGCTACCAGGAGAACCCCAGGGCTG
AGCCAACCCCATCCCCAATTTCCAGTGTGGCACCCTGCCTGAGACCGGTCCCAACGAGAA
ACTCTACATTGGCACCGAGTCGGGCACCATCCTGAAGGCGCTGTCCACGGCGAGCCGCAGCCT
CCTACCGCAGCCAGGGGGCATGCCTGGGGGCCCGGGACCCGTACTGTGGCTGGGACGGGAAGCAGCAACGTTG
CAGCACACTCGAGGACAGCTCCAACATGAGCCTCTGGACCCAGAACATCACCGCCTGTCCTGTGCGGAATGTG
ACACGGGATGGGGGCTTCGGCCCATGGTCACCATGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCT
TCCACATCGCCAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCC
CCGC
CTCCCAGGGTCCAGCGCCTGTGCTGGAAACAGCAGCCAGAGCCGCCCCTGCCCCTACAGCGAGATTCCCGTCA
CATCTCCTGCTTCTTGGGCTCTGGGCTCCTGACCCTAGCAGTGTACCTGTCTTGCCAGCACTGCCAGCGTCAG
TCCCAGGAGTCCACACTGGTCCATCCTGCCACCCCCAACCATTTGCACTACAAGGGCGGAGGCACCCCGAAGA
ATGAAAAGTACACACCCATGGAATTCAAGACCCTGAACAAGAATAACTTGATCCCTGATGACAGAGCCAACTT
AAGGCACAGAGCAGATGGAGATGGGACAGTGGAGCCAGTTTGGTTTCT
I15
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
CG10695I-04 SEQ m NO: 8 1130 as ~MW at 123700.9kD
FSPSPVAHHLVPGPPDTPAQQLRCGWTVGGWLLSLVRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPS
RLSLANVSLLQATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
EKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYD
GLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNELQSAFHL
EQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLT
RSLQDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHG
YLEELHVLPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCST
EDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIH
ANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGS
SKCSSNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCR
PLADPHGLQFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVR
RTCTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPEGWSP
SEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGIS
FLGSGLLTLAVYLSCQHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYP
~OOTNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
209829549 SEQ ID NO: 9 X1203
Sequence ~ORF Start: at I ~ORF Stop: end of
GAGCTCGATCCTGTGATTCCCCTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAGCCATCCACATCGC
CAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCCTGTGGCATCGGC
mmrraccmrrr_rrnrr~an
Att~rTC~CACTCAACCCTGCTCCCCGCCACGGGGGCCGCATCTGCGTGGGCAAGAGCC
CAAGTGCAGCAGCAACTGTGGAGGGGGCATGCGGTCGCGGCGTCGGGCCTGCGAGAACGGCAACTCCTGCCTG
GGCTGCGGCGTGGAGTTCAAGACGTGCAACCCCGAGGGCTGCCCCGAAGTGCGGCGCAACACCCCCTGGACGC
CCCGCGGACGGCTCC
ATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
CTGGAAACAGCAGCCAGAGCCGCCCCTGCGTCGAC
116
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209829549 SEQ ID NO: 10 401 as ~MW at 43284.SkD
PWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIG
SCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMRSRRRACENGNSCL
KTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGS
ALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNG~GLPCVGDAAEYQDC
VRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
AOSRSRHCEELLPGSSACAGNSSQSRPCVD
>.d, 209829553 SEQ ID NO: 11 03 by _
Sequence ORF Start: at 1 ~ ORF Stop: end of
CCCATGGTCACCATGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCTCTTGCCTGTGTC
TCCTGTGATTCCCCTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAGCCATCCACATCGC
AAGTTGCAGCAACCCTGCTCCCCGCCACGGGGGCCGCATCTGCGTGGGCAAGAGCC
AATGAGAACACGCCTTGCCCGGTGCCCATCTTCTGGGCTTCCTGGGGCTCCTGGAG
CTGGTGGAGGACCTCCTGCGCAGCGGGAGCACCTCCCCGCACACGGTGAGCG
CTAACCCGGAGTCCCGCAACGGGGGCCTGCCCTGCGTGGGCGATGCTGCCGAGTACCAGGACTGC
GGCTTGCCCAGTTCGGGGTGCTTGGTCCTGCTGGACCTCATGGTCTCCATGCTCAGCTTCCTGTG
CACTATCAACGCACCCGTTCCTGCACCAGCCCCGCACCCTCCCCAGGTGAGGACATCTGTCTCGG
CGGAGGAGGCACTATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
CTGGAAACAGCAGCCAGAGCCGCCCCTGCGTCGAC
209829553 ~SEQ ID NO: 12 X401 as ]MW at 43246.4kD
PCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIG
NPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCL
NPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGS
EDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPESRNGGLPCVGDAAEYQDC
AWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
RSRHCEELLPGSSACAGNSSQSRPCVD
117
CA 02486490 2004-12-03
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!e, 209829642 SEQ ID NO: 13 1203 by
Sequence ORF Start: at 1 ORF Stop: end of
TGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCTCTTGCCTGTGTC
CTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAGCCATCCACATCGC
TGCCCAGTTCGGGGTGCTTGGTCCTGCTGGACCTCATGGTCTCCATGCTCAGCTTCCTGTG
ATCAACGCACCCGTTCCTGCACCAGCCCCGCACCCTCCCCAGGTGAGGACATCTGTCTCGG
GGAGGCACTATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
209829642 ~SEQ ID NO: 14 X401 as BMW at 432$6.4kD
SPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIG
RSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCL
FKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGS
DALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYQDC
PVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
GAQSRSRHCEELLPGSSACAGNSSQSRPCVD
>.f, 209829670 SEQ ID NO:1$ ~ 1203 by _
Sequence ORh Start: at 1 ~~ORF Stop: end of
CAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCCTGTGGCATCGGC
CCGTTCCTGCACCAGCCCCGCACCCTCCCCAGGTGAGGACATCTGTCTCGG
TG,TGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
118
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209829670 ~SEQ ID NO: 16 ~40I as BMW at 43240.SkD
PWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIG
SCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCL
~VEVLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEY'QDC
.GAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
~SRSRHCEELLPGSSACAGNSSQSRPCVD
?g, CGI0695I-02 SEQ ID NO: 17 _ 4_23_3 by ,
Sequence ORF Start: ATG at 2 ORF Stop: TGA at 3281
ACCCTGGAGCCCGGGATTTCTCCCAGCTGGCTTT
AGGAGTGTCAGAACTACGTGCGAGTCCTGATCGTCGCCGGCCGGAAGGTGTTCATGTGTGGAACCAATGCCTT
TTCCCCCATGTGCACCAGCAGACAGGTGGGGAACCTCAGCCGGACTACTGAGAAGATCAATGGTGTGGCCCGC
TGCCCCTATGACCCACGCCACAACTCCACAGCTGTCATCTCCTCCCAGGGGGAGCTCTATGCAGCCACGGTCA
TCGACTTCTCAGGTCGGGACCCTGCCATCTACCGCAGCCTGGGCAGTGGGCCACCGCTTCGCACTGCCCAATA
TAACTCCAAGTGGCTTAATGAGCCAAACTTCGTGGCAGCCTATGATATTGGGCTGTTTGCATACTTCTTCCTG
CGGGAGAACGCAGTGGAGCACGACTGTGGACGCACCGTGTACTCTCGCGTGGCCCGCGTGTGCAAGAATGACG
CGAGGTCCCCTTCTACTATAACGAGCTGCAGAGTGCCTTCCACTTGCCAGAGCAGGACCTCATCTATGGAGTT
TTCACAACCAACGTAAACAGCATCGCGGCTTCTGCTGTCTGCGCCTTCAACCTCAGTGCTATCTCCCAGGCTT
ACTCTACATTGGCACCGAGTCGGG
CTGCGCAGCCTGCGCATCCTGCACAGCGCCCGCGCGCTCTTCGTGGGGCTGA
CACTGGAGAGGTGCGCCGCCTACCGCAGCCAGGGGGCATGCCTGGGGGCCCG
CGGGAAGCAGCAACGTTGCAGCACACTCGAGGACAGCTCCAACATGAGCCTC
TGGGGACAACTCAGGCTCTTGCCTGTGTCGAGCTCGATCCTGTGATTCCCC
ACAGCGAGATTCCCGTCATCCTGCCAGCCTCCAGCATGGAGGAGGCCACCGGCTGTGCAGGG
ACCCATTGCAGCAGACCAATGTGTACACGACTACTTACTACC
119
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WO 2004/000997 PCT/US2003/017512
CCAGGTCTCTCATGGTTATCTTCCAACCCACTGTCACGCTGACACTATGCTGCCATGCCTGGGCTGTGGACCT
ACTGGGCATTTGAGGAACTGGAGAATGGAGATGGCAAGAGGGCAGGCTTTTAAGTTTGGGTTGGAGACAACTT
CCTGTGGCCCCCACAAGCTGAGTCTGGCCTTCTCCAGCTGGCCCCAAAAAAGGCCTTTGCTACATCCTGATTA
ACCCAGGG
AATCTGGG
~AGAAGTCTGGCCTCTGTGCCTCTATGGAGACTATCTTCCAGTTGCTGCTCAACAGAGTTGTTGGCTGAGACCT
GCTTGGGAGTCTCTGCTGGCCCTTCATCTGTTCAGGAACACACACACACACACACTCACACACGCACACACAA
TCACAATTTGCTACAGCAACAAAAAAGACATTGGGCTGTGGCATTATTAATTAAAGATGATATCCAGTCTCC
CG1069$1-02 ~SEQ m N0:18 X1093 as BMW at 119865.3kD
MVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCALSKHPTVAFEDLQPWVSNFTYPGARDFSQLAL
DPSGNQLIVGARNYLFRLSLANVSLLQATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAF
SPMCTSRQVGNLSRTTEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQY
D1SKWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPG
EVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNF
QCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESG
TILKALSTASRSLHGCYLEELHVLPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGAR
DPYCGWDGKQQRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSP
RPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNE
NTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGCPEVRRNTPWTPWLPVNVT
QGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGP
WSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRT
RSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSR
PCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSCQHCQRQSQESTLVHPATPNHL
HYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQTNVYTTTYYPSPLNKHSFRPEASFGQRCFPNS
?h, CG1069$1-03 SEQ mNO:19 1203 by
Sequence ORF Start: at 7 ORF Ston: at 1198
GGATCCGGCCCATGGTCACCATGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCTCTTGCCTGTGTC
GAGCTCGATCCTGTGATTCCCCTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAACCATCCACATCGC
CAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCCTGTGGCATCGGC
TCTTCTGGGCTTCCTGGGGCTCCTGGAG
ATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
120
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
CG106951-03 ~SEQ ID NO: 20 397 as MW at 42882.1kD
PWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIGFQ
SCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCLGC
umrr'rpFt~rpF~mR1 TpwTPwLPVNVTOGGAROEORFRFTCRAPLADPHGLOFGRRRTETRTCPADGSGS
SWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKC
ELLPGSSACAGNSSQSRPC
V2i, SNP13382456 of SEQ ID NO: 21 _ 6408 bp~
106951-Ol, DNA Sequence ORF Start: ATG at 1400 Stop: TGA at 5456
Pos: 5770 SNP Change: C to T
pCATGCTGCCTCTTCCAACTTGATTTTTACCCCAGACTGGGCTACCAGACTGGTATGCC:CACAC:A'1'GCCCG'1.d
egree.1"1'
TGCACATTATGAGTTTCAGCATTTCTGTTGCCCTAGAAAGTCTATCTTTGAGATCTTGCACTGTTTCTCTTTT
TACAGTGTCTCATAAACTCCCTTCTTGGATTCAGAACCACCCTTTCTTTCCCATTATCCTGTCAAACTGCTTC
TGCTTTATCTGGGTTTTCCTTTACCCAGAATTTTATTATGTAAAATGCTTCACTCAGACTTTGTTCTAATTAT
CCAATTTTTGGCATACTCTAGAAAGTCTTTTGATATTTTCCTTCCTCCAACTTATCTATTTTTATTTCATAGT
TCTCTTTGGTTATCTCTTAGAATCACACTTTCCTGGTTTTAATTTTTCAAATCCTTTGTCTTTCTCACTCGTT
CTTAGGTCACCTTTTTTTACATTTTCAAATATATTTTTTGTTCAGCAGAGGGCTCCCTTCCCATCCCTCTTGC
GATCACTTAAACTTGTAAACAATTCGGCCTCGCTCCTTGTGATTGCGCTAAACCTTCCGTCCTCAGCTGAGAA
CGCTCCACCACCTCCCCGGATCGCTCATCTCTTGGCTGCCCTCCCACTGTTCCTGATGTTATTTTACTCCCCG
TATCCCCTACTCGTTCTTCACAATTCTGTAGGGTGCGTATTACTAACCCCAGTTTACAGCTGAGGAAACTGAG
GCTTGGAGAGGTTCGCTCGGTATCGTACAGTTTGCAAGGTTAACCCTAATCCGGCCAGTTCTGGCTTTCCAGC
CCAGCCCAGCAGCCTAGCCTCCCTCTCTGCCGCTGCAGGTTATAACGGCTCTCCCCCGTTTTACACGAGGTCC
CTTCCCCTTCAAATCCACAGGCAGGAAGATCGTTCCGAACTGACGGGGCTGGGGAATGTGGGAGTCCGGAGTG
CATTCGAGATGGGGTGACCGAGAACGGCAAGGCGGGATGTGGCAAACGGCGGCAAGTGCTCGGAGTCCTAGGT
CTTGCCGCCGGAATGCCGGCCGGGGAAGGGGCTTCGGCCCACCGGGCTGGTCACCACACTCGGCAGGCCCGGG
TCCAGCGGCTGCTGGGAGCCCGAGCGCAGCGGGCGCGGGCCCGGGTGGGGACTGCACCGGAGCG
AAGGTGTGGATGGACAGTAGGGGGCTGGCTTCTCTCAC
GCTAGGACTGCAGAGGGGCCTATCATGGTGCTTGCAGG
AGCAAGCACCCCACCGTGGCCTTTGAAGACCTGC
CGTGGGAGCCAGGAACTACCTCTTCAGACTCAGCCTTGCCAATGTCTCTCTTCTTCAGGCCACA
GTGGGCCACCGCTTCGCACTGCCCAATATAACTCCAAGTGGCT
TATTGGGCTGTTTGCATACTTCTTCCTGCGGGAGAACGCAGTG
CGCGTGGCCCGCGTGTGCAAGAATGACGTGGGGGGCCGATTCC
AGGCCCGGCTCAACTGCTCCCGCCCGGGCGAGGTCCCCTTCTA
(~CCA(iA(tCAC~GACCTCATCTATGGAGTTTTCACAACCAACGTA
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TCGAGGACAGCTCCAACATGAGCCTCTGGACCCAGAACATC
TGGGGGCTTCGGCCCATGGTCACCATGGCAACCATGTGAGC
TGTCGAGCTCGATCCTGTGATTCCCCTCGACCCCGCTGTGG
TCGCCAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCA
AATGAGAACACGCCTTGCCC
TGCGCAGCGGGAGCACCTCCCCGCACACGGTGAGCGGGGGCTGGGCCGCCTGGGGCCCGTGGTCGTCCTGCTC
CCGGGACTGCGAGCTGGGCTTCCGCGTCCGCAAGAGAACGTGCACTAACCCGGAGCCCCGCAACGGGGGCCTG
CCCTGCGTGGGCGATGCTGCCGAGTACCAGGACTGCAACCCCCAGGCTTGCCCAGTTCGGGGTGCTTGGTCCT
GCTGGACCTCATGGTCTCCATGCTCAGCTTCCTGTGGTGGGGGTCACTATCAACGCACCCGTTCCTGCACCAG
AAGTGCACTGACGACGGAGCCCAGAGCCGAAGCCGGC
TGCCAGCCTCCAGCATGGAGGAGGCCACCGGCTGTGCAGGGTTCAATCTCATCCAC
CTCCTGCTTCTTGGGCTCTGGGCTCCTGACCCTAGCAGTGTACCTGTCTTGCCAGC
CAGGAGTCCACACTGGTCCATCCTGCCACCCCCAACCATTTGCACTACAAGGGCGG
nnaar_mnrnrnrrrAmrrAA~r'TC~AAGACCCTGAACAAGAATAACTTGATCCCTGAT
AACACAGCTTCCGGCCCGAGGCCTCACCTGGACAACGGTGCTTCCCCAACAGCTGATACCGCCGTCCTGGGGA
CTTGGGCTTCTTGCCTTCATAAGGCACAGAGCAGATGGAGATGGGACAGTGGAGCCAGTTTGGTTTTCTCCCT
CTGCACTAGGCCAAGAACTTGCTGCCTTGCCTGTGGGGGGTCCCATCCGGCTTCAGAGAGCTCTGGCTGGCAT
TGACCATGGGGGAAAGGGCTGGTTTCAGGCTGACATATGGCCGCAGGTCCAGTTCAGCCCAGGTCTCTCATGG
TTATCTTCCAACCCACTGTCACGCTGACACTATGCTGCCATGCCTGGGCTGTGGACCTACTGGGCATTTGAGG
AATTGGAGAATGGAGATGGCAAGAGGGCAGGCTTTTAAGTTTGGGTTGGAGACAACTTCCTGTGGCCCCCACA
AGCTGAGTCTGGCCTTCTCCAGCTGGCCCCAAAAAAGGCCTTTGCTACATCCTGATTATCTCTGAAAGTAATC
AATCAAGTGGCTCCAGTAGCTCTGGATTTTCTGCCAGGGCTGGGCCATTGTGGTGCTGCCCCAGTATGACATG
GGACCAAGGCCAGCGCAGGTTATCCACCTCTGCCTGGAAGTCTATACTCTACCCAGGGCATCCCTCTGGTCAG
ATCCAGTCTCC
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V2i, SNP133S2456 of SEQ ID NO: 22 .1352 as MW at 145674.11cD
106951-Ol, Protein Sequence SNP Change: no cha
KPAGEGASAHRAGHHTRQARGGSRPSSRGMQGAPSRSSARLEAGGCSARRGRSAPAPSSFSLPLPSFSPFACN
SSPTAPSLLLLPRSPPPCSLRAPGRELVGARGLVPEPSSAEPGGSAAHPAAAGSPSAAGAGPGGDCTGALRAG
3RSCAAAPFPDRPPAHLVSSRRSAPPGSREPRGTGHLHPPLGVSGSSWCLACVSWMPCGFSPSPVAHHLVPGP
PDTPAQQLRCGWTVGGWLLSLVRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEP
SSEQQLCALSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQATEWAS
SEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRTTEKINGVARCPYDPRHNS
TAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLFAYFFLRENAVEHDC
GRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIA
ASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQ
PVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHVLPPGRREPLR
SLRILHSAR.ALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQNITACP
VRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWAL
CSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRA
CGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEG
CAGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVAT
VHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRAN
PLNKHSFRPEASPGQRCFPNS
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 2B.
Table 2B. Comparison of the NOV2 protein sequences.
NOV2a MPAGEGASAHRAGHHTRQARGGSRPSSRGMQGAPSRSSARLEAGGCSARRGRSAPAPSSF
NOV2b ____________________________________________________________
NOV2c ____________________________________________________________
NOV2d ____________________________________________________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g ____________________________________________________________
NOV2h -___________________________________________________________
NOV2a SLPLPSFSPFACNSSPTAPSLLLLPRSPPPCSLRAPGRELVGARGLVPEPSSAEPGGSAA
NOV2b ____________________________________________________________
NOV2C ____________________________________________________________
NOV2d ____________________________________________________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g ____________________________________________________________
NOV2h ____________________________________________________________
NOV2a HPAAAGSPSAAGAGPGGDCTGALRAGGRSCAAAPFPDRPPAHLVSSRRSAPPGSREPRGT
NOV2b ____________________________________________________________
NOV2C ____________________________________________________________
NOV2d ____________________________________._______________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g ____________________________________________________________
NOV2h ____________________________________________________________
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NOV2a GHLHPPLGVSGSSWCLACVSWMPCGFSPSPVAHHLVPGPPDTPAQQLRCGWTVGGWLLSL
NOV2b ---------------------MPCGFSPSPVAHHLVPGPPDTPAQQLRCGWTVGGWLLSL
____________________________________________________________
'NOV2c
'NOV2d ____________________________________________________________
INOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g ___________________________________-________________________
NOV2h ____________________________________________________________
NOV2a VRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCA
NOV2b VRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCA
NOV2c ____________________________________________________________
NOV2d _____________________________________-______________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g -------------------~GPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCA
NOV2h _____________________________________________.______________
NOV2a LSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQA
NOV2b LSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQA
NOV2c ______________________________________________-_____________
NOV2d ____________________________________________________________
NOV2e ___-________________________________________________________
NOV2f ____________________________________________________________
NOV2g LSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQA
NOV2h ____________________________________________________________
NOV2a TEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
NOV2b TEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
____________________________________________________________
NOV2c
NOV2d ____________________________________________________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g TEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
NOV2h ____________________________________________________________
NOV2a TEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNS
NOV2b TEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNS
NOV2c ____________________________________________________________
NOV2d ____________________________________________________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g TEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNS
NOV2h ____________________________________________________________
NOV2a KWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTF
NOV2b KWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTF
NOV2c ____________________________________________________________
NOV2d ____________________________________________________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g KWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTF
NOV2h _______-____________________________________________________
NOV2a MKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAF
NOV2b MKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAF
NOV2c ____________________________________________________________
NOV2d ____________________________________________________________
NOV2e ____________________________________________________________
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NOV2f _____________________________!______________________________
NOV2g MKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAF
NOV2h _____________________________-___________________________
NOV2a NGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVT
NOV2b NGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVT
NOV2C ______________________________________-____________-________
NOV2d ____________________________________________________________
NOV2e _____________-______________________________________________
NOV2f ____________________________________________________-_______
NOV2g NGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVT
NOV2h ____________________________________________________________
NOV2a PEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHV
NOV2b PEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHV
NOV2c ____________________________________________________________
NOV2d ____________________________________________-_______________
NOV2e ________________________________-____________-___-__________
NOV2f ____________________________________________________________
NOV2g PEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHV
NOV2h _____________________________________-______________________
NOV2a LPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQ
NOV2b LPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQ
NOV2c _______-____________________________________________________
NOV2d _____________________________________-_-____________________
NOV2e ____________________________________________________________
NOV2f _________________________________-__________________________
NOV2g LPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQ
NOV2h ________________________________-_______________________-___
NOV2a QRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2b QRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2c --------------------___________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2d ------------------_____________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2e -------------------____________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2f ------------------__-__________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2g QRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2h ------------------_______-_______GpWSPWQPCEHLDGDNSGSCLCRARSC
NOV2a DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2b DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGTGFQVRQRSCSNPAPRHG
NOV2c DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2d DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2e DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2f DSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2g DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2h DSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2a GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2b GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2c GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMRSRRRACENGNSCLGCG
NOV2d GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2e GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2f GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCLGCG
NOV2g GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2h GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCLGCG
'NOV2a VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
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NOV2b VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2c VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2d VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2e VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2f VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
'NOV2g VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2h VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
INOV2a ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2b ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2c ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2d ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2e ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2f ETRTCPADGSGSCDTDALVEVLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2g ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2h ETRTCPADGSGSCDTDALVEVLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2a CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2b CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2c CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2d CTNPESRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2e CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2f CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2g CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2h CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2a PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2b PAPSP---------------------EGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2C PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2d PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2e PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2f PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2g PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2h PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2a AGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSC
NOV2b AGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSC
NOV2c AGNSSQSRPCVD-_______________________________________________
NOV2d AGNSSQSRPCVD-_______________________________________________
NOV2e AGNSSQSRPCVD-_______________________________________________
NOV2f AGNSSQSRPCVD-_______________________________________________
NOV2g AGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSC
NOV2h AGNSSQSRPC-_________________________________________________
NOV2a QHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQ
NOV2b QHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQ
NOV2c ____________________________________________________________
NOV2d ____________________________________________________________
NOV2e ____________________________________________________________
NOV2f ____________________________________________________________
NOV2g QHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQ
NOV2h ____________________________________________________________
NOV2a TNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
NOV2b TNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
NOV2c ________________________________
NOV2d ________________________________
NOV2e ________________________________
NOV2f ________________________________
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NOV2g TNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
NOV2h ________________________________
NOV2a (SEQ ID NO: 6)
NOV2b (SEQ ID NO: 8)
NOV2c (SEQ ID NO: 10)
NOV2d (SEQ ID NO: 12)
NOV2e (SEQ ID NO: 14)
NOV2f (SEQ ID NO: 16)
NOV2g (SEQ ID NO: 18)
NOV2h (SEQ ID NO: 20)
Further analysis of the NOV2a protein yielded the following properties shown
in Table
2C.
Table 2C. Protein Sequence Properties NOV2a
SignalP analysis: ' No Known Signal Sequence Predicted
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 11; pos.chg 1; neg.chg 1
H-region: length 5; peak value -8.91
PSG score: -13.31
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -7.65
possible cleavage site: between 53 and 54
»> Seems to have no N-terminal signal peptide
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 1
Tentative number of TMS(s) for the threshold 0.5: 3
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood = -4.83 Transmembrane 259 - 275
PERIPHERAL Likelihood = 1.54 (at 232)
ALOM score: -4.83 (number of TMSs: 1)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 266
Charge difference: -3.5 C(-2.5) - N( 1.0)
N >= C: N-terminal side will be inside
» > membrane topology: type 2 (cytoplasmic tail 1 to 259)
MITDISC: discrimination of mitochondrial targeting seq
R content. 7 Hyd Moment(75): 4.89
Hyd Moment(95): 4.42 G content: 7
D/E content: 2 S/T content: 8
Score. -1.94
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 104 LRAIPG
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite. none
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content of basic residues: 9.3%
NLS Score: -0_47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memY~RL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: too long tail
Dileucine motif in the tail: found
LL at 81
LL at 82
LL at 83
LL at 237
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: nuclear
Reliability: 94.1
COIL: Lupas's algorithm to detect coiled-coil regions
1 total: 0 residues
Final Results (k = 9/23):
47.8 %: nuclear
26.1 %: mitochondrial
8.7 %: cytoplasmic
4.3 %: Golgi
4.3 %: plasma membrane
4.3 %: extracellular, including cell wall
4.3 %: peroxisomal
» prediction for CG106951-O1 is nuc (k=23)
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 ZD.
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Table
2D.
Geneseq
Results
for
NOV2a
NOV2a Identities/
Geneseq Protein/Organism/LengthResidues/similarities Expect
[Patent for the
Identifier#, Date] Match Value
Matched Region
Residues
AAE18212Human MOL4 protein - 1..1352 1352/1352 0.0
Homo (100%)
sapiens,1352 aa. 1..1352 1352/1352
(100%)
[W0200206339-A2, 24-JAN-2002]
AAG68293Human semaphorin G-like202..13521150/1151 0.0
NHP (99%)
protein SEQ ID NO:l 1..1151 1150/1151
0 - Homo (99%)
Sapiens, 1151 aa.
[W0200188133-A2, 22
NOV-2001]
AAG68294Human semaphorin G-like202..13521135/1151 0.0
NHP (98%)
protein SEQ ID N0:12 1..1136 1135/1151
- Homo (98%)
sapiens,1136 aa.
[W0200188133-A2, 22
NOV-2001]
AAG68290Human semaphorin G-like~ 260..13521092/1093 0.0
NHP (99%)
protein SEQ ID N0:4 1..1093 1092/1093
- Homo (99%)
Sapiens, 1093 aa.
[W0200188133-A2, 22
NOV-2001]
AAG68292Human semaphorin G-like260..13521077/1093 0.0
NHP (98%)
protein SEQ ID N0:8 1..1078 1077/1093
- Homo (98%)
sapiens, 1078 aa.
[W0200188133-A2, 22-NOV-2001]
In a BLAST search of public sequence databases, the NOV2a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 2E.
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Table
2E.
Public
BLASTP
Results
for
NOV2a
NOV2a
Protein Identities!
AccessionProtein/Organism/LengthResidues/Similarities E~Pect
for the
Number Matched PortionValue
R sidues
Q9P283 Hypothetical protein 151..13521202/1202 (100%)0.0
KIAA1445 -
Homo sapiens (Human),12021..1202 120211202 (100%)
as
(fragment).
Q60519 Semaphorin SB precursor260..13521021/1093 (93%)0.0
(Semaphorin G) (Sema 1..1093 1053/1093 (95%)
G) - Mus
musculus (Mouse), 1093
aa.
Q13591 Semaphorin SA precursor299..1336616/1043 (59%)0.0
(Semaphorin F) (Sema 30..1071781/1043 (74%)
F) - Homo
Sapiens (Human),1074
aa.
Q62217 Semaphorin SA precursor299..1336617/1046 (58%)0.0
(Semaphorin F) (Sema 30..1074776/1046 (73%)
F) - Mus
musculus (Mouse), 1077
aa.
Q8BXLT8 Sema domain - Mus musculus299..1109507/811 (62%) 0.0
(Mouse), 844 aa. 30..839 632/811 (77%)
PFam analysis predicts that the NOV2a protein contains the domains shown in
the Table
2F.
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Table 2F.
Domain
Analysis
of NOV2a
Identities/
Pfam DomainNOV2a Match RegionSimilarities Expect Value
for the Matched
Region
Sema 327..738 217/491 (44%) 7e-202
372/491 (76%)
PSI 756..803 18/67 (27%) 2.Se-14
40/67 (60%)
tsp_1 869..920 23/54 (43%) 3.Se-12
3 8/54 (70%)
tsp_I 927..971 ; 17/53 (32%) 4 3e-06
31/53 (58%)
tsp 1 1058..1108 24/53 (45%) 9.1e-11
3 4/53
( 64%)
tsp 1 1115..1165 23/53 (43%) 5.9e-08
( 35/53
66%)
tsp_1 1170..1210 17/53 (32%) 0.0034
( 51 %)
27/53
Example 3.
The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 3A.
3A. NOV3
Sa, CG121295-O1 SEQ ID N_O: _23 ~~750 by
Sequence ORF Start: ATG at 41 ORF Ston: TGA at 701
_GAATGGATTATTTGCTCATGATTTTCTCTCTGCTG
AGGCGCTGAGCTCAGCGCGGTGGGTGAGAACGGCG
TGTGCTAGCCAAAAAGACAAGAAGTGCTGGAATTTTTGCCAAGC
AGTGAGAGGAAGAAAAATCAGAAGAAGTTCAGAGGAACACC
TTGGTGACAGACCTTCGGGGCCTGTCTGAAGCCA
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CG121295-Ol ~SEp ID NO: 24 X220 as BMW at 25403.9kD
FSLLFVACQGAPETAVLGAELSAVGENGGEKPTPSPPWRLRRSKRCSCSSLMDKECWFCHLDIIW
LSLDNRHWPYGLGSPRSKRALENLLPTKATDRENRCQCASQKDKKCWNFCQAGKELRAEDIMEKD
KDCSKLGKKCIYQQLVRGRKIRRSSEEHLRQTRSETMRNSVKSSFHDPKLKGKPSRERYVTHNRAH
Further analysis of the NOV3a protein yielded the following properties shown
in Table
3B.
Table 3B. Protein Sequence Properties NOV3a
SignalP analysis: Cleavage site between residues 1 ~ and 19
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 2; pos.chg 0; neg.chg 1
H-region: length 17; peak value 0.00
PSG score: -4.40
GvH: von Heijne's method for signal seq_ recognition
GvH score (threshold: -2.1): 1.68
possible cleavage site: between 17 and 18
»> Seems to have no N-terminal signal peptide
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 1
Tentative number of TMS(s) for the threshold 0.5: 1
Number of TMS(s) for threshold 0.5: 0
PERIPHERAL Likelihood = 6.31 (at 67)
ALOM score: -1.59 (number of TMSs: 0)
MITDISC: discrimination of mitochondrial targeting seq
R content: 0 Hyd Moment(75): 4.56
Hyd Moment(95): 7.21 G content: 1
D/E content: 2 S/T content: 1
Score: -7.31
Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: PPWRLRR (3) at 43
pat7: PWRLRRS (4) at 44
pat7: PRSKRAL (5) at 96
bipartite: KKCIYQQLVRGRKIRRS at 161
content of basic residues: 18.6
NLS Score: 1.05
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
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VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: nuclear
Reliability: 94.1
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
69.6 %. nuclear
13.0 %. mitochondrial
8.7 %: extracellular, including cell wall
8.7 %. cytoplasmic
» prediction for CG121295-O1 is nuc (k=23)
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.
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Table
3C.
Geneseq
Results
for
NOV3a
NOV3a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
ABU03518Angiogenesis-associated 1..220 211/220 (95%)e-125
human protein
sequence #63 - Homo Sapiens,1..212 212/220 (95%)
212 aa.
[W0200279492-A2,10-OCT-2002]
ABP65215Hypoxia-regulated protein1..220 211/220 (95%)e-125
#89 - Homo
Sapiens, 212 aa. [WO200246465-A2,1..212 212/220 (95%)
13-JUN-2002]
AAG64862Heart muscle cell differentiation1..220 211/220 (95%)e-125
related
protein SEQ 1D NO: 65 1..212 212/220 (95%)
- Homo
Sapiens, 212 aa. [W0200148151-Al,
05-JUL-2001 ]
AAB99933Human ETl protein sequence1..220 ~ 211/220 e-125
SEQ ID (95%)
N0:65 - Homo Sapiens, 1..212 212/220 (95%)
212 aa.
[W0200148150-Al, 05-JLJL-2001]
AAB00197Preproendothelin-1 - Homo1..220 211/220 (95%)e-125
Sapiens,
212 aa. [W0200055314-A2, 1..212 212/220 (95%)
21-SEP-2000]
In a BLAST search of public sequence databases, the NOV3a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 3D.
Table
3D.
Public
BLASTP
Results
for
NOV3a
NOV3a
Protein Identities/
AccessionProtein/Organism/LengthResidues/Similarities Expect
for the
Number Matched PortionValue
Residues
P05305 Endothelin-1 precursor 1..220 211/220 (95%)e-124
(ET-1) -
Homo Sapiens (Human), 1..212 212/220 (95%)
212 aa.
P17322 Endothelin-1 precursor 1_.219 148/220 (67%)3e-80
(ET-1) -
Bos taurus (Bovine}, 1..202 167/220 (75%)
202 aa.
P09558 Endothelin-1 precursor 1..219 145/221 (65%)7e-78
(ET-1) - Sus
scrofa (Pig), 203 aa. 1..203 168/221 (75%)
P22387 Endothelin-1 precursor 1..219 147/220 (66%}le-77
(ET-1) -
Mus musculus (Mouse), 1..202 1651220 (74%)
202 aa.
Q9BG76 Preproendothelin-1 - 1..219 142/220 (64%)9e-76
Ovis aries
(Sheep), 202 aa. 1..202 164/220 (74%)
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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
endothelin 48..78 26/31 (84%) 8.6e-20
31/31 (100%)
Example 4.
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 4A.
4A. NOV4
'4a, CG124756-Ol S~EQ ID NO: 25 ! 1076 by _
. Sequence ORF Start: ATG at 75 ' ORF Stop: TGA at 834
TGATGATGAAGATCCCATGGGGCAGCATCCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATC
CCAGGCCCAGCTCAGCTGCACCGGGCCCCCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCC
AAAGGTGGCCCAGGGGCCCCTGGAGCCCCAGGCCCCAAAGGTGAATCGGGAGACTACAAGGCCACCCAGAAAA
TCGCCTTCTCTGCCACAAGAACCATCAACGTCCCCCTGCGCCGGGACCAGACCATCCGCTTCGACCACGTGAT
CACCAACATGAACAACAATTATGAGCCCCGCAGTGGCAAGTTCACCTGCAAGGTGCCCGGTCTCTACTACTTC
ATGCCTACAACACCTTCCAGGTCACCACCGGTGGCATGGTCCTCAAGCTGGAGCAGGG
GCAGGCCACCGACAAGAACTCACTACTGGGCATGGAGGGTGCCAACAGCATCTTTTCC
(GCAACGCTCACTCTACCCCCAACACCACCCCTTGCCCAGCCAATGCACACAGTAGGGCTTGGTGAATGCTGCT
GAGTGAATGAGTAAATAAACTCTTCAAGGCCAAGGG
IO
CG124756-O1 SEQ ID NO: 26 253 as ~MW at 26721.S1cD
GSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
GIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVI
EPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQG
TDKNSLLGMEGANSIFSGFLLFPDMEA
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V4b, CG124756-02 SEQ ID NO: 27 816 by _
A Sequence _ __ __ ~ ORF Start: ATG at 48 _ O__RF_Sto_p: T_GA at 807 _
~GTAACCTTCACATTGTCTTCTCCACAGGAGGCGTCTGACACAGTATGATGATGAAGATCCCATGGGGCAG
.'CCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATCTCCCAGGCCCAGCTCAGCTGCACCGGG
:CCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCCCCGATGGCCAACCTGGGACCCCAGGGA
CCTGGAGCC
CGGGTTCCTGCTCTTTCCAGATATGGAG
4b, CG124756-OZ ~SEQ ID NO: 28 X253 as BMW at 26721.SkD
PWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
DPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVI
IFSGFLLFPDMEA
V4c, SNP13382475 of SEQ ID NO: 29 1076 by _
124756-Ol, DNA Sequence O~' Start: ATG at 75 ORF Stop: TGA at 834
:. . _ .__ .
SNP Pos: 302 SNP Chance. G to T
TCCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATC
CCCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCC
TAAGGGAGACCCAGGGATTCCTGGGAATCCAGGAAAAGTCGGCCCCAAGGGCCCCATGGGCCCT
ACAACAATTATGAGCCCCGCAGTGGCAAGTTCACCTGCAAGGTGCCCGGTCTCTACTACTTC
CAGCTCTCGAGGGAACCTGTGCGTGAACCTCATGCGTGGCCGGGAGCGTGCACAGAAGGTGG
GACTATGCCTACAACACCTTCCAGGTCACCACCGGTGGCATGGTCCTCAAGCTGGAGCAGGG
CTTTCCAGATATGGAGGCCTGACCTGTGGGCTGCTTCACATCCACCCCGGCTCCCCCTGCCA
TCTACCCCCAACACCACCCCTTGCCCAGCCAATGCACACAGTAGGGCTTGGTGAATGCTGCT
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V4c, SNP13382475 of SEQ ID NO: 30 253 as ~MW at 26707.SkD
124756-Ol, Protein Sequence ~SNp pos: 76 SNP Chance: Glu to
MKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
_DKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVI
iMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQG
iVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA
V4d, SNP13382476 of SEQ ID NO: 31 1076.,bp.,...
124756-01, DNA Sequence ORF Start: ATG at 75 O_R_F _S_top:_YTGA_at 8_34
SNP Pos: 433 SNP~~Chan~e: A to~G
TGATGATGAAGATCCCATGGGGCAGCATCCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATC
CCAGGCCCAGCTCAGCTGCACCGGGCCCCCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCC
TCAACGTCCCCCTGCGCCGGGACCAGACCATCCGCTTCGACCACGTGAT
TCACCTTCTGTGACTATGCCTACAACACCTTCCAGGTCACCACCGGTGGCATGGTCCTCAAGCTGGAGCAGGG
GGAGAACGTCTTCCTGCAGGCCACCGACAAGAACTCACTACTGGGCATGGAGGGTGCCAACAGCATCTTTTCC
GGGTTCCTGCTCTTTCCAGATATGGAGGCCTGACCTGTGGGCTGCTTCACATCCACCCCGGCTCCCCCTGCCA
GCAACGCTCACTCTACCCCCAACACCACCCCTTGCCCAGCCAATGCACACAGTAGGGCTTGGTGAATGCTGCT
[GAGTGAATGAGTAAATAAACTCTTCAAGGCCAAGGG
NOV4d, SNP13382476 of SEQ ID NO: 32 ~ 2_53 as ~MW at 26749.6kD _
CG124756-Ol, Protein Sequence SNP Pos: 120 Y ~ SNP Change:~Gln to Arg
MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
FGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKAT_RKIAFSATRTINVPLRRDQTIRFDHVI
TNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVZ'FCDYAYNTFQVTTGGMVLKLEQG
ENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 4B.
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Table 4B. Comparison of the NOV4 protein sequences.
NOV4a MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKG
NOV4b MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKG
NOV4a EKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQ
NOV4b EKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQ
NOV4a KIAFSATRTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNL
NOV4b KIAFSATRTINVPLRRDQTIRFDHVITNr4rINNYEPRSGKFTCKVPGLYYFTYHASSRGNL
NOV4a CVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANS
NOV4b CVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANS
NOV4a IFSGFLLFPDMEA
NOV4b IFSGFLLFPDMEA
NOV4a (SEQ ID NO: 26)
NOV4b (SEQ ID NO: 28)
Further analysis of the NOV4a protein yielded the following properties shown
in Table
4C.
Table 4C. Protein Sequence Properties NOV4a
SignaIP analysis: Cleavage site between residues 28 and 29
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 4; pos.chg 1; neg.chg 0
H-region: length 18; peak value 11.91
PSG score: 7.51
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 4.21
possible cleavage site: between 27 and 28
» > Seems to have a cleavable signal peptide (1 to 27)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 28
Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) .. fixed
PERIPHERAL Likelihood = 2.60 (at 232)
ALOM score: 2.60 (number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al_)
Center position for calculation: l3
Charge difference: -3.0 C(-1.0) - N( 2.0)
N >= C: N-terminal side will be inside
MITDISC: discrimination of mitochondrial targeting seq
R content: 0 Hyd Moment(75): 6.93
Hyd Moment(95): 5.45 G content: 2
D/E content: 1 S/T content: 1
Score. -5.61
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Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 9.5%
NLS Score: -0.47
'KDEL: ER retention motif in the C-terminus: none
iER Membrane Retention Signals: none
iSKL: peroxisomal targeting signal in the C-terminus: none
IIPTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSTTE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic _
Reliability: 76.7
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23).
22.2 %: extracellular, including cell wall
22.2 %. vacuolar
22.2 %: mitochondrial
22.2 %: endoplasmic reticulum
ll.l %: Golgi
» prediction for CG124756-01 is exc (k=9)
__..... . . . . . . . _ ._ . .. .
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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.
Table
4D.
Geneseq
Results
for
NOV4a
NOV4a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
Ident'erDate] Match the Matched Value
ResiduesRegion
AAM40607Human polypeptide SEQ 1..253 253/253 (100%)e-151
1D NO 5538
- Homo Sapiens, 255 aa. 3..255 253/253 (100%)
[W0200153312 Al, 26-JLJL-2001]
AAM38821Human polypeptide SEQ I ..253 253/253 (100%)e-151
ID NO 1966
- Homo Sapiens, 253 aa. 1..253 253/253 (100%)
[W0200153312-Al, 26-JUL-2001]
ABB57231Mouse ischaemic condition3..253 201/253 (79%)e-117
related
protein sequence SEQ 1..253 218/253 (85%)
ID N0:599 -
Mus musculus, 253 aa.
[W0200188188-A2, 22 NOV-2001]
AAU32411Novel human secreted 1..248 203/267 (76%)e-103
protein #2902 -
Homo Sapiens, 309 aa. 3..269 212/267 (79%)
[WO200179449-A2, 25-OCT-2001]
AAU30709Novel human secreted 23..248 195/243 (80%)e-102
protein #1200 -
Homo Sapiens, 287 aa. 2..244 199/243 (81%)
[W0200179449-A2, 25-OCT-2001]
In a BLAST search of public sequence databases, the NOV4a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 4E.
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Table
4E.
Public
BLASTP
Results
for
NOV4a
Protein NOV4a Identities)
AccessionProtein/Organism/LengthResidues/Similarities Expect
for the
Number Matched PortionValue
Residues
C1HUQB complement subcomponent1..253 253/253 (100%)e-151
Clq
chain B precursor [validated]1..253 253/253 (100%)
-
human, 253 aa.
P02746 Complement Clq subcomponent,3..253 251/251 (100%)e-150
B
chain precursor - Homo 1..251 251/251 (100%)
sapiens
(Human), 251 aa.
P14106 Complement Clq subcomponent,3..253 2011253 (79%)e-117
B
chain precursor - Mus 1..253 219/253 (86%)
musculus
(Mouse), 253 aa.
I49560 complement Clq B chain 3..253 201/253 (79%)e-117
precursor -
mouse, 253 aa. 1..253 ' 218/253
(85%)
P31721 Complement Clq subcomponent,3..252 197/252 (78%)e-115
B
chain precursor - Rattus1..252 217/252 (85%)
norvegicus
(Rat), 253 aa.
PFam analysis predicts that the NOV4a protein contains the domains shown in
the Table
4F.
Table 4F. Domain Analysis of NOV4a
Identities/
Pfam Domain NOV4a Match Region Similarities Expect Value
for the Matched Region
Collagen 51..110 35/60 (58%) 8.7e-09
45/60 (75%)
Clq 123..247 69/138 (50%) 2.4e-72
124/138 (90%)
Example 5.
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table SA.
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able SA. NOVS Sequence Analysis
fOVSa, CG50353-Ol SEQ 1D NO: 33 ~ ' 1628 by
ANA Sequence OItF Start: ATG at 1 ORF Stop: TGA at 1048
TGAACCGGAAAGCGCGGCGCTGCCTGGGCCACCTCTTTCTCAGCCTGGGCATGGTCTGTCTCCTAGCATGTG
CTTCTCCTCAGTGGTAGCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCG
GCGATCTGCCAGAGCCGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGT
CGGG
GCGCACGGAGATGTACACGTGCAAGTGAGCCCCGTGTGCACACCACCCTCCCGCTGCAAGTCAGATTGCTGGG
T PTT P~ml~l~T /'~I~P~TTTI~I~T T l~P~ml~l~l~P~Tl~m/'Il"~!'tml'fP~I~T l1/~T
mlYlYm~ ~n ~~mm~mr~mmmm~m~nm~ ~n iv~-w .~r~nm,. .r
CCCAATGCTGCTCCACCCTCCCCCAGACACAGCCCAGGTCCCTCCGCGGCTGGAGCGAAGCCTTCTGCAGCAG
GAACTCTGGACCCCTGGGCCTCATCACAGCAATATTTAACAATTTATTCTGATAAAAATAATATTAATTTATT
(AGGATGATTTTGTTGCTAGGACAAGGAGCCGTGTAGAAGTGTACATAACTATTCTTTATGCAGATATTTCTAC
TAGCTGATTTTGCAGGTACCCACCTTGCAGCACTAGATGTTTAAGTACAAGAGGAGACATCTTTTATGCATAT
ATAGATATACACACACGAAAAA
a, CG50353-O1 SEQ ID NO: 34 349 as MW at 38980.7kD
LFLSLGMVCLLACGFSSVVALGATVICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDEC
SALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAITAACTHGNLSDCGCDKEKQGQYHRDEG
YGIGFAKVFVDAREIMKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLP
KYNAAVQVEVVRASRLRQPTFLRIKQLRSYRKPMKTDLVYIEKSPNYCEEDPVTGSVGTQGR
5b, 228753443 SEQ ID NO: 35 966 by
Sequence ORF Start: at 1 ORF Stop: end of sequence ~~
CTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
TCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
CACAGTTTCGGGAGCT
TCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
TGT
142
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
228753443 ~SEQ ID NO: 36 322 as MW at 36054.9kD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
KKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
>c, 169475673 SEQ ID NO:_37 J966 by
Sequence ~pRF Start: at 1 J' ORF Ston: end of
TCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGTCAGTTTCAGTTCCG
CTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGAAT
169475673 ~SEQ ID NO: 38 X322 as BMW at 36054.9kD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
VYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
VSd, 228753459 SEQ ID NO: 39 f 966 by
__. __ .. _ _ ~ "~",~~,.~, ..~_____.._ _ _.__~._.. ~ ~"
~1 Sequence ORF Start: _at 1 _ ~ ORF Stop: end of sequence
TCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
TTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
CTGCTCCGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
GCCCGGACTCTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGAAT
GTAAGTGCCACGGCGTGTCAGGCTCGTGCACCACCAAGACGTGCTGGACCACACTGCCACAGTTTCGGGAGCT
CCCACCTTCCTGAAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
143
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
Sd, 228753459 SEQ m NO: 40 322 as MW at 36054.9kD
CQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
KKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
VSe, 228753462 SEQ ID NO: 41 966 by
Sequence ORF Start: at 1 ~y ORF Stop: end of sequence
TCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
.TGGCCGCTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
228753462 SEQ m NO: 42 322 as MW at 36083.OkD
TVICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
YAIIAAGVVHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
IKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
if, 228753446 SEQ ID NO: 43 985 by
___ __
Sequence ~ORF Start: at 2 ~ ~~: ORF Stop: end of
AGGAGAAGGCTCACAAATGGGCCTGGACG
TCCTGGAG
CCAGCCGCAACAAGCGGCCCACCTTCCTGAAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACG
CCTGGTGTACATCGAGAAGTCGCCCAACTACTGCGAGGAGGACCCGGTGACCGGCAGTGTGGGCACCCAGG
CGCGCCTGCAACAAGACGGCTCCCCAGGCCAGCGGCTGTGACCTCATGTGCTGTGGGCGTGGCTACAACAC
ACCAGTACGCCCGCGTGTGGCAGTGCAACTGTAAGTTCCACTGGTGCTACTATGTCAAGTGCAACACGTGC
144
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
228753446 1SE0 ID NO: 44 X328 as BMW at 36733.6kD
EFALRSLGATVICNKIPGLAPRQR.AICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKE
VGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA
TKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVR
RNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNT
ig, 228753465 SEQ ID NO: 45 966 by
Sequence ~gp' Start: at 1 ~ ORF Stop: end of sequence
~TCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
T
CAAGGACAAGTACAACGAGGCCGTTCACGTGGAGCCTGTGCGTGCCAGCCGCAACAAGCGG
AAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
ACTGCGAGGAGGACCCGGTGACCGGCAGTGTGGGCACCCAGGGCCGCGCCTGCAACAAGAC
TGTCAAGTGCAACACGTGCAGCGAGCGCACGGAGATGT
g, 228753465 SEQ ID NO: 46 322 as ~MW at 36173.OkD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCDCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
KKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
FHWCCYVKCNTCSERTEMYTCKLE
Sh q 28753438 O~ ~Ovy47 ~~~~966 b "...~ p
..d.~...~...._~..._.._...~._.~~____...._._
Se,~.uence,.
.._......__...____._._....._..___.__.._...__..at...l..._.....~..___._._._~~_Sto
.._~.._end of sequence,.w., .._..._.....____..__
CTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
TGGCCGCTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
GCTGCGTTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
TCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
TGTCAAGTGCAACACGTGCAGCGAGCGCACGGAGATGT
145 '
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
228753438 SEQ 117 NO: 48 322 as ~MW at 35998.9kD
PRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
ITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
DTDLVYIEKSTNCCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
TCSERTEMYTCKLE
228753449 ~SEO ID NO: 49
Sequence ~ORF Start: at 1 ORF Stop: end of
AGATCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
GCCGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGTCAGTTTCAGTTCCG
CAATGGCCGCTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
GAGGCTGCGTTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
CTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
GCCCGGACTCTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGGAT
ACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
ACGCCCGCGTG
228753449 ~SEQ ID NO: 50 X322 as BMW at 35926.8kD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LHNNEAGRKILEENMKLGCKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
KKPLSYRKPMDTDLVYIEKSTNCCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
VSj, CG503S3-02 SEQ ID NO:_5_l 966 by
A Sequence ~ORF Start: at 7 ORF Stop: at 961
GGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
ACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGTCAGTTTCAGTTCCG
CTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
CTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
GCCCGGACTCTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGAAT
CCCACCTTCCTGAAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
TGTCAAGTGCAACACGTGCAGCGAGCGCACGGAGATGT
146
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
Sj, CG50353-02 ~SEQ ID NO: 52 318 as MW at 35569.4kD
CNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREA
IAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNAR
nnaFanRxrr.FFnmrtKr.FCKCHGVSGSCTTKTCWTTLPOFRELGYVLKDKYNEAVHVEPVRASRNKRPT
TEMYTCK
CG50353-03 SEQIDN0:53 X1057
ANA Sequence ORF Start: ATG at 1 ORF Stop: TGA at 1048
TGAACCGGAAAGCGCGGCGCTGCCTGGGCCACCTCTTTCTCAGCCTGGGCATGGTCTGTCTCCTAGCATGTG
CTTCTCCTCAGTGGTAGCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCG
GCGATCTGCCAGAGCCGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGT
AAGTGGGGAGCCGGGACGGTGCGTTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGC
CTGTACCCATGGCAACCTGAGCGACTGTGGCTGCGACAAAGAGAAGCAAGGCCAGTACCACCGGGACGAGGGC
TGGAAGTGGGGTGGCTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTCGTGGACGCTCGGG
AGATCATGAAGAACGCGCGGCGCCTCATGAACCTGCATAACAATGAGGCCGGCAGGAAGGTTCTAGAGGACCG
rama~ArrmarAGTGC_AAGTGCCACGGCGTGTCTGGCTCCTGCACCACCAAAACCTGCTGGACCACGCTGCCC
ATCGCAAGCCCATGAAGACGGACCT
TGCAACAAGACGGCTCCCCAGGCCAGCGGCTGTGACCTCATGTGCTGTGGGCGTGGCTACAACACCCACC
ACGCCCGCGTGTGGCAGTGCAACTGTAAGTTCCACTGGTGCTGCTATGTCAAGTGCAACACGTGCAGCGA
CACGGAGATGTACACGTGCAAGTGAGCCCCGT
CG50353-03 SEQ ID NO: 54 349 as ~MW at 38980.7kD
LSLGMVCLLACGFSSVVALGATVICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDEC
LGERTVFGKELKVGSRDGAFTYAIIAAGVAHAITAACTHGNLSDCGCDKEKQGQYHRDEG
IGFAKVFVDAREIMKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLP
NAAVQVEVVRASRLRQPTFLRIKQLRSYRKPMKTDLVYIEKSPNYCEEDPVTGSVGTQGR
LMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK
147
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
BSI, SNPI3382474 of SEQ ID NO: 55 1628 bp_ ___
0353-O1, DNA Sequence ORF Start: ATG at I OR_F Stop: T_GA at1048
SNP Pos: 951 SNP Chance: G to T
CGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGT
CCATGGCAACCTGAGCGACTGTGGCTGCGACAAAGAGAAGCAAGGCCAGTACCACCGGGACGAGGGC
TGGGGTGGCTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTCGTGGACGCTCGGG
TGAAGAACGCGCGGCGCCTCATGAACCTGCATAACAATGAGGCCGGCAGGAAGGTTCTAGAGGACCG
TGAAGACGGACCT
GAACTCTGGACCCCTGGGCCTCATCACAGCAATATTTAACAATTTATTCTGATAAAAATAATATTAATTTATT
TAATTAAAAAGAATTCTTCCACCTCGTCGGGATCCGTTTTCTGCAATCAAAGTGGACTGCTTGCTTTCCTAGC
AGGATGATTTTGTTGCTAGGACAAGGAGCCGTGTAGAAGTGTACATAACTATTCTTTATGCAGATATTTCTAC
TAGCTGATTTTGCAGGTACCCACCTTGCAGCACTAGATGTTTAAGTACAAGAGGAGACATCTTTTATGCATAT
SNP13382474 of ~ SEQ ID NO: 56 349 as MW at 38989.7kD
301, Protein Sequence SNP Pos: 317 ~ SNP Change: Gln to His
2CLGHLFLSLGMVCLLACGFSSWALGATVICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDEC
~RWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAITAACTHGNLSDCGCDKEKQGQYHRDEG
iADIRYGIGFAKVFVDAREIMKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLP
iLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYRKPMKTDLVYTEKSPNYCEEDPVTGSVGTQGR
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table SB.
I48
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
Table SB. Comparison of the NOVS protein sequences.
NOVSa MNRKARRCLGHLFLSLGMVCLLACGFSSWALGATVICNKIPGLAPRQRAICQSRPDAII
NOVSb ___-_________-_______________RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOV5c -----------------------------RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSd --------------------------__-RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSe --------------------------___RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOV5f -- .-----------------__S~FALRSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSg ----------------------------_RSLGATVICNKTPGLAPRQRAICQSRPDAII
NOV5h ----------------------------_RSI,GATVICNKTPGLAPRQRAICQSRPDAII
NOVSi ---------------------------__RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSj --------------------------__-__I,GATVICNKIPGLAPRQRAICQSRPDAII
NOVSk MNRKARRCLGHLFLSLGMVCLLACGFSSWALGATVICNKTPGLAPRQRAICQSRPDAII
NOVSa VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAIT
NOVSb VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOV5c VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSd VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSe VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVVHAIT
NOVSf VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSg VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSh VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSi VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSj VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSk VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAIT
NOVSa AACTHGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIMKNARRLM
NOV5b AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSc AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
'NOVSd AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
'NOVSe AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSf AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSg AACTQGNLSDCDCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSh AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSi AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSj AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSk AACTHGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIMKNARRLM
NOVSa NLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEV
NOVSb NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSc NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5d NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSe NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5f NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5g NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5h NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSi NLHNNEAGRKILEENMKLGCKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSj NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSk NLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEV
NOVSa VRASRLRQPTFLRIKQLRSYRKPMKTDLWIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSb VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSPNYCEEDpVTGSVGTQGRACNKTAPQ
NOVSc VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSd VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDpVTGSVGTQGRACNKTAPQ
NOVSe VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSPNYCEEDpWGSVGTQGRACNKTAPQ
NOVSf VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSg VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNXCEEDPVTGSVGTQGRACNKTAPQ
NOVSh VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSTNCCEEDPVTGSVGTQGRACNKTAPQ
I49
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
NOVSi VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSTNCCEEDPVTGSVGTQGRACNKTAPQ
NOVSj VR.ASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSk VRASRLRQPTFLRIKQLRSYRKPMKTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
'NOVSa ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK--
'NOVSb ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
INOVSc ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSd ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSe ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSf ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCYYVKCNTCSERTEMYTCKLE
NOVSg ASGCDLMCCGRGYNTHQYARVWQYNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSh ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSi ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSj ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK--
NOVSk ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK--
NOVSa(SEQID 34)
NO:
NOVSb(SEQID 36)
NO:
NOVSc(SEQID 38)
NO:
NOV5d(SEQID 40)
NO:
NOVSe(SEQID 42)
NO:
NOVSf(SEQID 44)
NO:
NOV5g(SEQID 46)
NO:
NOVSh(SEQID 48)
NO:
NOVSi(SEQID 50)
NO:
NOVSj(SEQID 52)
NO:
NOVSk(SEQID 54)
NO:
Further analysis of the NOVSa protein yielded the following properties shown
in Table
SC.
Table SC. Protein Sequence Properties NOVSa
SignalP analysis: Cleavage site between residues 32 and 33
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 7; pos.chg 4; neg_chg 0
H-region: length 32; peak value 10.30
PSG score: 5.90
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -0.60
possible cleavage site: between 27 and 28
» > Seems to have a cleavable signal peptide (1 to 27)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 28
Tentative number of TMS(s) for the threshold 0_5: 2
Number of TMS(s) for threshold 0.5: 0
PERIPHERAL Likelihood = 6.89 (at 151)
ALOM score: 0.05 (number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 13
Charge difference: -2.5 C( 3.0) - N( 5.5)
150
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
N >= C: N-terminal side will be inside
MITDISC: discrimination of mitochondrial targeting seq
R content: 6 Hyd Moment(75): 11.39
Hyd Moment(95): 26.83 G content: 5
D/E content: 1 S/T content: 5
Score: 1.59
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at G5 SRP~DA
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 14.6
NLS Score. -0.47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: NRKA
none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC. possible vacuolar targeting motif: found
TLPK at 217
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 55.5
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
65.2 &: mitochondrial
15I
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I3.0 %: Golgi j
8.7 %: extracellular, including cell wall
8.7 %: endoplasmic reticulum
4.3 %: cytoplasmic I
» prediction for CG50353-O1 is mit (Ic=23) ',
A search of the 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 SD.
Table SD. Geneseq Results for NOVSa
NOVSa Identities/
Geneseq Protein/Organism/Length . Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
ABJ10594Human novel protein NOVSa1..349 349/349 (100%)0.0
SEQ ID
NO: 16 - Homo Sapiens, ~ 1..349~ 349/349
349 aa. (100%)
[W0200259315-A2, Ol-AUG-2002]
AAY57598Human Wnt-7a protein 1..349 321/349 (91%)0.0
- Homo
Sapiens, 349 aa. [W09957248-Al,1..349 335/349 (95%)
11 NOV-1999]
AAY70737' Human Wnt-7a protein ' 1..349321/349 (91 0.0
- Homo %)
Sapiens, 349 aa. [W0200021555-Al,1..349 3351349 (95%)
20-APR-2000]
AAB19789Human Wnt-7a protein 1..349 321/349 (91%)0.0
involved in
kidney tubulogenesis 1..349 335/349 (95%)
- Homo sapiens,
349 aa. [W0200061630-Al,
19-OCT-2000]
AAE34043WNT-7A protein - Unidentified,1..349 317/349 (90%)0.0
349
aa. [W0200290992-A2, 1..349 333/349 (94%)
14 NOV-2002]
In a BLAST search of public sequence databases, the NOVSa protein was found to
have
homology to the proteins shown in the BLASTP data in Table SE.
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Table
5E. Public
BLASTP
Results
for NOVSa
NOVSa Identities/
Protein ~ Residues/SimilaritiesExpect
for
AccessionProtein/Organism/LengthMatch the Matched Value
Number ResiduesPortion
000755 Wnt-7a protein precursor1..349 321/349 (91 0.0
- Homo %)
Sapiens (Human), 349 1..349 335/349 (95%)
aa.
Q96H90 Hypothetical protein 1..349 317/349 (90%)0.0
- Homo Sapiens
(Human), 349 aa. 1..349 333/349 (94%)
AAH49093 Hypothetical protein 1..349 315/349 (90%)0.0
- Mus
musculus (Mouse), 433 85..433 332/349 (94%)
as
(fragment).
Q9DBY3 Wingless-related MMTV 1..349 315/349 (90%)0.0
integration
site 7A - Mus musculus 1..349 332/349 (94%)
(Mouse),
349 aa.
P24383 Wnt-7a protein precursor1..349 313/349 (89%)0.0
- Mus
musculus (Mouse), 349 1..349 330/349 (93%)
aa.
PFam analysis predicts that the NOVSa protein contains the domains shown in
the Table
SF.
Table~~SF. Domain Analysis of NOVSa
~~' Identities!
Pfam Domain NOVSa Match Region Similarities Expect Value
for the Matched Region
wnt 37..349 180/352 (51%) 3.2e-212
298/352 (85%)
Example 6.
The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 6A.
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6A. NOV6
ia, CG50709-03 SEQ ID NO: 57 ~93 by __ _
Sequence ORF Start: at 1 '~ORF ~Ston: end of
TTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCC
CTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGACTC
CTGAGCAACTTCCTGGGGTCCAAGAGAGGAAACAAGGACCTGCGGGCACGGGCAGACGCCCACAATACCCACG
TGGGCATCAAGGCTGTGAAGAGTGGCCTCAGGACCACGTGTAAGTGCCATGGCGTATCAGGCTCCTGTGCCGT
GCGCACCTGCTGGAAGCAGCTCTCCCCGTTCCGTGAGACGGGCCAGGTGCTGAAACTGCGCTATGACTCGGCT
GTCAAGGTGTCCAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCC
6a, CG50709-03 SEQ ID NO: 58 331 as MW at 36432.2kD
VLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
RTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
GSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
ATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
SRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
ib, 282997951 SEQ ID NO: 59 928 by
Sequence ORF Start: at 2 ~ ORF Stop: end of
CACCGGATCCCAGTGTGACCTGCTGAAGCTGTCCCGGCGGCAGAAGCAGCTCTGCCGGAGGGAGCCCGGCCTG
CTCTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGAC
TCAGGCTCCTGTGCC
TACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGC
ATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGA
GCAATGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCTCGAGGGC
154
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282997951 ~SEQ ID NO: 60 X309 as BMW at 34226.6kD
QCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVS
LTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTH
KAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGS
GLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVE
CG50709-OS ~SEQ ID NO: 61 ~ 1464
Sequence ~ORF Start: ATG at 38 ORF Stop: TAG at 1109
TTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCCACCTGAAGCAGTGTGACCTGCTGAAGCTGTC
GCCAGTTTCAGTTCCGGCATGAGCGCTGGAACTGTAGCCTGGAGGGCAGGACGGGCCTGCTCA
CAAAGAGACAGCTTTCCTGTACGCGGTGTCCTCTGCCGCCCTCACCCACACCCTGGCCCGGGC
GGGCGCATGGAGCGCTGCACCTGTGATGACTCTCCGGGGCTGGAGAGCCGGCAGGCCTGGCAG
GCGGTGACAACCTCAAGTACAGCACCAAGTTTCTGAGCAACTTCCTGGGGTCCAAGAGAGGAA
GCGGGCACGGGCAGACGCCCACAATACCCACGTGGGCATCAAGGCTGTGAAGAGTGGCCTCAG
AAGTGCCATGGCGTATCAGGCTCCTGTGCCGTGCGCACCTGCTGGAAGCAGCTCTCCCCGTTC
GCCAGGTGCTGAAACTGCGCTATGACTCGGCTGTCAAGGTGTCCAGTGCCACCAATGAGGCCT
AGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCCTCACCAAAGGCCTGGCCCCAAGGTCTGGGGA
ATGGAGGACTCACCCAGCTTCTGCCGGCCCAGCAAGTACTCACCTGGCACAGCAGGTAGGGTG
AGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGGGGCTATGACACCCAGAGCCGCCTGGTGGCCT
CTGCCAGGTGCAGTGGTGCTGCTACGTGGAGTGCCAGCAATGTGTGCAGGAGGAGCTTGTGTA
GGGCAGACTGTCATCACATGCATGCATAAACCGGCATGTGTGCCAATGCACACG
TTCCTTGGCCAGCCTTTTGCCTCCCTCGATACTCAACAAAGAGAAGCAAAGCCT
ATTCCATCTTGCTTC
CG50709-OS ~SEQ ID NO: 62 X357 as BMW at 38970.2kD
RPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPG
AETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCD
~SPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSC
..VRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCR
SKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
155
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277582109 ~SEp ID NO: 64 X364 as BMW at 39615.9kD
TGSTMRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCR
REPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMER
CTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGV
SGSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSP
SFCRPSKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG
277582117 SEQ ID NO: 65 X1024
Sequence ORF Start: at 2 ORF Stop: end of
TGCTGCGCACCTCGGCCTGCTTGAGTGCCAGTTTCA
TGG
TCAGGCTCCTGTGCCGTGCGCACCTGCTGGAAGCAGCTCTCCCCGTTCCGTGAGACGGGCCAGGTGCT
TGCGCTATGACTCGGCTGTCAAGGTGTCCAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGG
156
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277582117 ~SEQ ID NO: 66 341 as MW at 37431.2kD
Y H'CiL'1'CiKr:
VL'1'Yr'YCiLCi'1'AAAYAQCiGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQ
ERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLAR.ACSAGRMERCTCDDSPGLESRQAWQWGVCGDN
STKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVL
YDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASC
CCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG
NOV6f, CG50709-O1 SEQ ID NO: 67 ' _1021 by _
DNA Sequence ORF Start: at 3~~ORF Stop: TAG at 996-- J.._",~,
T_CCTGACCGGGCGGGAAGTCCTGACGCCCTTCCCAGGATTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGC
CCACCTGAAGCAGTGTGACCTGCTGAAGCTGTCCCGGCGGCAGAAGCAGCTCTGCCGGAGGGAGCCCGGCCTG
TGGGCCTGCTCAAGAGAGGCTTCAAAGAGACAGCTTTCCTGTACGCGGTGTC
CCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGAC
CG50709-O1 SEQ ID NO: 68 X331 as BMW at 36462.3kD
REVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
EGRMGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
FLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
SSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
TQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
5
ig, CG50709-02 SEQ ~ NO: 69 933 by
Sequence ORF Start: ATG at 274- ~~ ~ ORF Stop: TAG at 928
GCCTGGCTGAGACCCTGAGGGATGCTGCGCACCTCGGCCTGCTTGAGTGCCAGTTTCAGTTCCGGCATGAGCG
~CTGGAACTGTAGCCTGGAGGGCAGGACGGGCCTGCTCAAGAGAGGCTTCAAAGAGACAGCTTTCCTGTACGCG
GTGTCCTCTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTG
TGA
157
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CG50709-02 SEQ ID NO: 70 218 as MW at 24076.1kD
PGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKC
RTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYME
KYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
ih, CG50709-04 SEQ ID NO: 71 _,_849 by
Se uence _...__.._._ ___~__..__........._._._.. _- p: end of
q ~ Start. at 1 'Sto -_ .__.___..__.._
CGGCAGGCCTGGCAGTGGGGCGTGTGCGGTGACAACCTCAAGTACAGCACCAAGTTTCTGAG
CCTGCTGGAAGCAGCTCTCCCCGTTCCGTGAGACGGGCCAGGTGCTGAAACTGCGCTATGACTCGGCTGTCAA
GGTGTCCAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCCTCACC
anarrr~mar;~ce~CAAGC~TCTGGGGACCTGGTGTACATGGAGGACTCACCCAGCTTCTGCCGGCCCAGCAAGT
I~10V6h, CG50709-04 ~SEQ ID NO: 72 ~~283 as ~MW at 31272.41eD
Protein Sequence
KQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRMGLLKRGFKETAFLYAVSSA
ALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVG
IKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLT
KGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQV ___-
ii, CG50709-06 SEQ ID NO:_73 1093 by
Sequence ORF Start:~ATG at 14 ORF Stop: end of
CGGCACAGGGCGGGGCCCACCTGAAGCAGTGTGACCTGCTGAAGCTGTCCCGGCGGCAGAAGCAGCTCTGCCG
ACCCACGTGGGCATCAAGGCTGTGAAGAGTGGCCTCAGGACCACGTGTAAGTGCCATGGCGT
CAGT
I58
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CG50709-06 ~SEQ ID NO: 74 360 as MW at 39269.6kD
RPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPG
AETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCD
SPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSC
VRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCR
SKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG
CG50709-07 ~SEQIDN0:75 1024
Sequence ~ORF Start: at 2 ~ORF Stop: end of
TGAGCGCTGGAACTGTAGCCTGGAGGGCAGGACGGGCCTGCTCAAGAGAGGCTTCAAAGAGACA
TACGCGGTGTCCTCTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGG
TACCCACGTGGGCATCAAGGCTGTGAAGAGTGGCCTCAGGACCACGTGTAAGTGCCATG
CAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGG
CACCCAGCTTCTGCCGGCCCAGCAAGTACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTG
CAGCAGCCTGTGCTGCGGGCGGGGCTATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTG
CAGTGGTGCTGCTACGTGGAGTGCCAGCAATGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCACCTCGAGG
6j, CG50709-07 ~SEQ ID NO: 76 341 as MW at 37431.2kD
SYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQ
ERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDN
STKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVL
YDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASC
CCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG
159
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61c, SNP13381605 of SEQ ID NO: 77 993 by
)709-03, DNA Sequence ORF Start: a_t_1 ORF Stop: end of see
SNP Pos: 653 ~~ - SNP Change: C to T
CT
CAAGTACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGG
TGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCAC
SNP13381605 of SEQ m NO: 78 _ 331 as ~MW at 36458.3kD
-03, Protein Sequence ~Np pos: 218 ~ ~~SNP Change: Ser to Leu
LTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
CSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
LSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYD_LA
VKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
GYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH _ _~,.,_~m~-~~~~
S
I~OV61, SNP13381606 of SEQ ID NO: 79 993 by __ _ _
CG50709-03, DNA Sequence ORF Start: at 1 ORF Stop: endYof sequence
SNP Pos: 743 SNP Change: T to C
CTGACCGGGCGGGAAGTCCTGACGCCCTTCCCAGGATTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCC
GACCCTGAGGGATGCTGCGCACCTCGGCCTGCTTGAGTGCCAGTTTCAGTTCCGGCATGAGCGCTGGAAC
AGCCTGGAGGGCAGGACGGGCCTGCTCAAGAGAGGCTTCAAAGAGACAGCTTTCCTGTACGCGGTGTCCT
nr~ar~r~mrnr~~rnr~nr~t~r~rt~~f'CCC~C~C~CCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGA
CTC
CACG
CAAGTACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGG
GGCTATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT
160
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NOV6I, SNP13381606 of SEQ ID NO_: 80 331 as ,~MW_at 36416._2kD_
CG50709-03, Protein Sequence SNP Pos: 248 ~ SNP Change: Leu to Pro
PFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
LLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
RGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
EALGRLELWAPARQGSLTKG_PAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
VAFSCHCQVQWCCYVECQQCVQEELVYTCKH
~6m, SNP13378337 of SEQ ID NO_ : 81 _ 993 by
0709-03, DNA Sequence ORF Start: at l ORF Ston: end of
Pos: 764 SNP Change: T to C
ACAGCACCAAGTTT
ATCAGGCTCCTGTGCCGT
CGGTGTACATGGAGGACTCACCCAGCTTCTGCCGGCCCAG
TGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT
CAATGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCAC
SNP13378337 of SEQ ID NO: 82 331 as MW at 36416.2kD
-03, Protein Sequence SNP Pos: 255 ~ SNP Change: Leu to Pro
rPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
3LLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
EQ2GNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
VEALGRLELWAPARQGSLTKGLAPRSGD_PVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
LVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
161
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SNP13381607 of SEQ ID NO: 83 X993 by _
-03, DNA Sequence ORF Start: at 1 ~ORF Stop: end of
Pos: 799 SNP Change: C to T
AAGTGCCATGGCGTATCAGGCTCCTGTGCCGT
AGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCC
ATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT
6n, SNP13381607 of SEQ ID NO: 84 331 as ~MWat 36422.2kD_
1709-03, Protein Sequence SNP Pos: 267 SNP Change: Pro to Ser
EVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
GRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
SATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCR_SSKYSPGTAGRVCSREASCSSLCCGR
QSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
162
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SNP13378336 of SEQ )D NO: 86 331 as M_W at 36372.2kD
-03, Protein Sequence SNP Pos: 294 SNP Change: Tyr to
LTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
CSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
LSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
VKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
GCDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
6p, SNP13378335 of SEQ m NO: 87 =993 by
)709-03, DNA Sequence ORF Start: at 1 ORF Stop: end of
SNP Pos: 977 ISNP Change: T to C
CTGACCGGGCGGGAAGTCCTGACGCCCTTCCCAGGATTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCC
CTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGACTC
TCCGGGGCTGGAGAGCCGACAGGCCTGGCAATGGGGCGTGTGCGGTGACAACCTCAAGTACAGCACCAAGTTT
ATGACTCGGCT
AAAGGCCTGGCCCCAAGGTCTGGGGACCTGGTGTACATGGAGGACTCACCCAGCTTCTGCCGGCCCAG
ACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGG
TGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT
CAATGTGTGCAGGAGGAGCTTGCGTACACCTGCAAGCAC
T6p, SNP13378335 of SEQ ID NO:. 88 331_ as .MW at 36404.1kD _
0709-03, Protein Sequence ' SNP Pos. 326 SNP Change: Val to Ala
PGLAETLRDAAHLGLLECQFQFRHERWN
LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
SATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
QSRLVAFSCHCQVQWCCYVECQQCVQEELAYTCKH
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 6B.
Table 6B. Comparison of the NOV6 protein sequences.
NOV6a ------------------------------LTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6b ______________________________________________________TGSQCD
NOV6c ----MRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6d TGSTMRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6e -----------------------TGSSYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6f ------------------------------LTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6g ____________________________________________________________
NOV6h ________________________________________________________KQCD
NOV6i ----MRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
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NOV6j -----------------------TGSSYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
'NOV6a LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
'NOV6b LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
'NOV6c LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6d LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6e LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6f LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRMGLLKRGFKET
NOV6g _____________________________________________-______________
NOV6h LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRMGLLKRGFKET
NOV6i LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6j LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6a AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6b AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6c AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6d AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6e AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6f AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6g -----------------------MERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6h AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6i AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6j AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6a LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6b LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6c LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6d LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6e LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6f LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6g LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6h LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6i LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6j LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6a VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6b VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6c VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6d VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6e VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6f VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6g VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6h VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6i VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6j VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6a SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6b SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6c SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6d SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6e SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6f SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6g SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6h SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQV---------------------
NOV6i SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6j SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6a H---
~NOV6b LEG-
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NOV6cH---
NOV6dHLEG
NOV6eHLEG
NOV6fH---
NOV6gH---
NOV6h----
NOV6iHLEG
NOV6jHLEG
NOV6a(SEQ ID 58)
NO:
NOV6b(SEQ ID 60)
NO:
NOV6c(SEQ ID 62)
NO:
NOV6d(SEQ ID 64)
NO:
NOV6e(SEQ ID 66)
NO:
NOV6f(SEQ ID 68)
NO:
NOV6g(SEQ ID 70)
NO:
NOV6h(SEQ ID 72)
NO:
NOV6i(SEQ ID 74)
NO:
~NOV6j(SEQ ID ....7.6Y),
NO:...
Further analysis of the NOV6a protein yielded the following properties shown
in Table
6C.
Table 6C. Protein Sequence Properties NOV6a
SignalP analysis: No Known Signal Sequence Predicted
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region. length 5; pos.chg 1; neg.chg 1
H-region: length 21; peak value 7.18
PSG score: 2.78
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -5.38
possible cleavage site: between 22 and 23
» > Seems to have no N-terminal signal peptide
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 1
Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) .. fixed
PERIPHERAL Likelihood = 4.08 (at 90)
ALOM score: 4.08 (number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 6
Charge difference: 3.5 C( 4.5) - N( 1.0)
C > N: C-terminal side will be inside
» >Caution: Inconsistent mtop result with signal peptide
MITDISC: discrimination of mitochondrial targeting seq
R content: 1 Hyd Moment(75): 11.91
Hyd Moment(95): 8.21 G content: 5
D/E content: 2 S/T content. 3
Score: -6.56
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Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 53 CRR~EP
',NUCDISC: discrimination of nuclear localization signals
' pat4: none
pat7: none
bipartite: none
content of basic residues: 14.2%
NLS Score: -0.47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: TGRE
KKXX-like motif in the C-terminus: YTCK
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: found
KLSRRQKQL at 33
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
~NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction. nuclear
Reliability: 76.7
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23).
78.3 %: nuclear
13.0 %: mitochondrial
8.7 %: cytoplasmic
» prediction for CG50709-03 is nuc (k=23)
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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
ResiduesRegion
AAE17306Human WNT15 protein, 1..331 330/331 (99%)0.0
sbg389686WNT15a #2 - 31..361 330/331 (99%)
Homo
Sapiens, 361 aa. [WO200198342-Al,
27-DEC-2001]
AAE17305Human WNT15 protein, 1..331 330/331 (99%)0.0
sbg389686WNT15a #1 - 17..347 330/331 (99%)
Homo
sapiens, 704 aa. [W0200198342-Al,
27-DEC-2001 ]
ABB77769Amino acid sequence of 1..331 330/331 (99%)0.0
human Wnt
(ZwntS) polypeptide variant4..334 330/331 (99%)
- Homo
Sapiens, 334 aa. [W0200231148-A2,
18-APR-2002]
ABB77768Amino acid sequence of 1..331 330/331 (99%)0.0
human Wnt
(ZwntS) polypeptide - 31..361 330/331 (99%)
Homo Sapiens,
361 aa. [W020023I148
A2,
18-APR-2002]
ABB83080Wnt family related protein1..331 330/331 (99%)0.0
2 - Homo
sapiens, 363 aa. [W0200250278-A2,33..363 330/331 (99%)
27-JUN-2oo2]
s
In a BLAST search of public sequence databases, the NOV6a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 6E.
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Table -
6E.
Public
BLASTP
Results
for
NOV6a
NOV6a
Protein Identities/
AccessionProteinJOrganism/Length Residues/Similarities Expect
for the
Number Matched PortionValue
Residues
014905 Wnt-9b protein precursor1..331 331/331 (100%)0.0
(Wnt-15)
(Wnt-14b) - Homo sapiens27..357331/331 (100%)
(Human),
357 aa.
Q8C718 WNT14B - Mus musculus 1..330 310/330 (93%) 3 0:0
(Mouse),
359 aa. 29..358319/330 (95%)
035468 Wnt-9b protein precursor1..330 310/330 (93%) 0.0
(Wnt-15)
(Wnt-14b) - Mus musculus29..358319/330 (95%)
(Mouse),
359 aa.
014904 Wnt-9a protein precursor1..330 209/335 (62%) e-124
(Wnt-14) -
Homo Sapiens (Human), 33..364255/335 (75%)
365 aa.
Q8RSM2 Wnt-9a protein precursor1..330 208/335 (62%) e-123
(Wnt-14) -
Mus musculus (Mouse), 33..364255/335 (76%)
365 aa.
PFam analysis predicts that the N0V6a protein contains the domains shown in
the Table
6F.
Table 6F. Domain Analysis of NOV6a
Identities/
Pfam Domain NOV6a Match Region Similarities Expect Value
for the Matched Region
wnt ~ 28..330 132/354 (37%) 2.1e-104
234/354 (66%)
Example 7.
The N0V7 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 7A.
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7A. NOV7 Sequence Analysis
Via, CG53054-02 SEQ ID NO: 89 1128 by
Sequence ORF Start: ATG at 31 ORF Stop: TGA at 1102
ACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACCCTGGA
CGAGTGCCAGTTCC
CCACAACAACCTCGTGGGTGTGAAGGTGATCAAGGCTGGGGTGGAGACCACCTGCAAGTGC
rrcTCATGCACGGTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGC
CATCTCCCCACCACGGGGCCGTGCCTCGGGGGCAGGTGGCAGCGACCCGCTGCCCCGCACTCCAGAGCTGGTG
CACCTGGATGACTCGCCTAGCTTCTGCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTGCCACC
GTGAGAAGAACTGCGAGAGCATCTGCTGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTG
CG53054-02 SEQ 1D NO: 90 357 as ~MW at 39756.1kD
APLGYFLLLCSLKQALGSYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAET
VEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEA
DLENREAWQWGGCGDNLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVR
CWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCL
GRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG
lb,170251039 SEQ ID NO: 91 1029 by
Sequence OIZF Start: at 1 ~~RF Stop: end of
ATGCCATCTCCTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGCCGCAT
AAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGCCTCGGGGGCAGGTGGCAGCGACCCGCTGCCCCGCACTCCAGAGCTGGTGCACCT
TTCTGCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAG
TCTGCTGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT
rTGCTATGTGCtAGTGCAGGCAGTGCACGCAGCGTGAGGAGGTCTACACCTGCAAGGG
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170251039 ~SEQ ID NO: 92 X343 as BMW at 38208.1kD
IWWLTGSEPLTILPLTLEPEAGAQAHYKACDRLKLERKQRRMCRRDPGVVETLVEAVSMSALECQFQF
DTCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGD
SKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHL
TALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAGRFSPGTAGRRCHRE
ICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKGVD
~c,170251076 SEQ ID NO: 93 ~~~.~w1029 by .~.e~
Sequence ORF Start: at 1 ORF Stou: end of
TGTGCCG
CGCTTTGAGCGCTGGAACTGCACGCTGGAGGGCCGCTACCGGGCCAGCCTGCTCAAGCGAGGCTTCAAGGAGA
CTGTACCTGCGATGAGGCACCCGACCTGGAGAACCGTGAGGCCTGGCAGTGGGGGGGCTGCGGAGAC
AAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGAGACGGCACTCAAGGTGGGCAGCACCACCAATGAAGCTGCCGGCGAGGCAGGTGCCATCT
TGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT
ATGTGGAGTGCAGGCAGTGCACGCAGCGTGAGGAGGTCTACACCTGCAAGGG
170251076 ~SEQ ID NO: 94 X343 as BMW at 38194.1kD
PIWWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQF
WNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGD
SSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHL
FTALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAGRFSPGTAGRRCHRE
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7d, CG53054-Ol SEQ ID NO: 95~ ~c-1085 by ~__
Sequence ORF Start: ATG at 13 ORF Ston: TGA at 1078
CAAGGCCTGCGACCGGCTGAAGCTGGAGCGGAAGCAGCGGCGCATGTGCCGCCGGGACCCGGGCGTGGCAGAG
ACGCTGGTGGAGGCCGTGAGCATGAGTGCGCTCGAGTGCCAGTTCCAGTTCCGCTTTGAGCGCTGGAACTGCA
CTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGCCGCATGGAGCGCTGTACCTGCGATGAG
TGCCCGCTCAGCCATGAACCGCCACAACAACGA
GTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGCATCTGAAGCACAAGTATGAGTCGG
CACTCAAGGTGGGCAGCACCACCAATGAAGCTGCCGGCGAGGCAGGTGCCATCTCCCCACCACGGGGCCGTGC
CCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAGAAGAACTGCGAGAGCATCT
CG53054-O1 ~SEQ m NO: 96 X355 as BMW at 39194.1kD
ALLYSSLGVWCTCSPSYFGLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVE
VSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDL
NREGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQVIKAGVETTCKCHGVSGSCTVRTC
RQLAPFHEVGKHLKHKYESALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAG
FSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG
~e, CG53054-03 SEQ ~ NO: 97 1029 by
Sequence ORF Start: at 7 OR_F Stop: _at 1024 _
_CAGCTACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACCCTGGAGCCAG
3GCCGCCCAGGCGCACTACAAGGCCTGCGACCGGCTGAAGCTGGAGCGGAAGCAGCGGCGCATGTGCCG
TCTCCTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGCCGCAT
TTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGCACGGTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGCATCTG
CTCCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAG
AACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT
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CG53054-03 ~SEQ ID NO: 98 1339 as 1MW at 37835.8kD
WWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRF
CTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNL
KFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHLKH
CESICCGRGHNTQSRVUZ'RPCQCQVRWCCYVECRQCTQREEVYTCKG
NOV7f, CG53054-04 SEQ ID NO_: 99 _ 1631 by _ _
DNA Sequence ORF S_tar_t:~ATGaat 12 y ~,pRF Stop:mTGA at_~1107
GGCGCGGCAAGATGCTGGATGGGTCCCCGCTGGCGCGCTGGCTGGCCGCGGCCTTCGGGCTGACGCTGCTGCT
CGCCGCGCTGCGCCCTTCGGCCGCCTACTTCGGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACC
TGAGTGCGCTCGAGTGCCA
AAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTG
TGAAGCTGCCGGCGAGGCA
TAACACACAGAGCCGGGTGGTGACAAGG
CAGTGCACGCAGCGTGAGGAGGTCTACA
ACACCTGCACAGGCTGAGTTCCTGGGCTCGACCAGCCCAGCTGCGTGGGGTACAGGCATTGCACACAGT
AGTCCTAGCTGCATGGGGTGCAGGCATTGCACAGAGCATGAATGGGCCTACACCTGCCAAGGCTGAATCCCTG
GGCCCAGCCAGCCCTGCTGCACATGGCACAGGCATTGCACACGGTGTGAGGAGTGTACACCTGCAAGGGCTGA
CG53054-04 ~SEO ID NO: 100 X365 as IMW at 40319.7kD
GSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCR
GVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRM
TCDEAPDLENREAWQWGGCGDNLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHG
SCTVRTCWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHL
PSFCLAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 7B.
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Table 7B. Comparison of the NOV7 protein sequences.
NOV7a --------MAPLGYFLLLCSLKQALGSYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7b ------------------------GSSYPIWWLTGSEPLTILPLTLEPEAGAQAHYKACD
NOV?c ------------------------GSSYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7d -----------MALLYSSLGVWCTCSPSYFGLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7e --------------------------SYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7f MLDGSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7a RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7b RLKLERKQRRMCRRDPGVVETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7c RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7d RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7e RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7f RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7a ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV'7b ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7c ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7d ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREGWKWGGCSEDIEFGGMVSR
NOV7e ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7f ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7a EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7b EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7c EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7d EFADARENRPDARSAMNRHNNEAGRQVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7e EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7f EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7a GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7b GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7c GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7d GKHLKHKYESALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7e GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7f GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7a LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7b LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7c LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7d LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7e LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7f LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7a VYTCKG--
NOV7b VYTCKGVD
NOV7c VYTCKGVD
NOV7d VYTCKG--
NOV7e VYTCKG--
NOV7f VYTCKG--
NOV7a (SEQ ID NO: 90)
NOV7b (SEQ ID NO: 92)
NOV7c (SEQ ID NO: 94)
NOV7d (SEQ ID NO: 96)
NOV7e (SEQ ID NO: 98)
NOV7f (SEQ ID NO: 100)
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Further analysis of the NOV7a protein yielded the following properties shown
in Table
7C.
Table 7C. Protein Sequence Properties NOV7a
SignaIP analysis: Cleavage site between residues 19 and 20
.,..~...~~.....~..,......~.,...~..,..~,...~....
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 0; pos.chg 0; neg.chg 0
H-region: length 13; peak value 9.00
PSG score: 4.60
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 0.73
possible cleavage site: between 18 and 19
» > Seems to have a cleavable signal peptide (1 to 18)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 19
Tentative number of TMS(s) for the threshold 0_5: 0
number of TMS(s) .. fixed
PERIPHERAL Likelihood = 3.76 (at 114)
ALOM score: 3.76 (number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 9
Charge difference: 0.0 C( 1.0) - N( 1.0)
N >= C: N-terminal side will be inside
MITDISC: discrimination of mitochondrial targeting seq
R content: 0 Hyd Moment(75): 1.56
Hyd Moment(95): 3.50 G content: 3
D/E content: 1 S/T content: 4
Score: -6.15
Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 14.8&
NLS Score: -0.47
~KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
KICXX-like motif in the C-terminus: YTCK
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal~targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif.
type 1: none
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type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memY~RL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: nuclear
Reliability: 70_6
~ICOIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
55.6 %: extracellular, including cell wall
22.2 %: mitochondrial
11.1 %: vacuolar
11.1 %: nuclear
» prediction for CG53054-02 is exc (k=9)
A search of the NOV7a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several
homologous proteins shown in Table 7D.
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Table
7D.
Geneseq
Results
for
NOV7a
NOV7a Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the MatchedValue
ResiduesRegion
AAE34048WNT-14 protein - Unidentified,2..357 339/356 0.0
365 (95%)
aa. [W0200290992 A2, 13..365 343/356
(96%)
14 NOV-2002]
ABU55894Human WNT-14 protein 2..357 339/356 0.0
- Homo (95%)
sapiens, 365 aa. [W0200277204-A2,13..365 343/356
(96%)
03-OCT-2002]
ABG69638Human secreted protein 2..357 311/357 0.0
SCEP-18 - (87%}
Homo Sapiens, 366 aa. 13..366 327/357
(91 %)
[W0200248337-A2, 20-JUN-2002]
AA018744Human NOVB protein - 25..357 302/334 0.0
Homo Sapiens, (90%)
355 aa. [W0200257450-A2,22..355 316/334
(94%)
25-JUL=2002]
AAE17305Human WNT15 protein, 22..356 210/338 e-124
(62%)
sbg389686WNT15a #1 - 14..346 257/338
Homo (75%)
sapiens, 704 aa. [W0200198342
Al,
27-DEC-200 / ]
n. ..
In a BLAST search of public sequence databases, the NOV7a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 7E.
Table
7E.
Public
BLASTP
Results
for
NOV7a
~
NOV7a Identities/
Protein Residues/SimilaritiesExpect
for
AccessionProtein/Organism/LengthMatch the Matched Value
Number ResiduesPortion
014904 Wnt-9a protein precursor2..357 339/356 (95%)0.0
(Wnt-14) -
Homo Sapiens (Human), 13..365 343/356 (96%)
365 aa.
Q8RSM2 Wnt-9a protein precursor2..357 333/356 (93%)0.0
(Wnt-14) -
Mus musculus (Mouse), 13..365 340/356 (94%)
365 aa.
042280 Wnt-9a protein precursor25..356 283/333 (84%)e-173
(Wnt-14) -
Gallus gallus (Chicken},24..353 310/333 (92%)
354 aa.
Q8C718 WNT14B - Mus musculus 8..356 216/354 (61%)e-125
(Mouse),
359 aa. 12..358 ~ 264/354
(74%)
035468 Wnt-9b protein precursor8..356 216/354 (61%)e-125
(Wnt-15}
(Wnt-14b) - Mus musculus12..358 264/354 (74%)
(Mouse),
359 aa.
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PFam analysis predicts that the NOV7a protein contains the domains shown in
the Table
7F.
Table 7F. Domain Analysis of NOV7a
Identities/
Pfam Domain NOV7a Match Region Similarities Expect Value
for the Matched Region
wnt 50..356 1291359 (36%) 4.6e-103
234/359 (65%)
Example 8.
The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 8A.
Table 8A. NOV8 Sequence Analysis _
I~10V8a, CG53473-02 SEQ ID NO:101 514 by
DNA Sequence OItF Start: ATG at 37 , O1ZF Stop: TGA at 400
CGCGCGCCCGAACGAAGCCGCGGCCCGGGCACAGCCATGGCCCGGCGGGCGGGGGGCGCTCGGATGTTCGGCA
CCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTC
TACAGGAGGCTGCTGGTACAAATACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAG
TTGTGCCCACCCAGGGAAGGTGCTGAATGGGACCCTGTTGATGGCCATCAACAGGGTCCCATTCAGCACAGG
LVOVBb, CG53473-O1 SEQ ID N0:103 , 646 by
DNA Sequence ORF Start: ATG at 62 ORF Stop: TGA at 398
AGCGCGCCCGAACGAAGCCGCGGCCCGGGCACAGCATGGCCCGCGGCGGGAGGGCGCTCGGATGTTCGGCAGC
CTCCTGCACTTCGCCCTGCTCGCTGCCGGCGTCGTCCCGCTCAGCTGGGATCTCCCGGAGCCCCGCAGCCGAG
CCAGCAAGATCCGAGTGCACTCGCGAGGCAAGCTCTGGGCCATCGGTCACTTCATGGGCAAGAAGAGTCTGGA
GCCTTCCAGCCCATCCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTCAT
ACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAGAT
....~.~....-............ .,.n.a..~~wn mivmniv ~n mnmW w T ml~/~m!'nT TI~TI~T T
T
(TCTCTGTTACTCCATTACTGTGATTTCTGGCTGGGTCACCAGAAATATCGCTGATGCAGACACAGATTATGTT'--
CCTGCTGTATTTCCTGCTTCCCTGTTGAATTGGTGAATAAAACCTTGCTCTATACATACAAA
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CG53473-Ol ~SEQ ID NO: 104 112 as ~MW at 12402.SkD
RASKIRVHSRGKLWAIGHFMGKKSLEPSSPSPLGTAPHTSLRDQRL
QYRRLLVQILQK
SNP13376396 of SEQ ID NO: 107_x(514 by _
f-02, DNA Sequence ORF Start: ATG at 37 ~ ORF Stop: TGA at 400
SNP Pos:190 SNP Change: A to G
AGCCATGGCCCGGCGGGCGGGGGGCGCTCGGATGTTCGGCA
TCCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTC
TCCTCCTGCTAAAGAAGGCTCTGGGCGTGAGCCTCAGCCGCCCCGCACCCCAAATCCA
GGTACAAATACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAG
GGAAGGTGCTGAATGGGACCCTGTTGATGGCCATCAACAGGGTCCCATTCAGCACAGG
17S
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SNP1337639$ of SEQ m NO: 109 $14 by
3-02, DNA Sequence ORF Start: ATG at 37 ORF Stop: _TGA_at 400
SNP Pos: 2$3 SNP Change: C to A
TCCGAGTGCACTCGCGAGGCAACCTCTGGGCCACCGGTCACTTCATGGGCAAGAAGAGTCTG
TCTGCTCGGAATCCTCCTGCTAAAGAAGGCTCTGGGCGTGAGCCTCAGCCGCCCCGCACCCCAAATCCA
AGGAGGCTGCTGGTACAAATACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAG
8f, SNP13376394 of SEQ ID NO: 111 514 by
.473-02, DNA Sequence ORF Start: ATG at 37 ORF Stop: T_AA at 4_00
SNP Pos: 401 SNP-Change: G to A
CGCCCGAACGAAGCCGCGGCCCGGGCACAGCCATGGCCCGGCGGGCGGGGGGCGCTCGGATGTTCGGCA
CCTGCTCTTCGCCCTGCTCGCTGCCGGCGTCGCCCCGCTCAGCTGGGATCTCCCGGAGCCCCGCAGCCG
AGCAAGATCCGAGTGCACTCGCGAGGCAACCTCTGGGCCACCGGTCACTTCATGGGCAAGAAGAGTCTG
CTTCCAGCCCATCCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTC
TCTGCTCGGAATCCTCCTGCTAAAGAAGGCTCTGGGCGTGAGCCTCAGCCGCCCCGCACCCCAAATCCA
CAACAGGGTCCCATTCAGCACAGG
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 8B.
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Table 8B. Comparison of the NOV8 protein sequences.
NOVBa MARRAGGARMFGSLLLFALLAAGVAPLSWDLPEPRSRASKIRVHSRGNLWATGHFMGKKS
NOVBb ---------MFGSLLHFALLAAGWPLSWDLPEPRSRASKIRVHSRGKLWA-IGHFMGKKS
NOVSc ______________________________________________GHI,WAIGHFM-___
NOVBa LEPSSPSPLGTAPHTSLRDQRLQLSHDLLGILLLKKALGVSLSRPAPQIQYRRLLVQILQ
NOVBb LEPSSPSPLGTAPHTSLRDQRLQLSHDLLGILLLKKALGVSLSRPAPQIQYRRLLVQILQ
NOVBc ____________________________________________________________
NOVBa K
NOV8b K
NOVBc -
NOVBa (SEQ ID NO: 102)
NOVBb (SEQ ID NO: 104)
NOVBc (SEQ ID NO: 106)
Further analysis of the NOVBa protein yielded the following properties shown
in Table
8C.
Table 8C. Protein Sequence Properties NOVBa
SignalP analysis: Cleavage site between residues 27 and 28
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 9; pos.chg 3; neg.chg 0
H-region: length 20; peak value 10.93
PSG score: 6.53
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2_1): 2.23
possible cleavage site: between 26 and 27
» > Seems to have a cleavable signal peptide (1 to 26)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 27
Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) _. fixed
PERIPHERAL Likelihood = 1.85 (at 87)
ALOM score: 1.85 (number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 13
Charge difference: -1.5 C( 2.5) - N( 4.0)
N >= C: N-terminal side will be inside
MITDISC: discrimination of mitochondrial targeting seq
R content: 3 Hyd Moment(75): 12.45
Hyd Moment(95): 10.60 G content: 4
D/E content: 1 5/T content: 2
Score: -2.16
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 19 ARM~FG
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NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 14_9
NLS Score: -0.47
IKDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: ARRA
none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/NUClear discrimination
Prediction: nuclear
Reliability: 89
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
44.4 ~: extracellular, including cell wall
33.3 &: mitochondrial
22.2 ~s: nuclear
prediction for CG53473-02 is exc (k=9)
A search of the NOV 8a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several
homologous proteins shown in Table 8D.
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Table
SD.
Geneseq
Results
for
NOVBa
.
NOVBa Identities/
Geneseq Protein/Organism/Length ResidueslSimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAE17605Humanextracellularmessenger1..121 121/121 (100%)le-63
(XMES)-7 protein - Homo 1..121 121/121 (100%)
Sapiens,
121 aa. [W0200194587
A2,
13-DEC-2001]
ABP51992NOVNEURhomologous amino 1..121 114/121 (94%)3e-58
acid
sequence SEQ ID N0:29 1..121 114/121 (94%)
- Homo
Sapiens, 121 aa. [US2002068279-Al,
06-JUN-2002]
ABP51987NOVNEUR homologous amino4..121 1121118 (94%)7e-58
acid
sequence SEQ ID N0:24 1..118 112h 18 (94%)
- Homo
Sapiens, 118 aa. [US2002068279-A1,
06-JUN-2002]
ABP51989NOVNEURhomologous amino 4..121 111/11'8 le-56
acid (94%)
sequence SEQ ID N0:26 1..118 111/118 (94%)
- Homo
Sapiens, 118 aa. [US2002068279-A1,
06-JUN-2002]
ABI'S1990NOVNEUR homologous amino10..121 108/112 (96%)7e-56
acid
sequence SEQ ID N0:27 1..112 108/112 (96%)
- Homo
Sapiens, 112 aa. [US2002068279-Al,
06-JUN-2002]
In a BLAST search of public sequence databases, the NOVBa protein was found to
have
homology to the proteins shown in the BLASTP data in Table 8E.
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Table
8E.
Public
BLASTP
Results
for
NOVBa
NOVBa Identities/
Protein Residues!SimilaritiesExpect
for
AccessionProtein/Organism/Length Match the MatchedValue
Number ResiduesPortion
P08949 Neuromedin B-32 precursor1..121 1201121 2e-62
(99%)
[Contains: Neuromedin 1..121 120/121
B] - Homo (99%)
Sapiens (Human), 121 .
aa.
Q9CR53 Neuromedin B-32 precursor1..121 89/121 (73%)2e-43
[Contains: Neuromedin 1..121 991121 (81%)
B] - Mus
musculus (Mouse), 121
aa.
A37178 neuromedin B precursor 1..115 84/115 (73%)2e-4.1
- rat, 117 aa.
1..115 94/115 (81
%)
A28945 neuromedin B precursor 1..73 69/73 (94%)Se-33
- human, 76
1..73 69/73 (94%)
P01297 Neuromedin B-32 [Contains:25..56 30/32 (93%)2e-11
Neuromedin B] - Sus scrofa1..32 30/32 (93%)
(Pig), 32
aa.
PFam analysis predicts that the NOVBa protein contains the domains shown in
the Table
8F.
Table 8F. Domain Analysis of NOVBa
Identities/
Pfam Domain NOVBa Match Region Similarities Expect Value
for the Matched Region
Bombesin 47..56 8/10 (80%) 0.26
10/10 (100%)
Example 9.
The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 9A.
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9A. NOV9 Sequence Analysis
>a, CG55184-03 SEQ ID NO: I 13 614 by
Sequence ORF Start: ATG at 4 ORF Stop: TAG at 607
ACGGACTCCAAGGGCTCCTCTTCCTCCCCGCTGGGGATATCGGTCCGGGCGGCCAACTCCAAGGTCGCC
CGGCGGTGCGGAGCACCAACCACGAGCCATCCGAGATGAGCAACAAGACGCGCATCATTTACTTCGATC
CCTGGTGAATGTGGGTAATTTTTTCACATTGGAGTCTGTCTTTGTAGCACCAAGAAAAGGAATTTACAG
AGTTTTCACGTGATTAAAGTCTACCAGAGCCAAACTATCCAGGTTAACTTGATGTTAAATGGAAAACCA
TATCTGCCTTTGCGGGGGACAAAGATGTTACTCGTGAAGCTGCCACGAATGGTGTCCTGCTCTACCTAG
AAA(zc,ATAAGGTTTACCTAAAACTGGAGAAAGGTAATTTGGTTGGAGGCTGGCAGTATTCCACGTTTTC
CG55184-03 ~SEQ ID N0:114 X201 as BMW at 21807.9kD
MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPLGISVRAANSKVAF
SAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIYSFSFHVIKVYQSQTIQVNLMLNGKPV
ISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLEKGNLVGGWQYSTFSGFLVFPL_,v_,.~ . - ~"
fib, CGSSI84-O1 SEQ ID_NO: 115 _ .614 by
Sequence ORF Stazt:~ATG at 4 ORF Stop:YTAG at 607
AGACGCGCATCATTTACTTCGATC
AGCACCAAGAAAAGGAATTTACAG
TAAGGTTTACCTAAAACTGGAGAAAGGTAATTTGGTTGGAGGCTGGCAGTATTCCACGTTTTC
GTGTTCCCCCTATAGGATTC
CG55184-OI ~SEQ ID NO: I16 201 as MW at 21807.9kD
MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPLGISVRAANSKVAF
SAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIYSFSFHVIKVYQSQTIQVNLMLNGKPV
ISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLEKGNLVGGWQYSTFSGFLVFPL _-
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NOV9c, CGSS I84-02 SEQ ID NO: 117 522 bp- _ __~
DNA Sequence ORF Start: at 1 ~ OR_F Stop: end of sequence
CAGAACGACACGGAGCCCATTGTGCTGGAGGGCAAGTGTCTGGTGGTGTGCGACTCGAACCCGGCCACGGACT
CCAAGGGCTCCTCTTCCTCCCCGCTGGGGATATCGGTCCGGGCGGCCAACTCCAAGGTCGCCTTCTCGGCGGT
GCGGAGCACCAACCACGAGCCATCCGAGATGAGCAACAAGACGCGCATCATTTACTTCGATCAGATCCTGGTG
TTAAAGTCTACCAGAGCCAAACTATCCAGGTTAACTTGATGTTAAATGGAAAACCAGTAATATCTGC
GGGGGACAAAGATGTTACTCGTGAAGCTGCCACGAATGGTGTCCTGCTCTACCTAGATAAAGAGGAT
CG55184-02 ~SEQ ID NO: 118 174 as ~MW at 19080.6kD
KCLWCDSNPATDSKGSSSSPLGISVRAANSKVAFSAVRSTNHEPSEMSNKTRIIYFDQILV
FVAPRKGIYSFSFFiVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKED
VGGWQYSTFSGFLVFPL '~
Vie, CG55184-OS SEQ m NO: 121 45 b
-'_..~ ~_._._Jr.__
Sequence ~ORF Start: at 1 RF Ston: end of
. q
NOV9e, CG55I84-OS SEQ ID NO: 122 15 as MW at I588.71eD
Protein Se uence . ~,__~___~~;
_r.._._.___~._~.~ ___. __ ~ .._.____..._._~~ -:~
ANSKVAFSAVRSTNfi
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 9B.
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Table 9B. Comparison of the NOV9 protein sequences.
NOV9a MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPL
NOV9b MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLWCDSNPATDSKGSSSSPL
NOV9c ---------------------------QNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPL
NOV9d ____________________________________________________________
NOV9e ____________________________________________________________
NOV9a GISVRAANSKVAFSAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIY
NOV9b GISVRAANSKVAFSAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIY
NOV9c GISVRAANSKVAFSAVRSTNHEPSEMSNKTRTIYFDQILVNVGNFFTLESVFVAPRKGIY
NOV9d _____p,~7S~AFSAVRSTNH-______________________________________
NOV9e ______~JS~AFSAVRSTNH-______________________________________
NOV9a SFSFHVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLE
NOV9b SFSFHVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLE
NOV9c SFSFHVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKEDKVYLKL,E
NOV9d _______________________________-____________________________
NOV9e __-___________-_________________________________-___________
NOV9a KGNLVGGWQYSTFSGFLVFPL
NOV9b KGNLVGGWQYSTFSGFLVFPL
NOV9c KGNLVGGWQYSTFSGFLVFPL
NOV9d _____________________
NOV9e ___________________._
NOV9a (SEQ ID NO: 114)
NOV9b (SEQ ID NO: 116)
NOV9c (SEQ ID NO: 118)
NOV9d (SEQ ID NO: 120)
NOV9e (SEQ ID N0: 122)
Further analysis of the NOV9a protein yielded the following properties shown
in Table
9C.
Table 9C. Protein Sequence Properties NOV9a
SignalP analysis: Cleavage site between residues 28 and 29
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 6; pos.chg 2; ~neg.chg 0
Ii-region: length 23; peak value 10.04
PSG score: 5.64
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 0.95
possible cleavage site. between 27 and 28
» > Seems to have a cleavable signal peptide (1 to 27)
ALOM: Klein et al's method for TM region allocation
Tnit position for calculation: 28
Tentative number of TMS(s) for the threshold 0.5: 1
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Number of TMS(s) for threshold 0.5: 0
PERIPHERAL Likelihood = 5.67 (at 60)
ALOM score: 0.10 {number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 23
Charge difference: -5.0 C(-2.0) - N( 3.0)
N >= C: N-terminal side will be inside
MITDISC: discrimination of mitochondrial targeting seq
R content: 2 Hyd Moment(75): 11.01
Hyd Moment(95): 9.83 G content: 3
D/E content: l S/T content: 3
Score: -2.58
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 16 RRA~LS
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 9.5%
NLS Score: -0.47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: GSGR
none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
ACtinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif. none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: . none
(checking 71 PROSITE ribosomal protein motifs: none
'checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 94.1
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
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Final Results (k = 9/23):
33.3 ~: extracellular, including cell wall
33.3 ~: mitochondrial
22.2 ~: endoplasmic reticulum
11.1 ~: Golgi
» prediction for CG55184-03 is exc (k=9)
A search of the NOV9a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several
homologous proteins shown in Table 9D.
Table
9D.
Geneseq
Results
for
NOV9a
3
NOV9a ~ Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the Matched Value
ResiduesRegion
AAE16346Human cerebellin-like 1..201 201/201 (100%)e-11
protein, I
POLY10 -Homo Sapiens, 1..201 201/201 (100%)
201 aa.
[W0200185767-A2, 15 NOV-2001]
ABB84924Human PR01382 protein 1..201 ~ 201/201 e-111
sequence (100%)
SEQ ID N0:216 - Homo Sapiens,1..201 201/201 (100%)
201
aa. [W0200200690-A2,
03-JAN-2002]
ABB95530Human angiogenesis related1..201 201/201 (100%)e-111
protein
PRO1382 SEQ 1D NO: 216 1..201 201/201 (100%)
- Homo
Sapiens, 201 aa. [W0200208284-A2,
31-JAN-2002]
AA015422Human genset metabolic 1..201 201/201 (100%)e-111
gene
(GMG-8) protein - Homo 1..201 2011201 (100%)
Sapiens, 201
aa. [W0200255694-A2,
18-JUL-2002]
.
_
AAB66151Protein of the invention 1..201 201/201 (100%)e-111
#63 -
Unidentified, 201 aa. 1..201 201/201 (100%)
[W0200078961-A1, 28-DEG-2000]
In a BLAST search of public sequence databases, the NOV9a protein was found to
have
homology to the proteins shown in the BLASTP data in Table 9E.
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Table
9E.
Public
BLASTP
Results
for
NOV9a
Protein NOV9a Identities/
AccessionProtein/Organism/Length Residues!Similarities Expect
for the
Number Matched PortionValue
Residues
Q9NTU7 Cerebellin-like glycoprotein1..201 201/201 (100%)e-111
1
precursor - Homo sapiens1..201 201/201 (100%)
(Human),
201 aa.
Q8BME9 3 CEREBELLIN-like glycoprotein1..201 193/201 (96%) e-105
precursor - Mus musculus1..198 195/201 (96%)
(Mouse),
198 aa.
Q8BMF0 CEREBELLIN-like gIycoprotein1..201 192/201 (95%) e-104
precursor - Mus musculus1..198 1941201 (95%)
(Mouse),
198 aa.
Q8BGU2 Cerebellin 2 precursor 7..201 145/196 (73%) 2e-76
protein - Mus
musculus (Mouse), 224 31..224170/196 (85%)
aa.
P98087 Cerebellin-like glycoprotein7..201 144/196 (73%) 6e-76
1-
Rattus norvegicus (Rat),31..224169/196 (85%)
224 aa.
PFam analysis predicts that the NOV9a protein contains the domains shown in
the Table
9F.
Table 9F. Domain Analysis of NOV9a
Identities/
Pfam Domain NOV9a Match Region ~ Similarities Expect Value
for the Matched Region
Clq 72..198 48/137 (35%) 1.4e-48
' 113/137 (82%)
Example 10.
The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 10A.
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Oa, CG55274-OS SEQ ID NO: 124 ~86 as ~MW at 9590.OkD
i Sequence ,
~EFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDLKGKAKCAAWTLQKRLSKEDAT
ISKAKEPIEK
V l Ob, CG55274-O1 SEQ m N0:125 _ 280 by _
A Sequence ORF Start: ATG at 7 --~ Stop: TAG at 265
:ACCATGGCACTGCAGGCTGAATTCGACAAGGCTGCAGAAGACGTGAGGAAGCTGCCAACAAGACCAGCAG
~ATAAAGAACTGAAAAAACTCGATGGACTTTACAAACAAGCTATAATTGGAGACATTAATATTGAGTATCT
3AATGCTGGACTTTAAGGGCAAGGCCAAATGCGCAGCATGGACCCTCCAAAAAAGGTTGTCAAAGGAAGAT
SAC'GAGTGTCTCTATTTCTAAGGCAAAAGAGCCGATAGAAAAATAGGACATTTAGAATA
Ob, CG55274-Ol ~SEQ ID NO: I26 ~86 as MW at 9624.1kD
iSequence __
EFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDFKGKAKCAAWTLQKRLSKEDAT
SVSISKAKEPIEK
.Oc, CG55274-02 SEQ ID NO: 127 289 by
__ ._.. ._.___.__:__.__ .__._ __...__.._..__~~-._.._........ _ ._. ...___ ....
Sequence ~ORF Start. ATG at 17 ORF Sto : TAG at 272
3CCGCCACCACCATGGCACTGCAGGCTGATCGAGACAAGGCTGCAGAAGACGTGAGGAAGCTGCCAACA
AGATGAGAAAGAACTGAAAAAACTCGATGGACTTTACAAACAAGCTATAATTGGAGACATTAATATTG
CCTGGGAATGCTGGATTTAAAGGGCAAGGCCAAATGCGCAGCATGGACCCTCCAAAAAAGGTTGTCAAA
iATGCAACGAGTGTCTCTATTTCTAAGGCAAAAGAGCCGATAGAAAAATAGGACATTTAGAATACA
Oc, CG55274-02 ~SEQ ID NO: 128 a ~85 as -__~MW at 9528.9kD
i_.Sequence.....__. ..________. __._.._.... _..._.___ _ .. __. ..._____. .
_.._._ .. _....._.._.._....._.__ _ ... ._.__.___..._..._........_
nRnKAAFnVRKLPTRPDEKELKKLDGLYKOAIIGDINIEYLGMLDLKGKAKCAAWTLQKRLSKEDATS
ISKAKEPIEK
IS
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NOV 10e, CG55274-04 ~ ~ SEQ ID N0: 132 a 18 as MW at 2053.4kD
Protein Sequence
QAIIGDINIEYLGMLDFK
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table l OB.
Table lOB. Comparison of the NOV10 protein sequences.
NOVlOa MALQAEFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDLKGKAKCAA
NOVlOb MALQAEFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDFKGKAKCAA
NOVlOc MALQADRDKAAEDVRKLPTRPDE-KELKKLDGLYKQAIIGDINIEYLGMLDLKGKAKCAA
NOVlOd -----------------------------------QAIIGDINIEYLGMLDLKGK-----
NOVlOe ___________________________________QAIIGDINIEYLGMLDFK-______
NOVlOa WTLQKRLSKEDATSVSISKAKEPIEK
NOVlOb WTLQKRLSKEDATSVSISKAKEPIEK
NOVlOc WTLQKRLSKEDATSVSISKAKEPIEK
NOVlOd __________________________
NOVlOe __________________________
NOVlOa (SEQ ID NO: 124)
NOVlOb (SEQ ID NO: 126)
NOVlOc (SEQ ID NO: 128)
NOVlOd (SEQ ID NO: 130)
NOVlOe (SEQ ID NO: 132)
Further analysis of the NOVlOa protein yielded the following properties shown
in Table
lOC.
Table lOC. Protein Sequence Properties NOVlOa
SignalP analysis: No Known Signal Sequence Predicted
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 9; pos.chg 1; neg.chg 2
H-region: length 2; peak value 0.00
PSG score: -4.40
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -10.68
possible cleavage site: between 58 and 59
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»> Seems to have no N-terminal signal peptide
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 1
Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) _. fixed
PERIPHERAL Likelihood = 7.11 (at 36)
ALOM score: 7.11 (number of TMSs: 0)
MITDISC: discrimination of mitochondrial targeting seq
R content: 0 Hyd Moment(75): 3.71
Hyd Moment(95): 2.95 G content: 0
D/E content: 2 S/T content: 0
Score: -7.75
Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 19.8
NLS Score: -0.47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
yAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
~~checking 33 PROSITE prokaryotic DNA binding motifs: none
I!NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: nuclear
Reliability: 76.7
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
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(Final Results (k = 9/23):
82.6 %. nuclear
4.3 %: cytoskeletal
4.3 %: mitochondria!
4.3 %: cytoplasmic
4.3 %. peroacisomal
» prediction for CG55274-05 is nuc (k=23)
A search of the NOV l0a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several
homologous proteins shown in Table lOD.
Table ~
10D.
Geneseq
Results
for NOVlOa
NOVlOa Identities/
Geneseq Protein/Organism/Length Residues/SimilaritiesExpect
[Patent #, for
IdentifierDate] Match the MatchedValue
ResiduesRegion
AAB81814 Human endozepine-like 1..86 85/86 (98%)le-42
ENDOS SEQ
ID NO: 10 - Homo Sapiens,1..86 85/86 (98%)
86 aa.
[W0200125436-A2, 12-APR-2001]
ABU11538 Human MDDT polypeptide 1..86 64/86 (74%)le-26
SEQ ID 485
- Homo sapiens,100 aa. 13..97 70/86 (80%)
[W0200279449-A2, 10-OCT-2002)
AAB81811 Human endozepine-like 3..86 61/84 (72%)Se-25
END04 SEQ
ID NO: 8 - Homo Sapiens, 11..93 68/84 (80%)
96 aa.
[W0200125436-A2, 12-APR-2001]
ABJ05397 Frog acyl coenzyme A binding4..86 57/83 (68%)2e-23
protein
(ACBP) - Rana sp, 86 aa. 2..83 66183 (78%)
[W0200261096-Al, 08-AUG-2002]
ABJ05396 Duck acyl coenzyme A binding4..86 55/83 (66%)3e-23
protein
(ACBP) 2 - Anas sp, 86 2..83 66/83 (79%)
aa.
[W0200261096-Al, 08 AUG-2002)
In a BLAST search of public sequence databases, the NOVlOa 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 Identities/
Protein Residues/SimilaritiesExpect
for
AccessionProtein/Organism/Length Match the MatchedValue
Number ResiduesPortion
Q8N6N7 Similar to RII~EN cDNA 1..86 64/86 (74%)4e-26
9230116818
gene - Homo Sapiens (Human),1..85 70/86 (80%)
88 aa.
Q9D258 9230116B18Rik protein - 1..86 60186 (69%)3e-25
Mus musculus
(Mouse), 88 aa. 1..85 70/86 (80%)
A57711 diazepam-binding inhibitor1..86 58/86 (67%)2e-23
- laughing
frog, 88 aa. 1..85 68/86 (78%)
P45883 Acyl-CoA-binding protein 4..86 57/83 (68%)4e-23
homolog
(ACBP) (Diazepam binding 3..84 66/83 (78%)
inhibitor
homology (DBI) - Rana ridibunda
(Laughing frog) (Marsh
frog), 87 aa.
P45882 Acyl-CoA-binding protein 4..86 55/83 (66%)7e-23
(ACBP)
(Diazepam binding inhibitor)19..10066/83 (79%)
(DBI)
(Endozepine) (EP) - Anas
platyrhynchos
(Domestic duck), 103 aa.
PFam analysis predicts that the NOV l0a protein contains the domains shown in
the Table
l OF.
Table l OF. Domain Analysis of NOVlOa
Identities/
Pfam Domain NOVlOa Match Region Similarities Expect Value
for the Matched Region
ACBP 3..86 42/90 (47%) S.le-18
66/90 (73%)
Example 11.
The NOV 11 clone was analyzed, and the nucleotide and encoded polypeptide
sequences
are shown in Table 11 A.
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11A. NOVll
~la, GG55379-04 SEQ m,N0~133 6291 by _
Sequence ORF Start:~ATG at 1 ORF Stop: TAG at 3763
AAGGATGGGGACACCCTGCTGGAGCACGACCACTTACACCTGCTGCCCAATGGTTCCCTGTGGCTGTCCCAGC
CACTAGCACCCAATGGCAGTGACGAGTCAGTCCCTGAGGCTGTGGGGGTCATTGAAGGCAACTATTCGTGCCT
CTGCACCCGGAGTCTCAGACGGTGGAGGAGAACGGGACAGCTCGCTTTGAGTGCCACATTGAAGGGCTGCCAG
CTCCCATCATTACTTGGGAGAAGGACCAGGTGACATTGCCTGAGGAGCCTCGGCTCATCGTGCTTCCCAACGG
CGTCCTTCAGATCCTGGATGTTCAGGAGAGTGATGCAGGCCCCTACCGCTGCGTGGCCACCAACTCAGCTCGC
CCCCTTTTGTGTCCTGGGTCCGAGACGGGAAGCCCATCTCCACAGATGTCATCGTCCTGGGC
ACTAATTGCCAACGCGCAGCCCTGGCACTCCGGCGTCTATGTCTGCCGCGCCAACAAGCCCC
TTCGCCACTGCAGCCGCTGAGCTCCGTGTGCTGCTAGCGGCTCCCGCCATCACTCAGGCGCC
CGCTGGCCGTGGTGGTGCGCGAGGGGCTGCCCAGCGCCCCCACGCGGGTCACTGCTACG
CTCTCCACTACCAGAAGGCACGGGGTATGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGA
ACTACAGGTTCGGGACCTGGAACCCAACACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCC
AGCCGCACCTCCACCCCAGCACTGGTGCACACACTGGATGATGTCCCCAGTGCAGCACCCCAGCTCTCCCTGT
CCAGCCCCAACCCTTCGGACATCAGGGTGGCGTGGCTGCCCCTGCCCCCCAGCCTGAGCAATGGGCAGGTGGT
GAAGTACAAGATAGAATACGGTTTGGGAAAGGAAGGTGAGTGGGGGGATCAGATTTTCTCTACTGAGGTGCGA
CAGCAGCCGGCTTCGGGGCCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGT
CCCTTTTGCCCCTGCAGAGTTGAAGGTGCAGGCAAAGATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCT
CACCCCACCCAGATCTCTGGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATC
GCCTGCCAGGGGGCCGTGGAGACCAGGCTTGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTA
TGTGGAAGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCC
CCACGTCCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTT
AAGATTGTCAACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACC
GTTCTGGAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGC
CGTGGACATGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTC
TCCGACCTGCGACTGAGCCCCCTGACACCGTCCACGGTTCGGCTGCACTGGTGCCCCCCCACA
ATGGGTGGCGGTGTTTCTGAAGGCCGGAGTCACTCCAAAAGAAAGATCTCCTGGGCTCAACCAAG
TCAAATCCCCCTGCCCTCTAGGAGCCAGCCCAGGCCTGCCCAGATCCCCGGTCTCCTCCTCTGCCTAGCTCTT
CCCAGAGGATGTGGTTTGGGGCAGGCAGGTATGGATCACATAGGATGCGATACCTGTGGCCGTGTATGTCCAC
ATGTGTGCCTGTAGATACATCATCAAGCCCTTTGGAGCTTCCTAAGTTGCTTTGGCTGAGGGGAGAGGAAAAC
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TTCACTCCCCCCATACTCTTTGTGATACACATGTGACATGTGAAAGACATACGAGACATAGCTAC
CTGACAAAGACAGTCC
#CCCAGCTCCTTATTGCCCAAATAGAGAGGGTGGCCCTGGCTCCCCTCCGAGCAACTCTGCATTTAATTTTGTA
~TCCTCTCTCCTGTTTTCCTTCCTCTGCTATCTCTCACACCTCTCCCAGACTATGTCATCTTGTTCTCCTGCCT
#GGGTTCAAACTCTGCATCCTTCTCTAACAACGTGACTACCTCATGTCTGCTTCAAGGCCCCCGTGCCCTTCCT
#GTATCCGCGGCTGCCGCGCACTCGCCTGCCATCCTCCTGCCTCCTCTTCACTCAGTGCTTCTGCTTGCCCTGC
#TTGGGATCCAAGGGTTCTCCATGGATGGATCCAAGTCATAGAGGGGAATGTTTGAGACAGGGAAGGGGGCTGT
#GAGGCTTGACTTGATCTGGATTGGGGATGACAGGAATCTCACCCTCTGGGGTGCTGGCAAGGAGGTCTTTGCA
#CAGGAAAAGGGGTAGCTCATTTCAGTTTGTTTTTTCTTTAAATTGAATCCTCAAGTCATTTTCTGTTCACCTG
ECTCTGCCTTCTGGGCCAAATTCGAAATAACCAGTCCATTTTTCCTTTTTTTTTTTTTTTTTTTTTTTTAAATG
#GTGGAATGTCTCTCAGCACAGTTGCGGCTTCCTCAAACCCTGAAAGCATCTGTGTTTATTATACTCGGGTGTC
#ACTCACTGTTGATGTCTGCACCTACGTTTCCACCTCCTCCCCCTCCTTCAGCCAGCCTATGATAACACTAAAG
ATTATTAATGTTGGTTTTGTATCTCGTTAAAGACAGAATTGTCACTTGTAGTATTTCTGTAGCATTCAGCGCT
GCTGTGGCTAACACCACTGTGTATGTTTCATCATTGCTCTGAAGGTCAAAAGCCTCATTTTATTTTGCTGGTT
ATTATACCACATAGTCATTTTTCATGTTCTTGTTTAACAGGCACTGAGGTTCTGGTTTAAATTAAATAGCTGC
AAATGAGACAATTTATAACCCATTAGGTTGGGTGGAAAATTGTTTCTCAAAAGCAAATAAGTAATAAATCTGG
TATCTGCCTATAACTCACAGTTGATAAGAAAGTGGCCATTTCTCACTAGCACTATATATGATTTGGGCTCTGG
GTAATTTGGAAGTGTTAGGTTTGTGTCTTTGTAGCAGTATTTTTATTAGAAAAGAATCTATTGGCCTTTTACA
GGGTATTAATCCCTTTGTCACCTACCATTGATGCCTTAAGTTTTCTGAGTCTCAATTAAAAATCTTCCTTTTC
TTGATGCATGACAAGTGTAATCAGTACTTGCTCATTTATTTGTCTGTATTTAGTTTATGCTGTACTATTTAAT
#TATCCTTCCAGCGTTTTTTTTTTCTCCTTACAAATATGATACTCTTTAGTGTTAAGCTAAGGCATTGATTCAT
#GTATCTGTCCTTATAATGAATTAATAAACTATTTTCCAG #
la, CG55379-04 ~SEQ ID NO: 134 1254 as MW at 134608.7kD
VLNCSLGAAAAGPPTRVTWS
DTLLEHDHLHLLPNGSLWLSQPLAPNGSDESVPEAVGVIEGNYSCLAHGPLGVLASQTAWKLASLADFS
ESQTVEENGTARFECHIEGLPAPIITWEKDQVTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSAR
SQEALLSVAHRGSLASTRGQDVVIVAAPENTTVVSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLG
TRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASGEPRPA
VITQIGLQDAGYYQCVAENSAGMACAAASLAVVVREGLPSAPTRVTAT
~SSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQVRDLEPNTDYEFYWAYSQLGA
'STPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQVVKYKIEYGLGKEGEWGDQIFSTEVR
TQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLVVSWQPPP
'QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVKLVAFNKHEDG
VNTKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVNYTVRFSPWGLRNASLVT
'SSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPT
fGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQE
~DSLDMHSVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSPPAAHEL
~VHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRKISWAQPSGLSWAGSWAGCELPQAGPRPALTR
~PPAGTGQTLLLQALVYDAIKGNGRKKSPPACRNQVEAEVIVHSDFSASNGNPDLHLQDLEPEDPLPPEAP
SGVGDPGQGAAWLDRELGGCELAAPGPDRLTCLPEAASASCSYPDLQPGEVLEETPGDSCQLKSPCPLGA
aLPRSPVSSSA
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.lb, CG55379-Ol SEQ m NO:1_35 ~~3741 by
Sequence ORF Start: ATG at 1 ORF Stop: end of
AGCACCCAATGGCAGTGACGAGTCAGTCCCTGAGGCTGTGGGGGTCATTGAAGGCAACTATTCGTGCCT
rarrcmrrrrrmrnnrm~rmarceAC~ccAGACTGCTGTCGTCAAGCTTGCCAGTCTCGCAGACTTCTCT
TTACTTGGGAGAAGGACCAGGTGACATTGCCTGAGGAGCCTCGGCTCATCGTGCTTCCCAACGG
GATCCTGGATGTTCAGGAGAGTGATGCAGGCCCCTACCGCTGCGTGGCCACCAACTCAGCTCGC
CCCCTTTTGTGTCCTGGGTCCGAGACGGGAAGCCCATCTCCACAGATGTCATCGTCCTGGGC
ACTAATTGCCAACGCGCAGCCCTGGCACTCCGGCGTCTATGTCTGCCGCGCCAACAAGCCCC
ACTACCAGTGCGTGGCTGAGAACAGCGCGGGAATGGC
ACG
ATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCC
CAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACA
ATGCTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCT
CCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCC
TCTCTGGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGG
CCGTGGAGACCAGGCTTGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCA
CTAGTCCCTGGCCGGCTGTACGAGGTGAAGCTCGTGGCTTTCAACAAACATGAGGATGGCTATGCAGCAGTG
GGAAGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCC
CGTCCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAA
ATTGTCAACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACAGTTCT
GAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGG
CCTGCGACTGAGCCCCCTGACACCGTCCACGGTTCGGCTGCACTGGTGCCCCCCCACAGAGCCCAACGGGGAG
TGTGATCACGCTCCAGGAGAAGCTGTCA
TGAATTGGAGTCCCTT
AAGTGCTCAACCAAGCGGGCTGAGCTG
CATCTAACGGGAACCCTGACCTCCATCTCCAAGACCTGGAGCCTGAGGACCCCCTGCCTCCAG
TCTCATCTCGGGTGTTGGGGATCCAGGGCAGGGGGCAGCCTGGCTGGACAGGGAGTTGGGAGG
CCGGACCTCCAGCCAGGCGAGGTGCTAGAGGAGACCCCTGGAGATAGCTGCCAGCTCAAATCCCCCTGCCCTC
TAGGAGCCAGCCCAGGCCTGCCCAGATCCCCGGTCTCCTCCTCT
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:I lb, CG55379-01 ~SEQ ID N0:136 1247 as ~MW at 133821.8kD
~iARGDAGRGRGLILALTFCLLAARGELLLPQETTVELSCGVGPLQVILGPEQAAVLNCSLGAAAAGPPTRVTWS
KDGDTLLEHDHLHLLANGSLWLSQPLAPNGSDESVPEAVGVIEGNYSCLAHGPPRVLASQTAWKLASLADFS
LHPESQTVEENGTARFECHIEGLPAPIITWEKDQVTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSAR
QHFSQEALLSVAHRGSLASTRGQDWIVAAPENTTWSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLG
RTNLLIANAQPWHSGVYVCRANKPRTRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASGEPRPA
LRWLHNGAPLRPNGRVKVQGGGGSLVITQIGLQDAGWQCVAENSAGMACAAASLAVVVREGLPSAPTRVTAT
PLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQVRDLEPNTDYEFYWAYSQLGA
SRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQVVKYKIEYGLGKEDQIFSTEVRGNET
QLMLNSLQPNKWRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPTQ
ISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVKLVAFNKHEDGYAAV
WKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVNYTVRFSPWGLRNASLVTYYSS
GEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSWERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGE
IVEYLILYSSNHTQPEHQWTLLTTQGEGNIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLS
DSLDMHSVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSP.PAAHELESL
VHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRKVSAQPSGLSWAGSWAGCELPQAGPRPALTRALLP
PAGTGQTLLLQVLCSDQGNGRKKSPPACRNQVEAEVIVHSDFSASNGNPDLHLQDLEPEDPLPPEAPDLISGV
GDPGOGAAWLDRELGGCELAAPGPDRLTCLPEAASASCSYPDLqPGEVLEETPGDSCQLKSPCPLGASPGLPR
SPVSSS
',1 c, 258065951 SEQ ID NO: 137 ~ 1609 by
Sequence ~ ORF Start: at l ORF Stop: at 1609
ACGCCAC
ATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
ACAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACACAGC
TATGCTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
AGGCAAAGATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCTCACCCCACCCAGATCT
ATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
TGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCAGCTA
TCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
GAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGGA
CATGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTCCACACCCCCATCCGAC
CTGCGACTGAGCCCCCTGACACCGTCCACGGTTCGGCTGCACTGGTGCCCCCCCACAGAGCCCAACGGGGAGA
TCGTGGAGTATCTGATCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
ACTTCTTCAAGATGGGGGCGCGC
s
198
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
:1 lc, 2$806$9$1 ~SEQ ID NO:138 $36 as MW at $9$32.7kD
LPSAPTRVTATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
FYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
IFSTEVRGNETQLMLNSLQPNKWRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
VSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQL
FNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
RNASLWYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSD
LHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
Vlld, 209886264 SEQ ID NO: 139_ 1611 by
A Se uence ___.~__..__.___.___.._.~.__-__._...___-_
q ORF Start: at-1~ ~ ~ ORF Ston: end of
TGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGAACTA
CACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
CCCCAACCCTTCGGACATCAGGGTGGCGTGGCTGCCCCTGCCCCCCAGCCTGAGCAATGGGCAGGTGGTGAAG
TACAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACACAGC
TTATGTTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
GGCAAAGATGGAGTCCCTGGTCGTATCATGGCAGCCACCCCCTCACCCCACCCAGATCT
TATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
GGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCAGCTA
AGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
CCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
GTCAACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACACCAGTTCTG
GAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGGA
CATGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTCCACACCCCCATCCGAC
TCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
TGCTGAGGTCCATGGCCTGGAGAGCGACACTCGGTACTTCTTCAAGATGGGGGCGCGC
ld, 209886264 ~SEQ ID NO: 140 $37 as ~MW at $9607.71eD
LAVWREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
LEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
YGLGKEDQIFSTEVRGNETQLMLNSLQPNKWRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
QAKMESLWSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQL
LYEVKLVAFNKHEDGYAAWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
VRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSWERSTLPDRPSTPPSD
PLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
PGPFSRLQDVITLQEKLSDSVD
199
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
L 1 e, 209886345 SEQ ID NO: 141 ;1672 by _
Sequence ~ ORF Start: at 1 ORF Stop: at 1672
AGCTCCGCTGTGTTGGTGGCCTGGGAGCGGCCCGAGATGCCCAGCGAGCAGATCATCGGCTTCTCTCT
ACCAGAAGGCACGGGGCATGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGAACTA
TCGGGACCTGGAACCCAACACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
mccArrcCAGC~CTGGTGCACACACTGGATGATGTCCCCAGTGCAGCACCCCAGCTCTCCCTGTCCAG
TACAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACACAGC
TTATGTTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
GGCCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
GAGTTGAAGGTGCAGGCAAGGATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCTCACCCCACCCAGATCT
CTGGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
TGGAGACCAGGCTTGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCAGCTA
GTCCCTGGCCGGCTGTACGAGGTGAAGCTCGTGGCTTTCAACAAACATGAGGATGGCTATGCAGCAGTGTGGA
AGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
CCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
TTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGGA
ACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
r~me~cAT(~GCCTGGAGAGCGACACTCGGTACTTCTTCAAGATGGGGGCGCGC
le, 209886345 ~SEQ ID NO: 142 557 as ~MW at 61878.3kD
REGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
DYEFYVVAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
EDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
SLVVSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQL
LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
WGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSD
TVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
RT.WDVTTT.GFKLSDSVDSFSWSVITAPRAPPRPATRY
l lf, 209886357 SEQ ID NO. 143 1611 by
Sequence ORF Start: at 1 ORF Stop: end of
ACCGCGTCGCTGGCCGTGGTGGTGCGCGAGGGGCTGCCCAGCGCCCCCACGCGGGTCACTGCTACGCCAC
~rnnrmrrrrmam~mmar~mc~CCCTGGGAGCGGCCCGAGATGCCCAGCGAGCAGATCATCGGCTTCTCTCT
TCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
ATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
GCTTTCAACAAACATGAGGATGGCTATGCAGCAGTGTGGA
TGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
200
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
TGTGATCACGCTCCAGGAGAAGCTGTCAGACTCGG
lf, 209886357 SEQ ID N0:144 537 as MW at 59607.7kD
VWREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
PNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
LGKEDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
YEVKLVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
RFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSD
LTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
~lg, CG55379-02 SEQ.ID.NO: 145_............._........_............_1611 bp.
._....._...... ...
Sequence ORF Start: at 7 ORF Stop: at 1606
ACGCCAC
CCACTACCAGAAGGCACGGGGCATGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGAACTA
CAGGTTCGGGACCTGGAACCCAACACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
CCCCAACCCTTCGGACATCAGGGTGGCGTGGCTGCCCCTGCCCCCCAGCCTGAGCAATGGGCAGGTGGTGAAG
ATGTTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
CCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
GTTGAAGGTGCAGGCAAAGATGGAGTCCCTGGTCGTATCATGGCAGCCACCCCCTCACCCCACCCAGATCT
GGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
TGGCTATGCAGCAGTGTGGA
CGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
CACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
AGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACACCAGTTCTG
ATCTGATCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
CTTCAGTGCTGAGGTCCATGGCCTGGAGAGCGACACTCGGTACTTCTTCAAGATGGGGGCGCGC
201
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
lg, CG55379-02 SEQ m NO: 146 533 as MW at 59235.4kD
VWREGLPSAPTRWATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQV
PNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWKYK
LGKEDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAEL
.FQ~fESLWSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVP
EVKLVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVN
.FSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSDLR
~TPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGARTE
.lh, CG55379-03 SEQ ID NO: 147 .167_2 by ___
Sequence ORF Start: at 7 ORF Stop:-'at 1606
ATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
CAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
TCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
ATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCTCACCCCACCCAGATCT
CCTGGCCGGCTGTACGAGGTGAAGCTCGTGGCTTTCAACAAACATGAGGATGGCTATGCAGCAGTGTGGA
GCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
TGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
AACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACACCAGTTCTG
TGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTCCACACCCCCATCCGAC
TCTGATCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
TGTGATCACGCTCCAGGAGAAGCTGTCAGACTCGG
ACC
lh, CG55379-03 SEQ m N0:148 533 as MW at 59263.4kD
SLAVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQV
DLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWKYK
EYGLGKEDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAEL
VQARMESLWSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVP
RLYEVKLVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVN
TVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSWERSTLPDRPSTPPSDLR
SPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGARTE
202
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 11B.
Table 11B. Comparison of the NOV11 protein sequences.
NOV2la MARGDAGRGRGLLALTFCLLAARGELLLPQETTVELSCGVGPLQVILGPEQAAVLNCSLG
NOVllb MARGDAGRGRGLLALTFCLLAARGELLLPQETTVELSCGVGPLQVILGPEQAAVLNCSLG
NOV11C ____________________________________________________________
NoVlld ____________________________________________________________
NOVlle ____________________________________________________________
NOVllf ____________________________________________________________
NOVllg ____________________________________________________________
NOVllh ____________________________________________________________
NOVlla AAAAGPPTRVTWSKDGDTLLEHDHLHLLPNGSLWLSQPLAPNGSDESVPEAVGVIEGNYS
NOVllb AAAAGPPTRVTWSKDGDTLLEHDHLHLLANGSLWLSQPLAPNGSDESVPEAVGVIEGNYS
NOVllc ____________________________________________________________
NOVlld ____________________________________________________________
NOVlle ____________________________________________________________
NOVl2f ____________________________________________________________
NOVllg ____________________________________________________________
NOVllh -___________________________________________________________
NOVlla CLAHGPLGVLASQTAWKLASLADFSLHPESQTVEENGTARFECHIEGLPAPIITWEKDQ
NOVllb CLAHGPPRVLASQTAWKLASLADFSLHPESQTVEENGTARFECHIEGLPAPIITWEKDQ
NOVllc _____________________________________________-______________
NOVlld ____________________________________________________________
NOVlle ____________________________________________________________
NOVllf ____________________________________________________________
NOVllg ____________________________________________________________
NOVllh ____________________________________________________________
NOVlla VTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSARQHFSQEALLSVAHRGSLASTR
NOVllb VTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSARQHFSQEALLSVAHRGSLASTR
NOVllc ____________________________________________________________
NOVlld ____________________________________________________________
NOVlle ____________________________________________________________
NOVllf ____________________________________________________________
g ____________________________________________________________
NOV11
NOVllh ____________________________________________________________
NOVlla GQDWIVAAPENTTWSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLGRTNLLIAN
NOVllb GQDWIVAAPENTTWSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLGRTNLLIAN
NOVllc ____________________________________________________________
NOVlld ____________________________________________________________
NOVlle ____________________________________________________________
NOVllf ____________________________________________________________
NOVllg ____________________________________________________________
NOVlih ________________________________________~___________________
NOVlla AQPWHSGVYVCRANKPRTRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASG
NOVllb AQPWHSGWVCRANKPRTRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASG
NOVllc ____________________________________________________________
NOVlld ____________________________________________________________
NOVlle ____________________________________________________________
203
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
NOVl2f ____________________________________________________________
NOVllg ____________________________________________________________
NOVllh ____________________________________________________________
'NOVlla EPRPALRWLHNGAPLRPNGRVKVQGGGGSLVITQIGLQDAGYYQCVAENSAGMACAAASL
',NOVIlb EPRPALRWLHNGAPLRPNGRVKVQGGGGSLVITQIGLQDAGYYQCVAENSAGMACAAASL
'NOVllc __________._____________________________________________GTASL
',NOVlld _______________________________________________________GTASL
NOVlIe _______________________________________________________GTASL
INOVllf _______________________________________________________GTASL
NOVllg _________________________________________________________p,SL
NOVllh _________________________________________________________p~SL
NOVlla AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFA
NOVllb AWVREGLPSAPTRWATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFA
NOVllc AVWREGLPSAPTRVTATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFA
NOVlld AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQTIGFSLHYQKARGMDNVEYQFA
NOVlle AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVlIf AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVllg AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVllh AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVlla VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllb VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOV11C VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVlld VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVlle VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllf VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllg VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllh VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVlla SDIRVAWLPLPPSLSNGQWKYKIEYGLGKEGEWGDQIFSTEVRGNETQLMLNSLQPNKV
NOVllb SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllc SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVlld SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVlle SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllf SDIRVAWLPLPPSLSNGQVVKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllg SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllh SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVlla YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVllb YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVllc YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVlld YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVlle YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQARMESLWSWQPPPHPT
NOVllf YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHWFAPAELKVQAKMESLWSWQPPPHPT
NOVllg YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVllh YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQARMESLWSWQPPPHPT
NOVlla QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllb QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllc QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLWGRLYEVK
NOVlld QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLWGRLYEVK
NOVlle QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllf QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllg QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllh QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVlla LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
204
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
NOVIlb LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllc LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVlld LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVlle LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllf LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllg LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllh LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVlla TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllb TVKIVNYTVRFSPWGLRNASLVTYYSS-GEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllc TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVlld TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVlle TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllf TVKIVNYTVRFSPWGLRNASLWYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllg TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGWMDGPFGS
NOVllh TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVlla VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllb VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllc VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVlld WERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVlle VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllf WERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllg WERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
'NOVllh VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVlla TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSLDMH
'NOVllb TLLTTQGEGNIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSLDMH
',NOVllc TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSV---
',NOVlld TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSVD--
',NOVlle TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSVDSF
',NOVllf TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSVD--
',NOVllg TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDS----
',NOVllh TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDS----
INOVIIa SVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSPPA
INOVIIb SVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSPPA
~NOVllc ____________________________________________________________
_____________________
~iNOVlld _______________________________________
~~NOVlle SWSVITAPRAPPRPATRY-_________________________________________
I,NOVIIg ____________________________________________________________
INOVIIh '___________________________________________________________
i
NOVlla AHELESLVHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRKISWAQPSGLSWAGS
NOVllb AHELESLVHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRK-VSAQPSGLSWAGS
NOVllc ____________________________________________________________
NOVlld ______-_____________________________________________________
NOVlle ____________________________________________________________
NOVllf ____________________________________________________________
NOVllg ______________________________-_____________________________
NOVllh ____________________________________________________________
NOVlla WAGCELPQAGPRPALTRALLPPAGTGQTLLLQALWDAIKGNGRKKSPPACRNQVEAEVI
NOVllb WAGCELPQAGPRPALTRALLPPAGTGQTLLLQVLCSD--QGNGRKKSPPACRNQVEAEVI
NOVllc ____________________________________________________________
NOVIId ____________________________________________________________
NOVlle ____________________________________________________________
NOVllf ____________________________________________________________
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
NOVllg ____________________________________________________________
NOVllh ____________________________________________________________
NOVlla VHSDFSASNGNPDLHLQDLEPEDPLPPEAPDLISGVGDPGQGAAWLDRELGGCELAAPGP
NOVllb VHSDFSASNGNPDLHLQDLEPEDPLPPEAPDLISGVGDPGQGAAWLDRELGGCELAAPGP
NOVllc ____________________________________________________________
NOVlld ____________________________________________________________
NOVlle ____________________________________________________________
NOVllf ____________________________________________________________
NOVllg ____________________________________________________________
'NOVllh ____________________________________________________________
INOVIIa DRLTCLPEAASASCSYPDLQPGEVLEETPGDSCQLKSPCPLGASPGLPRSPVSSSA
NOVllb DRLTCLPEAASASCSYPDLQPGEVLEETPGDSCQLKSPCPLGASPGLPRSPVSSS-
NOVllc ________________________________________________________
NOVlld ________________________________________________________
NOVlle ________________________________________________________
NOVllf ________________________________________________________
NOVllg ________________________________________________________
NOVllh ________________________________________________________
NOVlla (SEQ ID NO: 134)
NOVllb (SEQ ID NO: 136)
NOVllc (SEQ ID NO: 138)
NOVlid (SEQ ID NO: 140)
NOVlle (SEQ ID NO: 142)
NOVllf (SEQ ID NO: l44)
NOVllg (SEQ ID NO: 146)
NOVllh (SEQ ID NO: 148)
Further analysis of the NOV l 1 a protein yielded the following properties
shown in Table
11C.
Table 11C. Protein Sequence Properties NOVlla
SignalP analysis: Cleavage site between residues 25 and 26
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 10; pos.chg 3; neg.chg 1
H-region: length 12; peak value 10.30
PSG score: 5.90
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 1.11
possible cleavage site. between 24 and 25
» > Seems to have a cleavable signal peptide (1 to 24)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 25
Tentative number of TMS(s) for the threshold 0.5: 1
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood =-11.89 Transmembrane 963 - 979
PERIPHERAL Likelihood = 0.90 (at 321)
ALOM score. -11.89 (number of TMSs: 1)
206
CA 02486490 2004-12-03
WO 2004/000997 PCT/US2003/017512
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 22
Charge difference. -5.0 C(-2.0) - N( 3.0)
N >= C. N-terminal side will be inside
» > membrane topology: type is (cytoplasmic tail 980 to 1254)
MITDISC: discrimination of mitochondrial targeting seq
R content. 4 Hyd Moment(75): 11.83
Hyd Moment(95): 5.63 G content: 5
D/E content: 2 S/T content: 1
Score: -4.74
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 33 ARG~EL
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: PVRLKKK (4) at 696
bipartite: none
content of basic residues: 8.2g
NLS Score: -0.13
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: ARGD
none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif.
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: too long tail
Dileucine motif in the tail: found
LL at 1097
LL at 1107
'checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 55.5
COIL. Lupas's algorithm to detect coiled-coil regions
total: 0 residues
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
CONTENANT LES PAGES 1 A 207
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