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CA 02495563 2005-02-O1
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THERAPEUTIC POLYPEPTIDES, NUCLEIC ACIDS ENCODING
SAME, AND METHODS OF USE
TECfINICAL FIELD
The present invention relates to novel polypeptides, and the nucleic acids
encoding
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 txeating diverse pathological conditions.
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BACKGROUND OF THE INVENTION
Eukaryotic cells are characterized by biochemical and physiological processes
which under normal conditions are exquisitely balanced to achieve the
preservation and
propagation of the cells. When such cells are components of multicellular
organisms such
as vertebrates, or more particularly organisms such as mammals, the regulation
of the
biochemical and physiological processes involves intricate signaling pathways.
Frequently,
such signaling pathways involve 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 level of synthesis and secretion of protein
effectors. In other
classes of pathologies the dysregulation is manifested as increased or up-
regulated level of
synthesis and secretion of protein effectors. In a clinical setting a subject
may be suspected
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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
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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SUMMARY 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 >D N0:2n, wherein n is an integer between l
and 38. The
novel nucleic acids and polypeptides are referred to herein as NOVla, NOVIb,
NOVlb,
NOVlc, NOV2a, NOV2b, NOV2c, NOV2d, NOV3a, NOV3b, etc. 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 1D N0:2n, wherein n is an
integer
between 1 and 38, 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. In another embodiment, the invention includes
the amino
acid sequences selected from the group consisting of SEQ >D NO:2n, wherein n
is an
integer between 1 and 38. 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 l and 38 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
ll~ N0:2n, wherein n is an integer between 1 and 38, or any other amino acid
sequence
selected from this group. The indention also comprises fragments from these
groups in
which up to 15% 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
N0:2n, wherein n is an integer between 1 and 38. 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-l, wherein n is an integer between l and 38. The variant polypeptide
where any
amino acid changed in the chosen sequence is changed to provide a conservative
substitution.
In another embodiment, the invention comprises a pharmaceutical composition
involving a polypeptide with an amino acid sequence selected from the group
consisting of
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SEQ ID NO:2n, wherein n is an integer between 1 and 38 and a pharmaceutically
acceptable carrier. Tn 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 38 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 1 and 38 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 38 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 38, 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.
In another embodiment, the invention involves a method for 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
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between 1 and 38, 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 l and 38, 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
)D NO:2n, wherein n is an integer between 1 and 38, 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
group consisting of SEQ TD N0:2n, wherein n is an integer between 1 and 38,
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.
CA 02495563 2005-02-O1
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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 ammo acid sequence selected from the group consisting of SEQ ID N0:2n,
wherein n is
an integer between 1 and 38 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 N0:2n, wherein n is an integer between 1 and 38; 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 l and 38 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 1 and 38; 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 38, 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 38 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 df 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
)D N0:2n, wherein n is an integer between l and 38, wherein the nucleic acid
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
m N0:2n, wherein n is an integer between 1 and 38 that encodes a variant
polypeptide,
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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
. lD N0:2n, wherein n is an integer between 1 and 38, wherein the nucleic acid
molecule
differs by a single nucleotide from a nucleic acid sequence selected from the
group
consisting of SEQ 1D NOS: 2n-1, wherein n is an integer between l and 38.
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
1D N0:2n, wherein n is an integer between 1 and 38, 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 >D N0:2n-1, wherein n is an
integer
between 1 and 38; a nucleotide sequence wherein one or more nucleotides in the
nucleotide
sequence selected from the group consisting of SEQ >D N0:2n-1, wherein n is an
integer
between 1 and 38 is changed from that selected from the group consisting of
the chosen
sequence to a different nucleotide provided that no more than 15°l0 of
the nucleotides are so
changed; a nucleic acid fragment of the sequence selected from the group
consisting of
SEQ >D N0:2n-1, wherein n is an integer between l and 38; and a nucleic acid
fragment
wherein one or more nucleotides in the nucleotide sequence selected from the
group
consisting of SEQ m N0:2n-1, wherein n is an integer between 1 and 38 is
changed from
that selected from the group consisting~of the chosen sequence to a different
nucleotide
provided that no more than 15°l0 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 SEQ
)D N0:2n, wherein n is an integer between 1 and 38, wherein the nucleic acid
molecule
hybridizes under stringent conditions to the nucleotide sequence selected from
the group
consisting of SEQ )D N0:2n-1, wherein n is an integer between 1 and 38, 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
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lD N0:2n, wherein n is an integer between 1 and 38, 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 15% 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 fD NO:2n, wherein n is an integer betweeml and 38. 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 38 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
sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ )D N0:2n, wherein n is an integer between 1 and 38 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.
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The invention further provides an antibody that binds immunospecifically to a
NOVX polypeptide. The NOVX antibody may be monoclonal, humanized, or a fully
human antibody. Preferably, the antibody has a dissociation constant for the
binding of the
NOVX polypeptide to the antibody less than 1 x 10-9 M. More preferably, the
NOVX
antibody neutralizes the activity of the NOVX polypeptide.
In a further aspect, the invention provides for the use of a therapeutic in
the
manufacture of a medicament for treating a syndrome associated with a human
disease,
associated with a NOVX polypeptide. Preferably the therapeutic is a NOVX
antibody.
In yet a further aspect, the invention provides a method of treating or
preventing a
NOVX-associated disorder, a method of treating a pathological state in a
mammal, and a
method of treating or preventing a pathology associated with a polypeptide by
administering a NOVX antibody to a subject in an amount sufficient to treat or
prevent.the
disorder.
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.
BRIEF DESCRIPTION OF THE DRAWINGS
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
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proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the
novel
sequences disclosed herein. Table A provides a summary of the NOVX nucleic
acids and
their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers
SEQ ID SEQ ID
NOVX ~ Internal NO NO
gomology
Assignment Identification(nucleic (amino
acid) acid)
NOVla ~CG121992-03 Kl ~ 2 Chordinprecursor-
Homo sapiens
NOVlb ~ CG121992-02 3 4 Chordin precursor-
Homo sapiens
NOVlc CG121992-04 5 6 Chordin precursor-
Homo sapiens
~
NOV2a CG186275-03 7 8 ADAM 22 precursor
(A
disintegrin and
metalloproteinase
domain 22)
(Metalloproteinase-like,
disintegrin-like,
and
cysteine-rich protein
2)
(Metalloproteinase-
disintegrin ADAM22-
' 3) -Homo sapiens
NOV3a ~ 260368272 , 9 10 f Beta-secretase
- Homo
__ sapiens
NOV3b A 260368280 11 12 Beta-secretase
- Homo
_ _ _ sap_iens
~
NOV3c ~ 267441066 13 14 ' Beta-secretase
- Homo
_ sapiens
NOV3d CG50586-03 15 16 Beta-secretase
- Homo
f
sapiens
NOV4a CG50637-Ol 17 18 Transmembrane protein
AMIGO - Homo
sapiens
~
NOV4b 277577082 19 20 Transmembrane protein
AMIGO - Homo
sapiens __
NOV4c 3 277577094 , 21 22 Transmembrane protein
AMIGO - Homo
sapiens
NOV4d 277577141 23 24 Transmembrane protein
AMIGO - Homo
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__ _ _ ~ sapiens_
' ~
NOVSa 306433917 ~ 25 26 Nephronectin - Homo
sapiens
NOVSb 306447063 ~ 27 28 Nephronectin - Homo
sapiens
NOVSc 306447071 29 30 Nephronectin - Homo
Sapiens
'
NOVSd 306447075 31 32 Nephronectin - Homo
~~~~ _ sap_ie_ns _
~
NOVSe a CG51117-09 33 34 Nephronectin - Homo
sapiens
NOVSf CG51117-14 35 36 Nephronectin - Homo
sapiens
NOVSg ' SNP13382208 37 38 Nephronectin - Homo
_. _~_ _,sapiens
NOV6a CG51923-01 39 ~40 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
_ ~N __ sapie_ns __
NOV6b i 305869563 41 r 42 v Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
' sapiens
NOV6c ' 305869567 43 44 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
sapiens
NOV6d 306076041 45 46 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
sapiens
NOV6e 317868343 47 48 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
. growth factor-like
domains 1) - Homo
_ Sapiens
NOV6f 317868367 49 50 Protocadherin Fat
2
precursor (hFat2)
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(Multiple epidermal
growth factor-like
domains 1) - Homo
Sapiens
NOV6g 317871203 51 52 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
Sapiens
NOV6h 317871219 53 54 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
_ Sapiens
NOV6i 317871243 55 56 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
Sapiens
NOV6j 317871246 57 58 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
__ sapiens
~
NOV6k 317999764 59 60 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
~
_ _ -_ sapiens _
~ ~ _ _ ~,
-_.--
NOV61 318176301 61 62 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
sapiens
NOV6m CG51923-02 63 64 Protocadherin Fat
2
precursor (hFat2)
(Multiple epidermal
growth factor-like
domains 1) - Homo
Sapiens
NOV6n CG51923-03 65 66 Protocadherin Fat
2
precursor (hFat2)
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(Multiple epidermal
' growth factor-like
domains 1) - Homo
sapiens
NOV7a CG52919-06 67 68 SEZ-6 - Homo sapiens
. 298521010 69 _70 SEZ-_6 _- Homo sapiens
NOV7b
_ CG52919-09 71 72 ~ SEZ-6 - Homo sapiens
NOV7c
NOVBa CG94946-Ol 73 74 ~ AGRIN precursor
-
Homo sapiens
(Human), 2026 as
NOVBb 308909220 75 76 AGRIN precursor -
Homo sapiens
(Human), 2026 as
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 and condition 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), vascular
calcification, fibrosis, 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, osteoarthritis, rheumatoid arthritis, osteochondrodysplasia,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer,
diabetes, metabolic
disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility,
glomerulonephritis, hemophilia, hypercoagulation, idiopathic thrombocytopenic
purpura,
immunodeficiencies, psoriasis, skin disorders, graft versus host disease,
A>DS, bronchial
asthma, lupus, Crohn's disease; inflammatory bowel disease, ulcerative
colitis, 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, schizophrenia, depression, asthma, emphysema, allergies, the
metabolic
syndrome X and wasting disorders associated with chronic diseases and various
cancers, as
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well as conditions such as transplantation, neuroprotection, fertility, or
regeneration (in
vitro and in vivo).
NOVX nucleic acids and their encoded polypeptides are useful in a variety of
applications and contexts. The various NOVX nucleic acids and polypeptides
according to
the invention are useful as novel members of the protein families according to
the presence
of domains and sequence relatedness to previously described proteins.
Additionally,
NOVX nucleic acids and polypeptides can also be used to identify proteins that
are
members of the family to which the NOVX polypeptides belong.
Consistent with other known members of the family of proteins, identified in
column 5 of Table 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.
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 vs. diseased
tissues, e.g.
detection of a variety of cancers.
Additional utilities for NOVX nucleic acids and polypeptides according to the
invention are disclosed herein.
NOVX clones
NOVX nucleic acids and their encoded polypeptides are useful in a variety of
applications and contexts. The various NOVX nucleic acids and polypeptides
according to
the invention are useful as novel members of the protein families according to
the presence
of domains and sequence relatedness to previously described proteins.
Additionally,
NOVX nucleic acids and polypeptides can also be used to identify proteins that
are
members of the family to which the NOVX polypeptides belong.
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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
small molecule drug target, (iii) an antibody target (therapeutic, diagnostic,
drug
targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy
(gene delivery/gene
ablation), and (v) a composition promoting tissue regeneration in vitro and in
vivo (vi) a
biological defense weapon.
In one specific embodiment, the invention includes an isolated poIypeptide
comprising an amino acid sequence selected from the group consisting of: (a) a
mature
form of the amino acid sequence selected from the group consisting of SEQ ID
NO: 2n,
wherein n is an integer between 1 and 38; (b) a variant of a mature form of
the amino acid
sequence selected from the group consisting of SEQ >D NO: 2n, wherein n is an
integer
between 1 and 38, wherein any amino acid in the mature form is changed to a
different
amino acid, provided that no more than 15% of the amino acid residues in the
sequence of
the mature form are so changed; (c) an amino acid sequence selected from the
group
consisting of SEQ ID NO: 2n, wherein n is an integer between l and 38; (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 38 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 lD NO: 2n, wherein n is an integer between 1 and 38; (b) a
variant of
a mature form of the amino acid sequence selected from the group consisting of
SEQ ID
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. NO: 2n, wherein n is an integer between 1 and 38 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 lD NO: 2n,
wherein n
is an integer between 1 and 38; (d) a variant of the amino acid sequence
selected from the .
group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 38,
in which
any amino acid specified in the chosen sequence is changed to a different
amino acid,
provided that no more than 15% of the amino acid residues in the sequence are
so changed;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide
comprising the
amino acid sequence selected from the group consisting of SEQ ll~ NO: 2n,
wherein n is an
integer between 1 and 38 or any variant of said polypeptide wherein any amino
acid of the
chosen sequence is changed to a different amino acid, provided that no more
than 10% of
the amino acid residues in the sequence are so changed; and (f) the complement
of any of
said nucleic acid molecules.
In yet another specific embodiment, the invention includes an isolated nucleic
acid
molecule, wherein said nucleic acid molecule comprises a nucleotide sequence
selected
from the group consisting of: (a) the nucleotide sequence selected from the
group
consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 38; (b) a
nucleotide sequence wherein one or more nucleotides in the nucleotide sequence
selected
from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between
1 and 38
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 >D
NO: 2n-1, wherein n is an integer between 1 and 38; and (d) a nucleic acid
fragment
wherein one or more nucleotides in the nucleotide sequence selected from the
group
consisting of SEQ m NO: 2n-1, wherein n is an integer between 1 and 38 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 mIZNAs) and fragments for use as PCR
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primers for the amplification and/or mutation of NOVX nucleic acid molecules.
As used
herein, the term "nucleic. acid molecule" is intended to include DNA molecules
(e.g.,
cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA
generated using nucleotide analogs, and derivatives, fragments and homologs
thereof. The
nucleic acid molecule may be single=stranded or double-stranded, but
preferably is
comprised double-stranded DNA.
A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a
"mature" form of a polypeptide or protein disclosed in the present invention
is the product
of a naturally occurnng polypeptide or precursor form or proprotein. The
naturally
occurring polypeptide, precursor or proprotein 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 step
of
post-translational modification other than a proteolytic cleavage event. Such
additional
processes include, by way of non-limiting example, glycosylation,
myristylation or
phosphorylation. In general, a mature polypeptide or protein may result from
the operation
of only one of these processes, or a combination of any of them.
The term "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-
CA 02495563 2005-02-O1
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stranded 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 less
than
about 5 kb, 4 kb, 3 kb, 2 kb, I kb, 0.5 kb or 0.1 kb of nucleotide sequences
which naturally
flank the nucleic acid molecule in genomic DNA of the cell/tissue from which
the nucleic
. . acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an
"isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of other cellular
material, or
culture medium, 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 )D N0:2n-1, wherein n is an integer between 1 and
38, 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 NO:2n-l, wherein n is an integer between 1 and
38, 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
CLOrrn~rG: 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
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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 >D N0:2n-l, wherein n is an integer
between 1 and
38, or a complement thereof. Oligonucleotides may be chemically synthesized
and may
also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a nucleic acid molecule that is a complement of the nucleotide
sequence shown
in SEQ )D NO:2fi-1, wherein n is an integer between 1 and 38, 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 >D N0:2n-1, wherein n
is an
integer between 1 and 38, is one that is sufficiently complementary to the
nucleotide
sequence of SEQ JD N0:2n-1, wherein n is an integer between 1 and 38, that it
can
hydrogen bond with few or no mismatches to the nucleotide sequence shown in
SEQ ID
N0:2n-l, wherein n is an integer between 1 and 38, 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'
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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.
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.,
Cu~~~IT
PROTOCOLS ~~r 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 occurnng
allelic variations and mutations of the nucleotide sequences set forth herein.
A homologous
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nucleotide sequence does not, however, include the exact nucleotide sequence
encoding
human NOVX protein. Homologous nucleic acid sequences include those nucleic
acid
sequences that encode conservative amino acid substitutions (see below) in SEQ
>D
N0:2n-1, wherein n is an integer between 1 and 38, as well as a polypeptide
possessirig
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-1, wherein n is an integer between 1 and 38; or an anti-sense strand
nucleotide
sequence of SEQ m N0:2n-1, wherein n is an integer between 1 and 38; or of a
naturally
occurring mutant of SEQ 1D N0:2n-1, wherein n is an integer between 1 and 38.
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
5. "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 38, that encodes a polypeptide
having a
NOVX biological activity (the biological activities of the NOVX proteins are
described
below), expressing the encoded portion of NOVX protein (e.g., by recombinant
expression
in vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Single Nucleotide Polymorphisms
Variant sequences are also included in this application. A variant sequence
can
include a single nucleotide polymorphism (SNP). A SNP can, in some instances,
be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP
originates as a cDNA. A SNP can arise in several ways. For example, a SNP may
be due to
a substitution of one nucleotide for another at the polymorphic site. Such a
substitution can
be either a transition or a transversion. A SNP can also arise from a deletion
of a
nucleotide or an insertion of a nucleotide, relative to a reference allele. In
this case, the
polymorphic site is a site at which one allele bears a gap with respect to a
particular
nucleotide in another allele. SNPs occurring within genes may result in an
alteration of the
amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may
also be
silent, when a codon including a SNP encodes the same amino acid as a result
of the
redundancy of the genetic code. SNPs occurring outside the region of a gene,
or in an
intron within a gene, do not result in changes in any amino acid sequence of a
protein but
may result in altered regulation of the expression pattern. Examples include
alteration in
temporal expression, physiological response regulation, cell type expression
regulation,
intensity of expression, and stability of transcribed message.
SeqCalling assemblies produced by the exon linking process were selected and
extended using the following criteria. Genomic clones having regions with 98%
identity to
all or part of the initial or extended sequence were identified by BLASTN
searches using
the relevant sequence to query human genomic databases. The genomic clones
that
resulted were selected for further analysis because this identity indicates
that these clones
contain the genomic locus for these SeqCalling assemblies. These sequences
were
analyzed for putative coding regions as well as for similarity to the known
DNA and
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protein sequences. Programs used for these analyses include Grail, Genscan,
BLAST,
IiIVIMER, FASTA, Hybrid and other relevant programs.
Some additional genomic regions may have also been identified because selected
SeqCalling assemblies map to those regions. Such SeqCalling sequences may have
overlapped with regions defined by homology or exon prediction. They may also
be.
included because the location of the fragment was in the vicinity of genomic
regions
identified by similarity or exon prediction that had been included in the
original predicted
sequence. The sequence so identified was manually assembled and then may have
been
extended using one or more additional sequences taken from CuraGen
Corporation's human
SeqCalling database. SeqCalling fragments suitable for inclusion were
identified by the
CuraTools~ program SeqExtend or by identifying SeqCalling fragments mapping to
the
appropriate regions of the genomic clones analyzed.
The regions defined by the procedures described above were then manually
integrated and corrected for apparent inconsistencies that may have arisen,
for example,
from miscalled bases in the original fragments or from discrepancies between
predicted
exon junctions, EST locations and regions of sequence similarity, to derive
the final
sequence disclosed herein. When necessary, the process to identify and analyze
SeqCalling
assemblies and genomic clones was reiterated to derive the full length
sequence (Alderborn
et al., Determination of Single Nucleotide Polymorphisms by Real-time
Pyrophosphate
DNA Sequencing. Genome Research. 10 (8) 1249-1265, 2000).
Variants are reported individually but any combination of all or a select
subset of
variants are also included as contemplated NOVX embodiments of the invention.
NOVX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between ~ and
38, due to
degeneracy of the genetic code and thus encode the same NOVX proteins as that
encoded
by the nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between
1 and 38.
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 >D N0:2n,
wherein n
is an integer between 1 and 38.
In addition to the human NOVX nucleotide sequences of SEQ ID N0:2rz-1, wherein
n is an integer between 1 and 38, it will be appreciated by those skilled in
the art that DNA
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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 of the NOVX genes. Any and
all such
nucleotide variations and resulting amino acid polymorphisms in the NOVX
polypeptides,
which are the result of natural allelic variation and that do not alter the
functional activity
of the NOVX polypeptides, are intended to be within the scope of the
invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species,
and
thus that have a nucleotide sequence that differs from a human SEQ ID N0:2n-1,
wherein n
is an integer between 1 and 38, 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 3~. In another embodiment, the nucleic acid is at
least 10, 25,
'50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In
yet another
embodiment, an isolated nucleic acid molecule of the invention hybridizes to
the coding
region. As used herein, the term "hybridizes under stringent conditions" is
intended to
describe conditions for hybridization and washing under which nucleotide
sequences at
least 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., paralogs) can be obtained by
low, moderate or
high stringency hybridization with all or a portion of the particular human
sequence as a
probe using methods well known in the art for nucleic acid hybridization and
cloning.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to
no other sequences. Stringent conditions are sequence-dependent and will be
different.in
27
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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
excess, at Tm, 50% of the probes are occupied at equilibrium. Typically,
stringent
conditions will be those in which the salt concentration is less than about
1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and
the
temperature is at least about 30 °C for short probes, primers or
oligonucleotides (e.g., 10 nt
to 50 nt) and at least about 60 °C for longer probes, primers and
oligonucleotides.
Stringent conditions may also be achieved with the addition of destabilizing
agents, such as
formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel, et al., (eds.), CURRENT.PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons,
N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences
at least about
65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically
remain
hybridized to each other. A non-limiting example of stringent hybridization
conditions are
hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm
DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at
50°C. An
isolated nucleic acid molecule of the invention that hybridizes under
stringent conditions to
a sequence of SEQ ID N0:2fa-1, wherein n is an integer between 1 and 38,
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-1, wherein n
is an
integer between 1 and 38, or fragments, analogs or derivatives thereof, under
conditions of
moderate stringency is provided. A non-limiting example of moderate stringency
hybridization conditions are hybridization in 6X SSC, 5X 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
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nr MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER
arm EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule comprising the nucleotide sequences of SEQ ID. N0:2n-1, wherein n is
an integer
between 1 and 38, or fragments, analogs or derivatives thereof, under
conditions of low
stringency, is provided. A non-limiting example of low stringency
hybridization conditions
are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM
EDTA,
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X
SSC, 25 mM
Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions
of low
stringency that may be used are well known in the art (e.g., as employed for
cross-species
. hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS
IN
MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER
AND
EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981.
Proc Natl Acad Sci IJSA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may
. exist in the population, the skilled artisan will further appreciate that
changes can be
introduced by mutation into the nucleotide sequences of SEQ ID N0:2~a-1,
wherein n is an
integer between 1 and 38, thereby leading to changes in the amino acid
sequences of 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 m N0:2n, wherein n is
an integer
between l and 38. 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. Annino
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 38, yet retain biological activity. In one embodiment,
the isolated
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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 1D N0:2n, wherein n is an integer between 1 and 38.
Preferably,
the protein encoded by the nucleic acid molecule is at least about 60%
homologous to SEQ
)D N0:2n, wherein n is an integer between 1 and 38; more preferably at least
about 70%
homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 38; still
more
preferably at least about 80% homologous to SEQ ID N0:2n, wherein n is an
integer
between l and 38; even more preferably at Ieast about 90% homologous to SEQ ID
N0:2n,
wherein n is an integer between 1 and 38; and most preferably at least about
95%
homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 38.
An isolated nucleic acid molecule encoding a NOVX protein homologous to the
protein of SEQ JD N0:2n, wherein n is an integer between 1 and 38, can be
created by
introducing one or more nucleotide substitutions, additions or deletions into
the nucleotide
sequence of SEQ >D NO:2n-1, wherein n is an integer between 1 and 38, 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 N0:2n-l, wherein n is an integer
between 1 and 38, 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 1D N0:2n-l, wherein n is an integer between 1 and 38,
the
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encoded protein can be expressed by any recombinant technology known in the
art and the
activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side
chain
interactions. Substituted amino acids.may be fully conserved "strong" residues
or fully
conserved "weak" residues. The "strong" group of conserved amino acid residues
may be
any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILE, 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' L1T' region. See, e.g.,
PCT applications
WO00/44895, W099/32619, WO01/75164, W001/92513, WO 01/29058, W001/89304,
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 ribonucleotides. 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.
In another example, a hairpin RNAi product is a siRNA. The regulatory
sequences
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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 Hl. One example of
a
vector system is the GeneSuppressor~ RNA Interference kit (commercially
available from
Imgenex). The U6 and Hl promoters are members of the type III class of Pol III
promoters.
The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1
for H1
promoters is adenosine. The termination signal for these promoters is defined
by five
consecutive thymidines. The transcript is typically cleaved after the second
uridine.
Cleavage at this position generates a 3' W 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
25~ 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.
A NOVX mRNA region to be targeted by siRNA is generally selected from a
desired NOVX sequence beginning 50 to100 nt downstream of the start codon.
Alternatively, 5' or 3' UTRs and regions nearby the start codon can be used
but are
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generally avoided, as these may be richer in regulatory protein binding sites.
UTR-binding
proteins andlor 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 G/C-content of approximately 30% to 70%, and
more
preferably of about 50%. If the sequence AA(Nl9)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 (N19)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 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
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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' (N19)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 dug 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 ~tg single-stranded sense NOVX
siRNA will have no effect on NOVX silencing, and 0.84 p.g antisense siRNA has
a weak
ilencing effect when compared to 0.84 ,ug of duplex siRNAs. Control
experiments again
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
CA 02495563 2005-02-O1
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fraction of lamin A/C knockdown in cells is determined the next day by such
techniques as r
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
:10 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.
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 RNAi 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
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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 siRNA'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 E.~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
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
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addition of mRNAs. Then NOVX mRNAs are added and the incubation continued for
an
additional 60 min. 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 32P-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
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-purified
(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 ~M) 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.
Cell Culture
A cell culture known in the art to regularly express NOVX is propagated using
standard conditions. 24 hours before transfection, at approx. 80% confluency,
the cells are
trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3 X 105
cells/ml) and
transferred to 24-well plates (500 ml/well). Transfection is performed using a
commercially available lipofection kit and NOVX expression is monitored using
standard
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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 w.
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 117 N0:2n-1, wherein n is an integer between 1 and
38, or
fragments, analogs or derivatives thereof. An "antisense" nucleic acid
comprises a
nucleotide sequence that is complementary to a "sense" nucleic acid encoding a
protein
(e.g., complementary to the coding strand of a double-stranded cDNA molecule
or
complementary to an mRNA sequence). In specific aspects, antisense nucleic
acid
molecules are provided that comprise a sequence complementary to at least
about 10, 25,
50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only. a
portion
thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and
analogs of
a NOVX protein of SEQ ID N0:2n, wherein n is an integer between 1 and 38, or
antisense
nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID N0:2n-1,
wherein n is an integer between 1 and 38, 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
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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 510, 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 oligonueleotide) 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) uraeil, 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-methyleytosine, N6-adenine, 7-
methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil,
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, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively,
the antisense nucleic acid can be produced biologically using an expression
vector into
which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed
from the inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of
interest, described further in the following subsection).
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The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated in situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding a NOVX protein to thereby inhibit expression of the
protein (e.g.,
by inhibiting transcription and/or translation). The hybridization can be by
conventional
. nucleotide complementarity to form a stable duplex, or, for example, in the
case of an
antisense nucleic acid molecule that binds to DNA duplexes, through specific
interactions
in the major groove of the double helix. An example of a route of
administration of
antisense nucleic acid molecules of the invention includes direct injection at
a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to target
selected cells and
then administered systemically. For example, for systemic administration,
antisense
molecules can be modified such that they specifically bind to receptors or
antigens
expressed on a selected cell surface (e.g., by linking the antisense nucleic
acid molecules to
peptides'or antibodies that bind to cell surface receptors or antigens). The
antisense nucleic
acid molecules can also be 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 poI 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) or
a chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987. FEBS Lett. 215:
327-330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified
bases, and nucleic acids whose sugar phosphate backbones are modified or
derivatized.
These modifications are carried out at least in part to enhance the chemical
stability of the
modified nucleic acid, such that they 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
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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 ll~ NO:2n-1,
wherein n
is an integer between 1 and 38): For example, a derivative of a Tetrahymena L-
19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in a NOVX-encoding
mRNA.
See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to
Cech, et al.
NOVX mRNA can also be used to select a catalytic RNA having a specific
ribonuclease
activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993)
Science
261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the NOVX nucleic acid
(e.g., the
NOVX promoter and/or enhancers) to form triple helical structures that prevent
transcription of the NOVX gene in target cells. See, e.g., Helene, 1991.
Anticancer Drug .
Des. 6: 569-84; Helene, et al. 1992. Ann. N.Y. Acad. Sci. 660: 27-36; Maher,
1992.
Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the stability;
hybridization,
or solubility of the molecule. For example, the deoxyribose phosphate backbone
of the
nucleic acids can be modified to.generate peptide nucleic acids. See, e.g.,
Hyrup, et al.,
1996. Bioorg Med Claern 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
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restriction enzymes when used in combination with other enzymes, e.g., SI
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 polymerases) to interact with
the DNA
portion while the PNA portion would provide high binding affinity and
specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths selected
in terms of
base stacking, number of bonds between the nucleotide bases, and orientation
(see, Hyrup,
et al., 1996. supra). The synthesis of PNA-DNA chimeras can be perfoimed as
described
in Hyrup, et al., 1996. supra and Finn, et al.,1996. Nucl Acids Res 24: 3357-
3363. For
example, a DNA chain can be synthesized on a solid support using standard
phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used
between the
PNA and the 5' end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17:
5973-5988.
PNA monomers are then coupled in a stepwise manner to produce a chimeric
molecule
with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra.
Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and
a 3' PNA
segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-
11124.
In tither embodiments, the oligonucleotide may include other appended groups
such
as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acad. Sci. U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
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. BioTechniques 6:958-976) or intercalating
agents (see, e.g.,
Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be
conjugated to
another molecule, e.g., a peptide, a hybridization triggered cross-linking
agent, a transport
agent, a hybridization-triggered cleavage agent, and the like.
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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 m N0:2n, wherein n is an integer between 1 and 38. 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 >D N0:2n, wherein n is an integer between 1
and 38,
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
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 preparations of NOVX proteins having less than about 30%
(by dry
weight) of non-NOVX proteins (also referred to herein as a "contaminating
protein"), more
preferably less than about 20% of non-NOVX proteins, still more preferably
less than about
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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 ll~ N0:2n, wherein n is an
integer
between 1 and 38) 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 )D
N0:2ra, wherein n is an integer between 1 and 38. In other embodiments, the
NOVX
protein is substantially homologous to SEQ ID N0:2n, wherein n is an integer
between 1
and 38, and retains the functional activity of the protein of SEQ )D N0:2n,
wherein n is an
integer between 1 and 38, yet differs in amino acid sequence due to natural
allelic variation
or mu_tagenesis, 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 N0:2n, wherein n is an integer
between
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1 and 38, and retains the functional activity of the NOVX proteins of SEQ ID
NQ:2n,
wherein n is an integer between 1 and 38.
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. J Mol Biol 48: 443-453. Using GCG GAP software with
the following settings for nucleic acid sequence comparison: GAP creation
penalty of 5.0
and GAP extension penalty of 0.3, the coding region of the analogous nucleic
acid
sequences referred to above exhibits a degree of identity preferably of at
least 70%, 75%,
80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA
sequence
of SEQ ID N0:2ra-1, wherein n is an integer between 1 and 38.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I,
in the case of
nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing
the number of matched positions by the total number of positions in the region
of
comparison (i.e., the window size), and multiplying the result by 100 to yield
the
percentage of sequence identity. The term "substantial identity" as used
herein denotes a
characteristic of a polynucleotide sequence, wherein the polynucleotide
comprises a
sequence that has at least 80 percent sequence identity, preferably at least
85 percent
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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 38, 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
portions of a NOVX protein. Within the fusion protein, the term "operatively-
linked" is
intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide
are fused
in-frame with one another. The non-NOVX polypeptide can be fused to the N-
terminus or
C-terminus of the NOVX polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which
the
NOVX sequences are fused to the C-terminus of the GST (glutathione S-
transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
NOVX
polypeptides.
In another embodiment, the fusion protein is a NOVX protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host
cells), expression and/or secretion of NOVX can be increased through use of a
heterologous signal sequence.
In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion
protein in which the NOVX sequences are fused to sequences derived from a
member of
the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of
the
invention can be incorporated into pharmaceutical compositions and
administered to a
subject to inhibit an interaction between a NOVX ligand and a NOVX protein on
the
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surface of a cell, to thereby suppress NOVX-mediated signal transduction
ira.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-imrnunoglobulin fusion proteins of the invention can be used as
imTnunogens to
produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in
screening
assays to identify molecules that inhibit the interaction of NOVX with a NOVX
ligand.
A NOVX chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as
appropriate, alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic
ligation. In another embodiment, the fusion gene can be synthesized by
conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of
gene fragments can be carried out using anchor primers that give rise to
complementary
overhangs between two consecutive gene fragments that can subsequently be
annealed and
reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al.
(eds:) C~1~1T
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many
expression vectors are commercially available that already encode a fusion
moiety (e.g., a
GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an
expression
vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists
The invention also pertains to variants of the NOVX proteins that function as
either
NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein
can be generated by mutagenesis (e.g., discrete point mutation or truncation
of the NOVX
protein). An agonist of the NOVX protein can retain substantially the same, or
a subset of,
the biological activities of the naturally occurring form of the NOVX protein.
An
antagonist of the NOVX protein can inhibit one or more of the activities of
the naturally
occurring form of the NOVX protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which includes
the NOVX
protein. Thus, specific biological effects can be elicited by treatment with
~a variant of
4~
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limited function. In one embodiment, treatment of a subject with a variant
having a subset
of the biological activities of the naturally occurring form of the protein
has fewer side
effects in a subject relative to treatment with the naturally occurring form
of the NOVX
proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i.e.,
mimetics) or as NOVX antagonists can be identified by screening combinatorial
libraries of
mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein
agonist or
antagonist activity. In one embodiment, a variegated library of NOVX variants
is
generated by combinatorial mutagenesis at the nucleic acid level and is
encoded by a
variegated gene library. A variegated library of NOVX variants can be produced
by, for
example, enzymatically ligating a mixture of synthetic oligonucleotides into
gene
sequences such that a degenerate set of potential NOVX sequences is
expressible as
individual polypeptides, or alternatively, as a set of larger fusion proteins
(e.g., for phage
display) containing the set of NOVX sequences therein. There are a variety of
methods
which can be used to produce libraries of potential NOVX variants from a
degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can
be
performed in an automatic DNA synthesizer, and the synthetic gene then ligated
into an
appropriate expression vector. Use of a degenerate set of genes allows for the
provision, in
one mixture, of all of the sequences encoding the desired set of potential
NOVX sequences.
Methods for synthesizing degenerate oligonucleotides are well-known within the
art. See,
e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev.
Biochem. 53: 323;
Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res.
1 l: 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 S1
nuclease,
and ligating the resulting fragment library into an expression vector. By this
method,
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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 fox screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of NOVX
proteins. The most widely used techniques, which are amenable to high
throughput
analysis, for screening large gene libraries typically include cloning the
gene library into
replicable expression vectors, transforming appropriate cells with the
resulting library of
vectors, and expressing the combinatorial genes under conditions in which
detection of a
desired activity facilitates isolation of the vector encoding the gene whose
product was
detected. Recursive ensemble mutagenesis (REM), a new technique that enhances
the
frequency of functional mutants in the libraries, can be used in combination
with the
screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan,
1992. Proc.
Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein
Engineering
6:327-331.
Anti-NOVX Antibodies
Included in the invention are antibodies to NOVX proteins, or fragments of
NOVX
proteins. The term "antibody" as used herein refers to immunoglobulin
molecules and
immunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that
contain an antigen binding site that specifically binds (immunoreacts with) an
antigen.
Such antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, single
chain, Fab, Fab' and Fcabp2 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
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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 1D
N0:2n;
wherein n is an integer between 1 and 38, 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, a:g., a
hydrophilic region. A hydrophobicity analysis of the human NOVX protein
sequence will
indicate which regions of a NOVX polypeptide are particularly hydrophilic and,
therefore,
are likely to encode surface residues useful for targeting antibody
production. As a means
for targeting antibody production, hydropathy plots showing regions of
hydrophilicity and
hydrophobicity may be generated by any method well known in the art,
including, for
example, the Kyte Doolittle or the Hopp Woods methods, either with or without
Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. I7SA78:
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-NOVX antibody of the present invention is said to
specifically bind to
antigen NOVX when the equilibrium binding constant (KD) is <_1 p.M, preferably
<_ 100
nM, more preferably < 10 nM, and most preferably <_ 100 pM to about 1 pM, as
measured
by assays including radioligand binding assays or similar assays known to
skilled artisans.
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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
the native protein, a synthetic variant thereof, or a derivative of the
foregoing. An
appropriate immunogenic preparation can contain, for example, the naturally
occurring
immunogenic protein, a chemically synthesized polypeptide representing the
immunogenic
protein, or a recombinantly expressed immunogenic protein. Furthermore, the
protein may
be conjugated to a second protein known to be immunogenic in the mammal being
immunized. Examples of such immunogenic proteins include but are not limited
to
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin
inhibitor. The preparation can further include an adjuvant. Various adjuvants
used to
increase the immunological response include, but are not limited to, Freund's
(complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface active
substances (e.g.,
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium
parvum,
or similar immunostimulatory agents. Additional examples of adjuvants which
can be
employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the immunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
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chromatography. Purification of immunoglobulins is discussed, for example, by
D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA,
Vol. 14, No. 8
(April 17, 2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs)
of the monoclonal antibody are identical in all the molecules of the
population. MAbs thus
contain an antigen binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridorna
method, a
mouse, hamster, or other appropriate host animal, is typically immunized with
an
immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies
that will specifically bind to the immunizing agent. Alternatively, the
lymphocytes can be
immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof
or a fusion protein thereof. Generally, either peripheral blood lymphocytes
are used if cells
of human origin are desired, or spleen cells or lymph node cells are used if
non-human
mammalian sources 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
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medium such as HAT medium. More preferred immortalized cell lines are murine
myeloma lines, which can be obtained, for instance, from the Salk Institute
Cell
Distribution Center, San Diego, California and the American Type Culture
Collection,
Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines
also
have been described for the production of human monoclonal antibodies (Kozbor,
J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques
and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be
assayed
for the presence of monoclonal antibodies directed against the antigen.
Preferably, the
binding specificity of monoclonal antibodies produced by the hybridoma cells
is
determined by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such
techniques and assays are known in the art. The binding affinity of the
monoclonal
antibody can, for example, be determined by the Scatchard analysis of Munson
and Pollard,
Anal. Biochem., 107:220 (1980). It is an objective, especially important in
therapeutic
applications of monoclonal antibodies, to identify antibodies having a high
degree of
specificity and a high binding affinity for the target antigen.
After the desired hybridoma cells are identified, the clones can be subcloned
by
limiting dilution procedures and grown by standard methods (Goding,1986).
Suitable
culture media for this purpose include, for example, Dulbecco's Modified
Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo
as
ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified
from the culture medium or ascites fluid by conventional immunoglobulin
purification
procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography,
gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such
as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies
of the invention can be readily isolated and sequenced using conventional
procedures (e.g.,
by using oligonucleotide probes that are capable of binding specifically to
genes encoding
the heavy and light chains of 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
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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 marine sequences (U.S. Patent No..4,816,567; Morrison, Nature 368,
812-13
(1994)) or by covalently joining to the immunoglobulin coding sequence all or
part of the
coding sequence for a non-immunoglobulin polypeptide. Such a non-
immunoglobulin
polypeptide can be substituted for the constant domains of an antibody of the
invention, or
can be substituted for the variable domains of one antigen-combining site of
an antibody of
the invention to create a chimeric bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against
the administered immunoglobulin. Humanized forms of antibodies are chimeric
immunoglobulins, irnmunoglobulin 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
Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature,
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting
rodent CDRs or CDR sequences for the corresponding sequences of a human
antibody.
(See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues
of the
human immunoglobulin are replaced by corresponding non-human residues.
Humanized
antibodies can also comprise residues which are found neither in the recipient
antibody nor
in the imported CDR or framework sequences. In general, the humanized antibody
will
comprise substantially all of at least one, and typically two, variable
domains, in which all
or substantially ah of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the framework regions are those
of a human
immunoglobulin consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta,
Curr. Op.
Struct. Biol., 2:593-596 (1992)).
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Human Antibodies
Fully human antibodies essentially relate to antibody molecules in which the
entire
sequence of both the light chain and the heavy chain, including the CDRs,
arise from
human genes. Such antibodies are~termed "human antibodies", or "fully human
antibodies"
herein. Human monoclonal antibodies can be prepared by the trioma technique;
the human
B-cell hybridoma technique (see Kozbor, et al., 1983 Tm_m__unol Today 4: 72)
and the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In:
MONOCLONAL ANTIBODIES AND CANCER TxERAPY, Alan R. Liss, Inc., pp. 77-96).
Human
monoclonal antibodies may be utilized in the practice of the present invention
and may be
produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci
USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro
(see Cole, et
al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER TfIERAPY, 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 Biotechnology 14, 845-51 (1996));
Neuberger
(Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol.
13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals which are modified so as to produce fully human antibodies rather than
the
animal's endogenous antibodies in response to challenge by an antigen. (See
PCT
publication W094/02602). The endogenous genes encoding the heavy and light
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
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chromosomes containing the requisite human DNA segments. An animal which
provides
all the desired modifications is then obtained as progeny by crossbreeding
intermediate
transgenic animals containing fewer than the full complement of the
modifications. The
preferred embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse~ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This
animal produces B cells which secrete fully human immunoglobulins. The
antibodies can
be obtained directly from the animal after immunization with an immunogen of
interest, as,
for example, a preparation of a polyclonal antibody, or alternatively from
immortalized B
cells derived from the animal, such as hybridomas producing monoclonal
antibodies.
Additionally, the genes encoding the immunoglobulins with human variable
regions can be
recovered and expressed to obtain the antibodies directly, or can be further
modified to
obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous imrnunoglobulin heavy chain is disclosed
in U.S.
Patent No. 5,939,598. It can be obtained by a method including deleting the J
segment
genes from at least one endogenous heavy chain locus in an embryonic stem cell
to prevent
rearrangement of the locus and to prevent formation of a transcript of a
rearranged
immunoglobulin heavy chain locus, the deletion being effected by a targeting
vector
containing a gene encoding a selectable marker; and producing from the
embryonic stem
cell a transgenic mouse whose somatic and germ cells contain the gene encoding
the
selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression
vector that
contains a nucleotide sequence encoding a heavy chain into one mammalian host
cell in
culture, introducing an expression vector containing a nucleotide sequence
encoding a light
chain into another mammalian host cell, and fusing the two cells to form a
hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and the light
chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99!53049.
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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). Tn 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
F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the
antibody molecule
with papain and a reducing agent and (iv) F" fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies
that have binding specificities for at least two different antigens. In the
present case, one of
the binding specificities is for an antigenic protein of the invention. The
second binding
target is any other antigen, and advantageously is a cell-surface protein or
receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas)
produce a potential mixture of ten different antibody molecules, of which only
one has the
correct bispecific structure. The purification of the correct molecule is
usually
accomplished by affinity chromatography steps. Similar procedures are
disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-
3659
(1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least
part of the hinge, CH2, and CH3 regions. It is preferred to have the first
heavy-chain
constant region (CHl) containing the site necessary for light-chain binding
present in at
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least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if
desired, the immunoglobulin light chain, are inserted into separate expression
vectors, and
are co-transfected into a suitable host organism. For further details of
generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210
(1986).
According to another approach described in WO 96/27011, the interface between
a
pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
which are recovered from recombinant cell culture. The preferred interface
comprises at
least a part of the CH3 region of an antibody constant domain. In this method,
one or more
small amino acid side chains from the interface of the first antibody molecule
are replaced
with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical
or similar size to the large side chains) are created on the interface of the
second antibody
molecule by replacing 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')~ 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., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody Flab' )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.
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Various techniques for making and isolating bispecific antibody fragments
directly
from recombinant cell culture have also been described. For example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553
(1992), The leucine zipper peptides from the Fos. and Jun proteins were linked
to the Fab'
portions of two different antibodies by gene fusion. The antibody homodimers
were
reduced at the hinge region to form monomers and then re-oxidized to form the
antibody
heterodimers. This method can also be utilized for the production of antibody
homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
USA
90:6444-.6448 (1993) has provided an alternative mechanism for making
bispecific
antibody fragments. The fragments comprise a heavy-chain variable domain (VH)
connected to a light-chain variable domain (VL) by a linker which is too short
to allow
pairing between the two domains on the same chain. Accordingly, the VH and VL
domains
of one fragment are forced to pair with the complementary VL and VH domains of
another
fragment, thereby forming two antigen-binding sites. Another strategy for
making
bispecific antibody fragments by the use of single-chain 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), Fc~yRII
(CD32) and
Fc~yRHI (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
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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 vitxo using
known methods in synthetic protein chemistry, including those involving
crosslinking
agents. For example, immunotoxins can be constructed using a disulfide
exchange reaction
or by forming a thioether bond. Examples of suitable reagents for this purpose
include
iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for
example, in U.S.
Patent No. 4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing
interchain disulfide bond formation in this region. The homodimeric antibody
thus
generated can have improved internalization capability and/or increased
complement-mediated cell killing and antibody-dependent cellular cytotoxicity
(ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol.,148:
2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can
also be
prepared using heterobifunctional cross-linkers as described in Wolff et al.
Cancer
Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered
that has
dual Fc regions and can thereby have enhanced complement lysis and ADCC
capabilities.
See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g.,
an
enzymatically active toxin of bacterial, fungal, plant, or animal origin, or
fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above. 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, PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,
enornycin, and the
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tricothecenes. A variety of radionuclides are available for the production of
radioconjugated antibodies. Examples include 2i2Bi, isih l3iIn, 9oY, and
186Re.
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
dirnethyl 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 triarninepentaacetic acid (MX-DTPA}
is an
exemplary chelating agent for conjugation of radionucleotide to the antibody.
See
W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate
is administered to the patient, followed by removal of unbound conjugate from
the
circulation using a clearing agent and then administration of a "ligand"
(e.g., avidin) that is
in turn conjugated to a cytotoxic agent.
Immunoliposomes
The antibodies disclosed herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art,
such as
described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985);
Hwang et al.,
Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and
4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Patent No.
5,013,556.
Particularly.useful liposomes can be generated by the reverse-phase
evaporation
method with a lipid composition comprising phosphatidylcholine, cholesterol,
and
PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded
through
filters of defined pore size to yield liposomes with the desired diameter.
Fab' fragments of
the antibody of the present invention can be conjugated to the liposomes as
described in
Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-
interchange reaction. A
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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 ~ ..
In one embodiment, methods for the screening of antibodies that possess the
desired
specificity include, but are not limited to, enzyme linked immunosorbent assay
(ELISA)
and other immunologically mediated techniques known within the art. In a
specific
embodiment, selection of antibodies that are specific to a particular domain
of an NOVX
protein is facilitated by generation of hybridomas that bind to the fragment
of an NOVX
protein possessing such a domain. Thus, antibodies that are specific for a
desired domain
within an NOVX protein, or derivatives, fragments, analogs or homologs
thereof, are also
provided herein.
Antibodies directed against a NOVX protein of the invention may be used in
methods known within the art relating to the localization andlor quantitation
of a NOVX
protein (e.g., for use in measuring levels of the NOVX protein within
appropriate
physiological samples, for use in diagnostic methods, for use in imaging the
protein, and
the like). In a given' embodiment, antibodies specific to a NOVX protein, or
derivative,
fragment, analog or homolog thereof, that contain the antibody derived antigen
binding
domain, are utilized as pharmacologically active compounds (referred to
hereinafter as
"Therapeutics").
An antibody specific for a NOVX protein of the invention (e.g., a monoclonal
antibody or a polyclonal antibody) can be used to,isolate a NOVX polypeptide
by standard
techniques, such as immunoaffinity, chromatography or immunopreci~itation. An
antibody
to a NOVX polypeptide can facilitate the purification of a natural NOVX
antigen from
cells, or of a recombinantly produced NOVX antigen expressed in host cells.
Moreover,
such an anti-NOVX antibody can be used to detect the antigenic NOVX protein
(e.g., in a
cellular lysate or cell supernatant) in order to evaluate the abundance and
pattern of
expression of the antigenic NOVX protein. Antibodies directed against a NOVX
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,
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and radioactive materials. Examples of suitable enzymes include horseradish
peroxidase,
alkaline phosphatase, (3-galactosidase, or acetylcholinesterase; examples of
suitable
prosthetic group complexes include streptavidinlbiotin and avidin/biotin;
examples of
suitable fluorescent materials include urnbelliferone, 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 lasIysy' 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 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 ligand which may be absent or
defective in the
disease or pathology, binds the antibody as a surrogate effector ligand,
initiating a
receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates
generally
to the amount needed to achieve a therapeutic objective. As noted above, this
may be a
binding interaction between the antibody and its target antigen that, in
certain cases,
interferes with the functioning of the target, and in other cases, promotes a
physiological
response. The amount required to be administered will furthermore depend on
the binding
affinity of the antibody for its specific antigen, and will also depend on the
rate at which an
administered antibody is depleted from the free volume other subject to which
it is
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administered. Common ranges for therapeutically effective dosing of an
antibody or
antibody fragment of the invention may be, by way of nonlimiting example; from
about 0.1
mglkg 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.
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)
rnicrocapsules, respectively, in colloidal drug delivery systems (for example,
liposomes,
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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 rnicrocapsules. Examples of sustained-release matrices include
polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and'y
ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic
acid copolymers such as the LUPRON DEPOT ~ (injectable microspheres composed
of
lactic acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl
acetate and
lactic acid-glycolic acid enable release of molecules for 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 ira
vitro as well
as in vivo. For example, in vitro techniques for detection of an analyte mRNA
include
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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 irrimunofluorescence. 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. ~Tij ssen,
Elsevier
Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for
detection of an
analyte protein include introducing into a subject a labeled anti-an analyte
protein antibody.
For example; the antibody can' be labeled.with a radioactive marker whose
presence and
location in a subject can be detected by standard imaging techniques.
NOVX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding a NOVX protein, or derivatives, fragments,
analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule
capable of transporting another nucleic acid to which it has been linked. One
type of vector
is a "plasmid", which refers to a circular double stranded DNA loop into which
additional
DNA segments can be ligated. Another type of vector is a viral vector, wherein
additional
DNA segments can be ligated into the viral genome. Certain vectors are capable
of
autonomous replication in a host cell into which they are introduced (e.g.,
bacterial vectors
having a bacterial origin of replication and episomal mammalian vectors).
Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the genome of a
host cell upon
introduction into the host cell, and thereby are replicated along with the
host genome.
Moreover, certain vectors are capable of directing the expression of genes to
which they are
operatively-linked. Such vectors are referred to herein as '.'expression
vectors". In general,
expression vectors of utility in recombinant DNA techniques are often in the
form of
piasmids. In the.present specification, "plasmid" and "vector" can be used
interchangeably
as the plasmid is the most commonly used form of vector. However, the
invention is
intended to include such other forms of expression vectors, such as viral
vectors (e.g.,
replication defective retroviruses, adenoviruses and adeno-associated
viruses), which serve
equivalent functions.
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The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means
that the recombinant expression vectors include one or more regulatory
sequences; selected
on the basis of the host.cells to be used for expression, that is operatively-
linked to the
nucleic acid sequence to be expressed. Within a recombinant expression vector,
.
"operably-linked" is intended to mean that the nucleotide sequence of interest
is linked to
the regulatory sequences) in a manner that allows for,expression of the
nucleotide
sequence (e.g., in an in vitro transcription/translation system or in a host
cell when the
vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers
and
other expression control elements (e.g., polyadenylation signals). Such
regulatory
sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory
sequences include those that direct constitutive expression of a nucleotide
sequence in
many types of host cell and those that direct expression of the nucleotide
sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It will be
appreciated by
those skilled in the art that the 'design of the expression vector can depend
on such factors
as the choice of the host cell to be transformed, the level of expression of
protein desired,
etc. The expression vectors of the invention can be introduced into host cells
to thereby
produce proteins or peptides, including fusion proteins or peptides, encoded
by nucleic
acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins,
fusion
proteins, etc.).
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
Eschericlaia 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
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fusion vectors typically serve three purposes: (i) to increase expression of
recombinant
protein; (ii) to increase the solubility of the recombinant protein; and (iii)
to aid in the
purification of the recombinant protein by acting as a ligand in affinity
purification. Often,
in fusion expression vectors, a proteolytic cleavage site is introduced at the
junction of the
fusion moiety and the recombinant protein to enable separation of the
recombinant protein
from the fusion moiety subsequent to purification of the fusion protein. Such
enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin and
enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith
and
Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and
pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E
binding protein, or protein A, respectively, to the target recombinant
protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
One strategy to maximize recombinant protein expression in E. cvli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the
recombinant protein. See, e. g., Gottesman, GENE EXPRESSION TECHNOLOGY:
METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another
strategy is
to alter the nucleic acid sequence of the nucleic acid to be inserted into an
expression vector
so that the individual codons for each amino acid are those preferentially
utilized in E. coli
(see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such
alteration of nucleic
acid sequences of the invention can be carried out by standard DNA synthesis
techniques.
In another embodiment, the NOVX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include
pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,
Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells
(e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell.
Biol. 3: 2156-2165)
and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using~a mammalian expression vector. Examples of mammalian
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expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC
(Kaufman,
et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the
expression.vector's
control functions are often provided by viral regulatory elements. For
example, commonly
used promoters are derived from polyoma, adenovirus 2; cytomegalovirus, and
simian virus
40. For other suitable expression systems for both prokaryotic and eukaryotic
cells see,
e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR. CLONING: A LABORATORY
MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant'mammalian expression vector is capable
of directing expression of the nucleic acid preferentially in a particular
cell type (e.g.,
tissue-specific regulatory elements are used to express the nucleic acid).
Tissue-specific
regulatory elements are known in the art. Non-limiting examples of suitable
tissue-specific
promoters include the albumin promoter (liver-specific; .Pinkert, et al.,
1987. Genes Dev. 1:
268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol.
43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore,
1989. EMBO
J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;
Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the
neurofilament
promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477),
pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316
and European
Application Publication No. 264,166). Developmentally-regulated promoters are
also
encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science
249:
374-379) and the 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
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the forrri of a.recombinant plasmid, phagemid or attenuated virus in which
antisense nucleic
acids are produced under the control of a high efficiency regulatory region,
the activity of
which can be determined by the cell type into which the vector is introduced.
For a
discussion of the regulation of gene expression using antisense genes see,
e.g., Weintraub,
et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-
Trends in
Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms ."host cell"
and
"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.
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Cells stably transfected with the introduced nucleic acid can be identified by
drug selection
(e.g., cells that have incorporated the selectable marker gene will survive,
while the other
cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture,
can be used to produce (i.e., express) NOVX protein. Accordingly, the
invention further
provides methods for producing NOVX protein using the host cells of the
invention. In one
embodiment, the method comprises culturing the host cell of invention (into
which a
recombinant expression vector encoding NOVX protein has been introduced) in a
suitable
medium such that NOVX protein is produced. In another embodiment, the method
further
comprises isolating NOVX protein from the medium or the host cell.
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
mammal,
more preferably a mouse, in which an endogenous NOVX gene has been altered by
homologous recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic cell of the
animal, prior to
development of the animal.
A transgenic animal of the invention can be created by introducing
NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte
(e.g., by
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microinjection, retroviral infection)'and allowing the oocyte to develop in a
pseudopregnant
female foster animal. The human NOVX cDNA sequences, i.e., any one of SEQ ID
N0:2n-1, wherein n is an integer between 1 and 38, can be introduced as a
transgene into
the genome of a non-human animal: Alternatively, a non-human homologue of the
human
NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization
to the
human NOVX cDNA (described further supra) and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the transgene to
increase the
efficiency of expression of the transgene. A tissue-specific regulatory
sequences) can be
operably-linked to the NOVX transgene to direct expression of NOVX protein to
particular
cells. Methods for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become conventional in
the art and
are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and
4,873,191; and
Hogan,1986. In: MANIPULATING °THE MOUSE EMBRYO, Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y. Similar methods are used for production of
other
transgenic animals. A transgenic founder animal can be identified based upon
the presence
of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues
or
cells of the animals. A transgenic founder animal can then be used to breed
additional
animals carrying the transgene. Moreover, transgenic animals carrying a
transgene-encoding NOVX protein can further be bred to other transgenic
animals carrying
other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at
least a portion of a NOVX gene into which a deletion, addition or substitution
has been
introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The
NOVX gene
can be a human gene (e.g., the cDNA of any one of SEQ DJ N0:2n-1, wherein n is
an
l
integer between 1 and 38), but more preferably, is a non-human homologue of a
human
NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID
N0:2n-1, wherein n is an integer between 1 and 38, 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 ector 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
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alter the expression of the endogenous NOVX protein). In the homologous
recombination
vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-
termini by
additional nucleic acid of the NOVX gene to allow for homologous recombination
to occur
between the exogenous NOVX gene carned 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 AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152. A chimeric embryo can then be implanted into a suitable
pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the
homologously-recombined DNA in their germ cells can be used to breed animals
in which
all cells of the animal contain the homologously-recombined DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination
vectors and homologous recombinant animals are described further in Bradley,
1991. Curr.
Opin. Biotechnol. 2: 823=829; PCT International Publication Nos.: WO 90/11354;
WO
91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain selected systems that allow for regulated expression of the transgene.
One example
of such a system is the cre/loxP recombinase system of bacteriophage Pl. For a
description
of the crelloxP recombinase system, See, e.g., Lakso, et al., 1992. Proc.
Natl. Acad. Sci.
LISA 89: 6232-6236. Another example of a recombinase system is the FLP
recombinase
system of Saccharomyces cereviszae. 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 385: 810-
813. In brief,
a cell (e.g., a somatic cell) from the transgenic animal can be isolated and
induced to exit
the growth cycle and enter Go phase. The quiescent cell can then be fused,
e.g., through the
use of electrical pulses, to an enucleated oocyte from an animal of the same
species from
which the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it
develops to morula or blastocyte and then transferred to pseudopregnant female
foster
animal. The offspring borne of this female foster animal will be a clone of
the animal from
which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies
(also referred to herein as "active compounds") of the invention, and
derivatives, fragments,
analogs and homologs thereof, can be incorporated into pharmaceutical
compositions
suitable for administration. Such compositions typically comprise the nucleic
acid
molecule, protein, or antibody and a pharmaceutically acceptable carrier. As
used herein,
"pharmaceutically acceptable carrier" is intended to include any and all
solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and absorption
delaying
agents, and the like, compatible with pharmaceutical administration. Suitable
carriers are
described in the most recent edition of Remington's Pharmaceutical Sciences, a
standard
reference text in the field, which is incorporated herein by reference.
Preferred examples of
such carriers or diluents include, but are not limited to, water, saline,
finger's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous
vehicles
such as fixed oils may also be used. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any
~ conventional media or agent is incompatible with the active compound, use
thereof in the
compositions is contemplated. Supplementary active compounds can also be
incorporated
into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with
its intended route of administration. Examples of routes of administration
include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation), transdermal
(i.e., topical), transmucosal, and rectal administration. Solutions or
suspensions used for
parenteral, intradermal, or subcutaneous application can include the following
components:
a sterile diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols,
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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
ELTM (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable
under the conditions of manufacture and storage and must be preserved against
the
contaminating action of microorganisriis such as bacteria and fungi. The
carrier can be a
solvent or dispersion medium containing, for example, water, ethanol, polyol
(for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and
suitable
mixtures thereof. The proper fluidity can be maintained, for example, by the
use of.a
coating such as lecithin, by the maintenance of the required particle size in
the case of
dispersion and by the use of surfactants. Prevention of the action of
microorganisms can be
achieved by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases,
it will be
preferable to include isotonic agents, for example, sugars, polyalcohols such
as manitol,
sorbitol, sodium chloride in the composition. Prolonged absorption of the
injectable
compositions can be brought about by including in the composition an agent
which delays
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound
(e.g., a NOVX protein or anti-NOVX antibody) in the required amount in an
appropriate
solvent with one or a combination of ingredients enumerated above, as
required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active
compound into a sterile vehicle that contains a basic dispersion medium and
the required
other ingredients from those enumerated above. In the case of sterile powders
for the
preparation of sterile injectable solutions, methods of preparation are vacuum
drying and
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freeze-drying that yields a powder of the active ingredient plus any
additional desired
ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can
be enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral
therapeutic administration, the active compound can be incorporated with
excipients and
used in the form of tablets, troches, or capsules. Oral compositions can
also.be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is
applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible
binding agents, and/or adjuvant materials can be included as part of the
composition. The
tablets, pills, capsules, troches and the 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 earners that will
protect the compound against rapid elimination from the body, such as a
controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation
of such formulations will be apparent to those skilled in the art. The
materials can also be
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obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal
antibodies to .
viral antigens) can also be used as pharmaceutically acceptable carriers.
These can be
prepared according to methods known to hose 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
10' ~ produce the desired therapeutic effect in association with the required
pharmaceutical
carrier. The specification for the dosage unit forms of the invention are
dictated by and
directly dependent on the unique characteristics of the active compound and
the particular
therapeutic effect to be achieved, and the limitations inherent in the art of
compounding
such an active compound for the treatment of individuals.
~ The nucleic acid molecules of the invention can be inserted into vectors and
used as
gene therapy vectors. Gene therapy vectors can be delivered to a subject by,
for example,
intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by
stereotactic injection (see, e.g., Chen, et al:, 1994. Proc. Natl. Acad. Sci.
USA 91:
3054-3057). The pharmaceutical preparation of the gene therapy vector can
include the
gene therapy vector in an acceptable diluent, or can comprise a slow release
matrix in
which the gene delivery vehicle is imbedded. Alternatively, where the complete
gene
delivery vector can be produced intact from recombinant cells, e.g.,
retroviral vectors, the
pharmaceutical preparation can include one or more cells that produce the gene
delivery
system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy
applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic
lesion in a
NOVX gene, and to modulate NOVX activity, as described further, below. In
addition, the
NOVX proteins can be used to screen drugs or compounds that modulate the NOVX
protein activity or expression as well as to treat disorders characterized by
insufficient or
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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 dyslipidernias. 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
~25 parallel solid phase or solution phase libraries; synthetic library
methods requiring
deconvolution; the "one-bead one-compound" library method; and synthetic
library
methods using affinity chromatography selection. The biological library
approach is
limited to peptide libraries, while the other four approaches are applicable
to peptide,
non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam,
1997.
Anticancer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about 5 kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics,
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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. Chem..37:
2678;. Cho, et al., 1993. Science 261: 1303; Carrell, et al.,1994. Angew.
Chem. Int. Ed:-
Engl. 33: 2059; Carell, et al.,1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and
Gallop, et
al., 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on
chips (Fodor,
1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409),
spores (Ladner,
U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. 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: 30'1-310; 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 lash 355, iaC, or
3H, either
directly or indirectly, and the radioisotope detected by direct counting of
radioemission or
by scintillation counting. Alternatively, test compounds can be enzymatically-
labeled with,
for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic
label detected by determination of conversion of an appropriate substrate to
product. In
one embodiment, the assay comprises contacting a cell which expresses a
membrane-bound
form of NOVX protein, or a biologically-active portion thereof, on the cell
surface with a
known compound which binds NOVX to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the test compound
to interact
with a NOVX protein, wherein determining the ability of~the test compound to
interact with
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a NOVX protein comprises determining the ability of the test compound to
preferentially
bind to NOVX protein or a biologically-active portion thereof as compared to
the known
compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of NOVX protein, or a biologically-active
portion
thereof, on the'cell surface with a test compound and determining the ability
of the test..
compound to modulate (e.g., stimulate or inhibit) the activity-of the NOVX
protein or
biologically-active portion thereof. Determining the ability of the test
compound to
modulate the activity of NOVX or a biologically-active portion thereof can be
accomplished, for example, by determining the ability of the NOVX protein to
bind to or
interact with a NOVX target molecule. As used herein; a "target molecule" is a
molecule
with which a NOVX protein binds or interacts in nature, for example, a
molecule on the
surface of a cell which expresses a NOVX interacting protein, a molecule on
the surface of
a second cell, a molecule in the extracellular milieu; a molecule associated
with the internal
surface of a cell membrane or a cytoplasmic molecule. A NOVX target molecule
can be a
non-NOVX molecule or a NOVX protein or polypeptide of the invention. In one
embodiment, a NOVX target molecule is a component of a signal transduction
pathway
that facilitates transduction of an extracellular signal (e.g. a signal
generated by binding of
a compound to a membrane-bound NOVX molecule) through the cell membrane and
into
the cell. The target, for example, can be a second intercellular protein that
has catalytic
activity or a protein that facilitates the association of downstream signaling
molecules with
NOVX.
Determining the ability of the NOVX protein to bind to or interact with a NOVX
target molecule can be accomplished by one of the methods described above for
determining direct binding. In one embodiment, determining the ability of the
NOVX
protein to bind to or interact with a NOVX target molecule can be accomplished
by
determining the activity of the target molecule. For example, the activity of
the target
molecule can be determined by detecting induction of a cellular second
messenger of the
target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting
catalytic/enzymatic
activity of the target an appropriate substrate, detecting the induction of a
reporter gene
(comprising a NOVX-responsive regulatory element operatively linked to a
nucleic acid
encoding a detectable marker, e.g., luciferase), or detecting a cellular
response, for
example, cell survival, cellular differentiation, or cell proliferation.
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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/enzymatic activity of the
target
molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically-active portion thereof with a known compound which
binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test
compound, and
determining the ability of the test compound to interact with a NOVX protein,
wherein
determining the ability of the test compound to interact with a NOVX protein
comprises
determining the ability of the NOVX protein to preferentially bind to or
modulate the
activity of a NOVX target molecule.
The cell-free assays of the invention 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
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of such solubilizing agents include non-ionic detergents such as n-
octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton ° X-100, Triton~ X-114, Thesit~,
Isotridecypoly(ethylene glycol ether)n, 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.
Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example, either the NOVX protein or its
target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g.,
biotinylation
kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated
96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX
protein or
target molecules, but which do not interfere with binding of the NOVX protein
to its target
molecule, can be derivatized to the wells of the plate, and unbound target or
NOVX protein
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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 of
NOVX mRNA
or protein expression. The level of NOVX mRNA or protein expression in the
cells can be
determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait
proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent
No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et
al., 1993
Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that
bind to or '
interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX
activity. Such NOVX-binding proteins are also involved in the propagation of
signals by
the NOVX proteins as, for example, upstream or downstream elements of the NOVX
pathway.
The two-hybrid system is based on the modular nature of most transcription
factors,
which consist of separable DNA-binding and activation domains. Briefly, the
assay utilizes
two different DNA constructs. In one construct, the gene that codes for NOVX
is fused to a
gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-4). In
the other construct, a DNA sequence, from a library of DNA sequences, that
encodes an
unidentified protein ("prey" or "sample") is fused to a gene that codes for
the activation
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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 the NOVX
sequences
of SEQ ID N0:2n-1, wherein n is an integer between 1 and 38, or fragments or
derivatives
thereof, can be used to map the location of the NOVX genes, respectively, on a
chromosome. The mapping of the NOVX sequences to chromosomes is an important
first
step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the NOVX sequences. Computer analysis of
the
NOVX; sequences can be used to rapidly select primers that do not span more
than one
exon in the genomic DNA, thus complicating the amplification process. These
primers can
then be used for PCR screening of somatic cell hybrids containing individual
human
chromosomes. Only those hybrids containing the human gene corresponding to the
NOVX
sequences will yield an amplified fragment.
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Somatic cell hybrids are prepared by fusing somatic cells from different
mammals
(e.g., human and mouse cells). As hybrids of human and mouse cells grow and
divide, they
gradually lose human chromosomes in random order, but retain the mouse
chromosomes:
By using media in which mouse cells cannot grow, because they lack a
particular enzyme,
but in which human cells can, the one human chromosome that contains the gene
encoding
the needed enzyme will be retained. By using various media, panels of hybrid
cell lines
can be established. Each cell line in a panel contains either a single human
chromosome or
a small number of human chromosomes, and a full set of mouse chromosomes,
allowing
easy mapping of individual genes to specific human chromosomes. See, e.g.,
D'Eustachio,
et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only
fragments of
human chromosomes can also be produced by using human chromosomes with
translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
sequence to a particular chromosome. Three or more sequences can be assigned
per day
using a single thermal cycler. Using the NOVX sequences to design
oligonucleotide
primers, sub-localization can be achieved with panels of fragments from
specific
chromosomes.
Fluorescence irZ situ hybridization (FTSH) 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 Giernsa. A pattern
of light and
dark bands develops on each chromosome, so that the chromosomes can be
identified
individually. The FISH technique can be used with a DNA sequence as short as
500 or 600
bases. However, clones larger than 1,000 bases have a higher likelihood of
binding to a
unique chromosomal location with sufficient signal intensity for simple
detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get
good results at
a reasonable amount of time. For a review of this technique, see, Verma, et
al., H
CHROMOSOMES: A M~1~ of BAStc'I~c~QuES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding .regions of the genes actually are preferred for mapping purposes.
Coding
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sequences are more likely to be conserved within gene families, thus
increasing the chance
of cross hybridizations during chromosomal mapping.
Once a sequence has been~mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such
_data are found, e.g., in McKusick, MENDELIAN lNHERTTANCE.IN M.~1, available
on-line
through Johns Hopkins University Welch Medical Library). The relationship
between
genes and disease, mapped to theaame chromosomal region, can then be
identified through
linkage analysis (co-inheritance of physically adjacent.genes), described in,
e.g., Egeland,
et al., 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the NOVX gene, can be determined. If
a
mutation is observed in some or all of the affected individuals but not in any
unaffected
.individuals, then the mutation is likely to be the causative agent of the
particular disease.
Comparison of affected and unaffected individuals generally involves first
looking for
structural alterations in the chromosomes, such as deletions.or translocations
that are
visible from chromosome spreads or detectable using PCR'based on that DNA
sequence.
Ultimately, complete sequencing of genes from several individuals can be
performed to
confirm the presence of a mutation and to distinguish mutations from
polymorphisms.
Tissue Typing
The NOVX sequences of the invention can also be used to identify individuals
from
minute biological samples. In this technique, an individual's genomic DNA is
digested
with 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
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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
' 5 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 N0:2n-1, wherein n is an integer between l and 38, are
used, a
more appropriate number of primers for positive individual identification
would be
500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which
diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring clinical trials
are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically.
Accordingly, one aspect of the invention relates to diagnostic assays for
determining
NOVX protein and/or nucleic acid expression as well as NOVX activity, in the
context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby determine
whether an
individual is afflicted with a disease or disorder, or is at risk of
developing a disorder,
associated with aberrant NOVX expression or activity. The disorders include
metabolic
disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated
cachexia,
cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's
Disorder, immune
disorders, and hematopoietic disorders, and the various dyslipidemias,
metabolic
disturbances associated with obesity, the metabolic syndrome X and wasting
disorders
associated with chronic diseases and various cancers. The invention also
provides for
prognostic (or predictive) assays fox determining whether an individual is at
risk of
developing a disorder associated with NOVX protein, nucleic acid expression or
activity.
For example, mutations in a NOVX gene can be assayed in a biological sample.
Such
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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.
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., mIRNA, genomic DNA) that encodes NOVX protein such that
the
presence of NOVX is detected in the biological sample. An agent for detecting
NOVX
mlRlVA or genomic DNA is a labeled nucleic acid probe capable of hybridizing
to NOVX
mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length
NOVX
nucleic acid, such as the nucleic acid of SEQ ID N0:2n-1, wherein n is an
integer between
1 and 38, 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
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labeling include detection of a primary antibody using a fluorescently-labeled
secondary
antibody and end-labeling of a DNA probe with biotin such that it can be
detected with
fluorescently-labeled streptavidin. The term "biological sample" is intended
to include
. tissues, cells and biological fluids isolated from a subject, as well as
tissues, cells and fluids
present within a subject. That is, the detection method of the invention can
be used to
detect NOVX mRNA, protein, or genomic DNA in a biological sample in.vitro as
well as
in vivo. For example, in vitro techniques for detection'of NOVX mRNA include
Northern
hybridizations and in situ hybridizations. In vitro techniques for detection
of NOVX
protein include enzyme linked immunosorbent.assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence. In vitro techniques for
detection of
NOVX genomic DNA include Southern hybridizations: Furthermore, in vivo
techniques
for detection of NOVX protein include introducing into a subject a labeled
anti-NOVX
antibody. For example, the antibody can be labeled with a radioactive marker
whose
presence and location in a subject can be detected by standard imaging
techniques.
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.
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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"
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
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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, (vat 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.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in
a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
4,683,202),
such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain
reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et
al., 1994.
Proc. Natl. Acad. Sci. LISA 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. Acad. Sci. USA 87: 1874-1878),
transcriptional
amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177);
Q~3 Replicase (see, Lizardi, et al, 1988. BioTeclanology 6: 1197), or any
other nucleic acid
amplification method, followed by the detection of the amplified molecules
using
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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
' S identified by alterations in restriction enzyme cleavage patterns. For
example, sample and
control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and
compared. Differences in fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover; the use of sequence specific
ribozymes
(see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence
of specific
mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by
hybridizing
a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays
containing
hundreds or thousands of oligonucleotides probes: See, e.g., Cronin, et al.,
1996. Hunaan
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example,
genetic
mutations in NOVX can be identified in two dimensional arrays containing light-
generated
DNA probes as described in Cronin, et al., supra. Briefly, a first
hybridization array of
probes can be used to scan through long stretches of DNA in a sample and
control to
identify base changes between the sequences by making linear arrays of
sequential
overlapping probes. This step allows the identification of point mutations.
This is
followed by a second hybridization array that allows the characterization of
specific
mutations by using smaller, specialized probe arrays complementary to all
variants or
:- mutations detected. Each mutation array is composed of parallel probe sets,
one
complementary to the wild-type gene and the other complementary to the mutant
gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the
art can be used to directly sequence the NOVX gene and detect mutations by
comparing the
sequence of the sample NOVX with the corresponding wild-type (control)
sequence.
Examples of sequencing reactions include those based on techniques developed
by Maxim
and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc.
Natl. Acad.
Sci. USA 74: 5463. It is also contemplated that any of a variety of automated
sequencing
procedures can be utilized when performing the diagnostic assays (see, e.g.,
Naeve, et al.,
1995. Bioteclaniques 19: 448), including sequencing by mass spectrometry (see,
e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al.,1993. Appl. Biochem. Biotechnol. 38: 147-159).
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Other methods for detecting mutations in the NOVX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNAIRNA
or
RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In
general,
the art technique of "mismatch cleavage" starts by providing heteroduplexes of
formed by
hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded .
duplexes are treated with an agent that cleaves single-stranded regions of the
duplex such
as which will exist due to basepair mismatches between the control and sample
strands.
For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with S1 nuclease to~enzymatically digesting the mismatched regions. In
other
embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to digest
mismatched
regions. After digestion of the mismatched regions, the resulting material is
then separated
by size on denaturing polyacrylamide gels to determine the site of mutation.
See, e.g.;
Cotton, et al., 1988. Proc. IVatl. 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.
Genet. Anal.
Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX
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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.,199.1. 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
gradient gel electrophoresis (DGGE). See, eg:, Myers, et al., 1985. Nature
313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure
that it does
not completely denature, for example by adding a GC clamp of approximately 40
by of
high-melting GC-rich DNA by PCR. In a further embodiment, a temperature
gradient is
used in place of a denaturing gradient to identify differences in the mobility
of control and
sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265:
12753.
Examples of other techniques for detecting point mutations include, but are
not
limited to, selective oligonucleotide hybridization, selective amplification,
or selective
primer extension. For example, oligonucleotide primers may be prepared in
which the
known mutation is placed centrally and then hybridized to target DNA under
conditions
that permit hybridization only if a perfect match is found. See, e.g., Saiki,
et al., 1986.
Nature 324: 163; Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230.
Such allele
specific oligonucleotides are hybridized to PCR amplified target DNA or a
number of
different mutations when the oligonucleotides are attached to the hybridizing
membrane
and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on
selective
PCR amplification may be used in conjunction with the instant invention.
Oligonucleotides
used as'primers for specific amplification may carry the mutation of interest
in the center of
the molecule (so that amplification depends on differential hybridization;
see, e.g., Gibbs,
et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of
one primer
where, under appropriate conditions, mismatch can prevent, or reduce
polymerase
extension (see, e.g., Prossner,1993. Tibtech. 11: 238). In addition it may be
desirable to
introduce a novel .restriction site in the region of the mutation to create
cleavage-based
detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is
anticipated that in
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certain embodiments amplification may also be performed using Taq ligase for
amplification. See, e.g., Barany, 199.1. Proc. Natl. Acad. Sci. USA 88: 189.
In such cases,
ligation will occur only if there is a perfect match at the 3'-terminus of the
5' sequence,
making it possible to detect the presence of a known mutation at a specific
site by looking
for the presence or absence of amplification..
The methods described herein may be performed, for example, by utilizing . .
pre-packaged diagnostic kits comprising at least one probe nucleic acid or
antibody.reagent
described herein, which may be conveniently used, e.g:, in clinical settings
to diagnose
patients exhibiting symptoms or family history of a disease or illness
involving a NOVX
gene.
Furthermore, any cell type or tissue, preferably.peripheral blood
leukocytes,~.in
which NOVX is expressed may be utilized in the prognostic assays described
herein.
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 but are not limited to, e.g., those diseases, disorders and
conditions listed
above, and more particularly include those diseases, disorders, or conditions
associated
with homologs of a NOVX protein, such as those summarized in Table A.
In conjunction with such treatment, the pharmacogenornics (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.
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Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected persons.
See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985;
Linder, 1997.
Clin. Chem., 43: 254-266. In general, wo types of ~pharmacogenetic conditions
can be
differentiated. Genetic conditions transmitted as a single factor altering the
way drugs act
on the body (altered drug action) or genetic conditions transmitted as single
factors altering
the way the body acts on drugs (altered drug metabolism). These
pharmacogenetic
conditions can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited
enzymopathy in which the main clinical complication is hemolysis after
ingestion of
oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of
fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
determinant of both the intensity and duration of drug action. The discovery
of genetic
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 metabolizes (EM) and poor metabolizes (PM). The prevalence of PM is
different
among different populations. . For example, the gene coding for CYP2D6 is
highly
polymorphic and several mutations have been identified in PM, which all lead
to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite
frequently experience exaggerated drug response and side effects when they
receive
standard doses. If a metabolite is the active therapeutic moiety, PM show no
therapeutic
response, as demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. At the other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
Thus, the activity of 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
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phenotype. This knowledge, when applied to dosing or drug selection, can avoid
adverse
reactions or therapeutic failure and thus enhance therapeutic or prophylactic
efficiency
when treating a subject with a NOVX modulator, such as a modulator identified
by one of
the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity 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,
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peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified by
the screening assays described herein) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of the agent;
(ii) detecting
the level of expression of a NOVX protein, mRNA, or genomic DNA in the
preadministration sample; (iii) obtaining one or more post-administration
samples from, the
subject; (iv) detecting the level of expression or activity of the NOVX
protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the level of
expression or
activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration
sample
with the NOVX protein, mRNA, or genomic DNA in the post administration sample
or
samples; and (vi) altering the administration of the agent to the subject
accordingly. For
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 but are not limited to,
e.g., those
diseases, disorders and conditions listed above, and more particularly include
those
diseases, disorders, or conditions associated with homologs of a NOVX protein,
such as
those summarized in Table A.
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
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
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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., frorn biopsy tissue) and
assaying it irz 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.) andlor hybridization assays to detect expression of
mRNAs
(e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a
disease
or condition associated with an aberrant NOVX expression or activity, by
administering to
the subject an agent that modulates NOVX expression or at least one NOVX
activity.
Subjects at risk for a disease that is caused or contributed to by aberrant
NOVX expression
or activity can be identified by, for example, any or a combination of
diagnostic or
prognostic assays as described herein. Administration of a prophylactic agent
can occur
prior to the manifestation of symptoms characteristic of the NOVX aberrancy,
such that a
disease or disorder is prevented or, alternatively, delayed in its
progression. Depending
upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX
antagonist
agent can be used for treating the subject. The appropriate agent can be
determined based
on screening assays described herein. The prophylactic methods of the
invention are
further discussed in the following subsections.
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Therapeutic Methods
Another aspect of the invention pertains to methods of modulating NOVX
expression or activity for therapeutic purposes. The modulatory method of the
invention
involves contacting a cell with an agent that modulates one or more of the
activities of
NOVX protein activity associated with the cell. An agent that modulates NOVX
protein
activity can be an agent as described herein, such as a nucleic acid or a
protein, a
naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX
peptidomimetic, or other small molecule. In one embodiment, the agent
stimulates one or
more NOVX protein activity. Examples of such stimulatory agents include active
NOVX
protein and a nucleic acid molecule encoding NOVX that has been introduced
into the cell.
In another embodiment, the agent inhibits one or more NOVX protein activity.
Examples
of such inhibitory agents include antisense NOVX nucleic acid molecules and
anti-NOVX
antibodies. These modulatory methods can be performed in vitro (e.g., by
culturing the cell
with the agent) or, alternatively, ifa 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 ifa situations in which NOVX is
abnormally downregulated and/or in which increased NOVX activity is likely to
have a
beneficial effect. One example of such a situation is where a subject has a
disorder
characterized by aberrant cell proliferation and/or differentiation (e.g.,
cancer or immune
associated disorders). Another example of such a situation is where the
subject has a
gestational disease (e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays
are
performed to determine the effect of a specific Therapeutic and whether its
administration
is indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with
representative cells of the types) involved in the patient's disorder, to
determine if a given
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Therapeutic exerts the desired effect upon the cell type(s). Compounds for use
in thexapy
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. The
disorders include but are not limited to, e.g., those diseases, disorders and
conditions listed
above, and more particularly include those diseases, disorders, or conditions
associated
with homologs of a NOVX protein, such as those summarized in Table A.
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 diseases, disorders,
conditions and the like,
including but not limited to those listed herein.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of
the invention, or fragments thereof, may also be useful in diagnostic
applications, wherein
the presence or amount of the nucleic acid or the protein are to be assessed.
A further use
could be as an anti-bacterial molecule (i.e., some peptides have been found to
possess
anti-bacterial properties). These materials are further useful in the
generation of antibodies,
which immunospecifically-bind to the novel substances of the invention for use
in
therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do
not
limit the scope of the invention described in the claims.
EXAMPLES
Example A: Polynucleotide and Polypeptide Sequences, and Homology Data
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Example 1. NOV1, CG121992, CHORDIN
The NOVl clone was analyzed, and the nucleotide and encoded polypeptide
sequences are shown in Table IA.
Table 1A. NO_ V_1 Sequence Analysis _~
NOVla, CG121992-03~ SEQ >D NO: 1 .~_ 3628 by
DNA Sequence ORF Start: ATG at 247 ORF Stop: TAG at 3193
CCCGGGTCAGCGCCCGCCCGCCCGCGCTCCTCCCGGCCGCTCCTCCCGCCCCGCCCGGCCCGGCGCCG
ACTCTGCGGCCGCCCGACGAGCCCCTCGCGGCACTGCCCCGGCCCCGGCCCCGGCCCCGGCCCCCTCC
CGCCGCACCGCCCCCGGCCCGGCCCTCCGCCCTCCGCACTCCCGCCTCCCTCCCTCCGCCCGCTCCCG
CGCCCTCCTCCCTCCCTCCTCCCCAGCTGTCCCGTTCGCGTCATGCCGAGCCTCCCGGCCCCGCCGGC
CCCGCTGCTGCTCCTCGGGCTGCTGCTGCTCGGCTCCCGGCCGGCCCGCGGCGCCGGCCCCGAGCCCC
CCGTGCTGCCCATCCGTTCTGAGAAGGAGCCGCTGCCCGTTCGGGGAGCGGCAGGCTGCACCTTCGGC
GGGAAGGTCTATGCCTTGGACGAGACGTGGCACCCGGACCTAGGGGAGCCATTCGGGGTGATGCGCTG
CGTGCTGTGCGCCTGCGAGGCGCCTCAGTGGGGTCGCCGTACCAGGGGCCCTGGCAGGGTCAGCTGCA
AGAACATCAAACCAGAGTGCCCAACCCCGGCCTGTGGGCAGCCGCGCCAGCTGCCGGGACACTGCTGC
CAGACCTGCCCCCAGGAGCGCAGCAGTTCGGAGCGGCAGCCGAGCGGCCTGTCCTTCGAGTATCCGCG
GGACCCCGAGCATCGCAGTTATAGCGACCGCGGGGAGCCAGGCGCTGAGGAGCGGGCCCGTGGTGACG
GCCACACGGACTTCGTGGCGCTGCTGACAGGGCCGAGGTCGCAGGCGGTGGCACGAGCCCGAGTCTCG
CTGCTGCGCTCTAGCCTCCGCTTCTCTATCTCCTACAGGCGGCTGGACCGCCCTACCAGGATCCGCTT
CTCAGACTCCAATGGCAGTGTCCTGTTTGAGCACCCTGCAGCCCCCACCCAAGATGGCCTGGTCTGTG
GGGTGTGGCGGGCAGTGCCTCGGTTGTCTCTGCGGCTCCTTAGGGCAGAACAGCTGCATGTGGCACTT
GTGACACTCACTCACCCTTCAGGGGAGGTCTGGGGGCCTCTCATCCGGCACCGGGCCCTGGCTGCAGA
GACCTTCAGTGCCATCCTGACTCTAGAAGGCCCCCCACAGCAGGGCGTAGGGGGCATCACCCTGCTCA
CTCTCAGTGACACAGAGGACTCCTTGCATTTTTTGCTGCTCTTCCGAGGGCTGCTGGAACCCAGGAGT
GGGGGTAAGTGGGATGGGGGCAAAACACGTGAGAAGGTTAGGGAGAGCACCTGTCTCAGAAAGGCCCA
CATGTGCGGCCTTGCAGGACTAACCCAGGTTCCCTTGAGGCTCCAGATTCTACACCAGGGGCAGCTAC
TGCGAGAACTTCAGGCCAATGTCTCAGCCCAGGAACCAGGCTTTGCTGAGGTGCTGCCCAACCTGACA
GTCCAGGAGATGGACTGGCTGGTGCTGGGGGAGCTGCAGATGGCCCTGGAGTGGGCAGGCAGGCCAGG
GCTGCGCATCAGTGGACACATTGCTGCCAGGAAGAGCTGCGACGTCCTGCAAAGTGTCCTTTGTGGGG
CTGATGCCCTGATCCCAGTCCAGACGGGTGCTGCCGGCTCAGCCAGCCTCACGCTGCTAGGAAATGGC
TCCCTGATCTATCAGGTGCAAGTGGTAGGGACAAGCAGTGAGGTGGTGGCCATGACACTGGAGACCAA
GCCTCAGCGGAGGGATCAGCGCACTGTCCTGTGCCACATGGCTGGACTCCAGCCAGGAGGACACACGG
CCGTGGGTATCTGCCCTGGGCTGGGTGCCCGAGGGGCTCATATGCTGCTGCAGAATGAGCTCTTCCTG
AACGTGGGCACCAAGGACTTCCCAGACGGAGAGCTTCGGGGGCACGTGGCTGCCCTGCCCTACTGTGG
GCATAGCGCCCGCCATGACACGCTGCCCGTGCCCCTAGCAGGAGCCCTGGTGCTACCCCCTGTGAAGA
GCCAAGCAGCAGGGCACGCCTGGCTTTCCTTGGATACCCACTGTCACCTGCACTATGAAGTGCTGCTG
GCTGGGCTTGGTGGCTCAGAACAAGGCACTGTCACTGCCCACCTCCTTGGGCCTCCTGGAACGCCAGG
GCCTCGGCGGCTGCTGAAGGGATTCTATGGCTCAGAGGCCCAGGGTGTGGTGAAGGACCTGGAGCCGG
AACTGCTGCGGCACCTGGCAAAAGGCATGGCCTCCCTGATGATCACCACCAAGGGTAGCCCCAGAGGG
GAGCTCCGAGGGCAGGTGCACATAGCCAACCAATGTGAGGTTGGCGGACTGCGCCTGGAGGCGGCCGG
GGCCGAGGGGGTGCGGGCGCTGGGGGCTCCGGATACAGCCTCTGCTGCGCCGCCTGTGGTGCCTGGTC
TCCCGGCCCTAGCGCCCGCCAAACCTGGTGGTCCTGGGCGGCCCCGAGACCCCAACACATGCTTCTTC
GAGGGGCAGCAGCGCCCCCACGGGGCTCGCTGGGCGCCCAACTACGACCCGCTCTGCTCACTCTGCAC
CTGCCAGAGACGAACGGTGATCTGTGACCCGGTGGTGTGCCCACCGCCCAGCTGCCCACACCCGGTGC
AGGCTCCCGACCAGTGCTGCCCTGTTTGCCCTGAGAAACAAGATGTCAGAGACTTGCCAGGGCTGCCA
AGGAGCCGGGACCCAGGAGAGGGCTGCTATTTTGATGGTGACCGGAGCTGGCGGGCAGCGGGTACGCG
GTGGCACCCCGTTGTGCCCCCCTTTGGCTTAATTAAGTGTGCTGTCTGCACCTGCAAGGGGGGCACTG
GAGAGGTGCACTGTGAGAAGGTGCAGTGTCCCCGGCTGGCCTGTGCCCAGCCTGTGCGTGTCAACCCC
ACCGACTGCTGCAAACAGTGTCCAGTGGGGTCGGGGGCCCACCCCCAGCTGGGGGACCCCATGCAGGC
TGATGGGCCCCGGGGCTGCCGTTTTGCTGGGCAGTGGTTCCCAGAGAGTCAGAGCTGGCACCCCTCAG
TGCCCCCTTTTGGAGAGATGAGCTGTATCACCTGCAGATGTGGGGCAGGGGTGCCTCACTGTGAGCGG
GATGACTGTTCACTGCCACTGTCCTGTGGCTCGGGGAAGGAGAGTCGATGCTGTTCCCGCTGCACGGC
CCACCGGCGGCCAGCCCCAGAGACCAGAACTGATCCAGAGCTGGAGAAAGAAGCCGAAGGCTCTTAGG
GAGCAGCCAGAGGGCCAAGTGACCAAGAGGATGGGGCCTGAGCTGGGGAAGGGGTGGCATCGAGGACC
TTCTTGCATTCTCCTGTGGGAAGCCCAGTGCCTTTGCTCCTCTGTCCTGCCTCTACTCCCACCCCCAC
TACCTCTGGGAACCACAGCTCCACAAGGGGGAGAGGCAGCTGGGCCAGACCGAGGTCACAGCCACTCC
AAGTCCTGCCCTGCCACCCTCGGCCTCTGTCCTGGAAGCCCCACCCCTTTCCTCCTGTACATAATGTC
ACTGGCTTGTTGGGATTTTTAATTTATCTTCACTCAGCACCAAGGGCCCCCGACACTCCACTCCTGCT
GCCCCTGAGCTGAGCAGAGTCATTATTGGAGAGTTTTGTATTTATTAAAACATTTCTTTTTCAGTCAA
AAAAAAAAAAAAAAAAAAAAAAAA
NOVla, CG121992-03 SEQ >D NO: 2 982 as MW at 105031.2kD
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Protein Sequence ~ ~ _ _
MPSLPAPPAPLLLLGLLLLGSRPARGAGPEPPVLPIRSEKEPLPVRGAAGCTFGGKVYALDETWHPDL
'GEPFGVMRCVLCACEAPQWGRRTRGPGRVSCKNIKPECPTPACGQPRQLPGHCCQTCPQERSSSERQP
',SGLSFEYPRDPEHRSYSDRGEPGAEERARGDGHTDFVALLTGPRSQAVAR.ARVSLLRSSLRFSISYRR
LDRPTRIRFSDSNGSVLFEHPAAPTQDGLVCGVWRAVPRLSLRLLRAEQLHVALVTLTHPSGEVWGPL
IRHRALAAETFSAILTLEGPPQQGVGGITLLTLSDTEDSLHFLLLFRGLLEPRSGGKWDGGKTREKVR
ESTCLRKAHMCGLAGLTQVPLRLQILHQGQLLRELQANVSAQEPGFAEVLPNLTVQEMDWLVLGELQM
ALEWAGRPGLRISGHIAARKSCDVLQSVLCGADALIPVQTGAAGSASLTLLGNGSLIYQVQWGTSSE
WAMTLETKPQRRDQRTVLCHMAGLQPGGHTAVGICPGLGARGAHMLLQNELFLNVGTKDFPDGELRG
HVAALPYCGHSARHDTLPVPLAGALVLPPVKSQAAGHAWLSLDTHCHLHYEVLLAGLGGSEQGTVTAH
LLGPPGTPGPRRLLKGFYGSEAQGWKDLEPELLRHLAKGMASLMITTKGSPRGELRGQVHIANQCEV
GGLRLEAAGAEGVRALGAPDTASAAPPWPGLPALAPAKPGGPGRPRDPNTCFFEGQQRPHGARWAPN
YDPLCSLCTCQRRWICDPWCPPPSCPHPVQAPDQCCPVCPEKQDVRDLPGLPRSRDPGEGCYFDGD
RSWRAAGTRWHPWPPFGLIKCAVCTCKGGTGEVHCEKVQCPRLACAQPVRVNPTDCCKQCPVGSGAH
PQLGDPMQADGPRGCRFAGQWFPESQSWHPSVPPFGEMSCITCRCGAGVPHCERDDCSLPLSCGSGKE
SRCCSRCTAHRRPAPETRTDPELEKEAEGS
NOVlb, CG121992-02 SEQ m NO: 3 2829 by __
DNA Sequence ORF Start: ATG at 40 ORF Stop: TGA at 2410
CCTCCTCCCTCCCTCCTCCCCAGCTGTCCCGTTCGCGTCATGCCGAGCCTCCCGGCCCCGCCGGCCCC
GCTGCTGCTCCTCGGGCTGCTGCTGCTCGGCTCCCGGCCGGCCCGCGGCGCCGGCCCCGAGCCCCCCG
TGCTGCCCATCCGTTCTGAGAAGGAGCCGCTGCCCGTTCGGGGAGCGGCAGGCTGCACCTTCGGCGGG
AAGGTCTATGCCTTGGACGAGACGTGGCACCCGGACCTAGGGGAGCCATTCGGGGTGATGCGCTGCGT
GCTGTGCGCCTGCGAGGCGCCTCAGTGGGGTCGCCGTACCAGGGGCCCTGGCAGGGTCAGCTGCAAGA
ACATCAAACCAGAGTGCCCAACCCCGGCCTGTGGGCAGCCGCGCCAGCTGCCGGGACACTGCTGCCAG
ACCTGCCCCCAGGAGCGCAGCAGTTCGGAGCGGCAGCCGAGCGGCCTGTCCTTCGAGTATCCGCGGGA
CCCGGAGCATCGCAGTTATAGCGACCGCGGGGAGCCAGGCGCTGAGGAGCGGGCCCGTGGTGACGGCC
ACACGGACTTCGTGGCGCTGCTGACAGGGCCGAGGTCGCAGGCGGTGGCACGAGCCCGAGTCTCGCTG
CTGCGCTCTAGCCTCCGCTTCTCTATCTCCTACAGGCGGCTGGACCGCCCTACCAGGATCCGCTTCTC
AGACTCCAATGGCAGTGTCCTGTTTGAGCACCCTGCAGCCCCCACCCAAGATGGCCTGGTCTGTGGGG
TGTGGCGGGCAGTGCCTCGGTTGTCTCTGCGGCTCCTTAGGGCAGAACAGCTGCATGTGGCACTTGTG
ACACTCACTCACCCTTCAGGGGAGGTCTGGGGGCCTCTCATCCGGCACCGGGCCCTGGCTGCAGAGAC
CTTCAGTGCCATCCTGACTCTAGAAGGCCCCCCACAGCAGGGCGTAGGGGGCATCACCCTGCTCACTC
TCAGTGACACAGAGGACTCCTTGCATTTTTTGCTGCTCTTCCGAGGGCTGCTGGAACCCAGGAGTGGG
GGACTAACCCAGGTTCCCTTGAGGCTCCAGATTCTACACCAGGGGCAGCTACTGCGAGAACTTCAGGC
CAATGTCTCAGCCCAGGAACCAGGCTTTGCTGAGGTGCTGCCCAACCTGACAGTCCAGGAGATGGACT
GGCTGGTGCTGGGGGAGCTGCAGATGGCCCTGGAGTGGGCAGGCAGGCCAGGGCTGCGCATCAGTGGA
CACATTGCTGCCAGGAAGAGCTGCGACGTCCTGCAAAGTGTCCTTTGTGGGGCTGATGCCCTGATCCC
AGTCCAGACGGGTGCTGCCGGCTCAGCCAGCCTCACGCTGCTAGGAAATGGCTCCCTGATCTATCAGG
TGCAAGTGGTAGGGACAAGCAGTGAGGTGGTGGCCATGACACTGGAGACCAAGCCTCAGCGGAGGGAT
CAGCGCACTGTCCTGTGCCACATGGCTGGACTCCAGCCAGGAGGACACACGGCCGTGGGTATCTGCCC
TGGGCTGGGTGCCCGAGGGGCTCATATGCTGCTGCAGAATGAGCTCTTCCTGAATGTGGGCACCAAGG
ACTTCCCAGACGGAGAGCTTCGGGGGCACGTGGCTGCCCTGCCCTACTGTGGGCATAGCGCCCGCCAT
GACACGCTGCCCGTGCCCCTAGCAGGAGCCCTGGTGCTACCCCCTGTGAAGAGCCAAGCAGCAGGGCA
CGCCTGGCTTTCCTTGGATACCCACTGTCACCTGCACTATGAAGTGCTGCTGGCTGGGCTTGGTGGCT
CAGAACAAGGCACTGTCACTGCCCACCTCCTTGGGCCTCCTGGAACGCCAGGGCCTCGGCGGCTGCTG
AAGGGATTCTATGGCTCAGAGGCCCAGGGTGTGGTGAAGGACCTGGAGCCGGAACTGCTGCGGCACCT
GGCAAAAGGCATGGCCTCCCTGCTGATCACCACCAAGGGTAGCCCCAGAGGGGAGCTCCGAGGGCAGG
TGCACATAGCCAACCAATGTGAGGTTGGCGGACTGCGCCTGGAGGCGGCCGGGGCCGAGGGGGTGCGG
GCGCTGGGGGCTCCGGATACAGCCTCTGCTGCGCCGCCTGTGGTGCCTGGTCTCCCGGCCCTAGCGCC
CGCCAAACCTGGTGGTCCTGGGCGGCCCCGAGACCCCAACACATGCTTCTTCGAGGGGCAGCAGCGCC
CCCACGGGGCTCGCTGGGCGCCCAACTACGACCCGCTCTGCTCACTCTGCACCTGCCAGAGACGAACG
GTGATCTGTGACCCGGTGGTGTGCCCACCGCCCAGCTGCCCACACCCGGTGCAGGCTCCCGACCAGTG
CTGCCCTGTTTGCCCTGAGAAACAAGATGTCAGAGACTTGCCAGGGCTGCCAAGGAGCCGGGACCCAG
GAGAGGGGGGGCACTGGAGAGGTGCACTGTGAGAAGGTGCAGTGTCCCCGGCTGGCCTGTGCCCAGCC
TGTGCGTGTCAACCCCACCGACTGCTGCAAACAGTGTCCAGTGGGGTCGGGGGCCCACCCCCAGCTGG
GGGACCCCATGCAGGCTGATGGGCCCCGGGGCTGCCGTTTTGCTGGGCAGTGGTTCCCAGAGAGTCAG
AGCTGGCACCCCTCAGTGCCCCCGTTTGGAGAGATGAGCTGTATCACCTGCAGATGTGGGGCAGGGGT
GCCTCACTGTGAGCGGGATGACTGTTCACTGCCACTGTCCTGTGGCTCGGGGAAGGAGAGTCGATGCT
GTTCCCGCTGCACGGCCCACCGGCGGCCAGCCCCAGAGACCAGAACTGATCCAGAGCTGGAGAAAGAA
GCCGAAGGCTCTTAGGGAGCAGCCAGAGGGCCAAGTGACCA
NOVlb, CG121992-02 'SEQ m NO: 4 790 as MW at 84215.7kD
Protein Sequence
MPSLPAPPAPLLLLGLLLLGSRPARGAGPEPPVLPIRSEKEPLPVRGAAGCTFGGKVYALDETWHPDL
GEPFGVMRCVLCACEAPQWGRRTRGPGRVSCKNIKPECPTPACGQPRQLPGHCCQTCPQERSSSERQP
SGLSFEYPRDPEHRSYSDRGEPGAEERARGDGHTDFVALLTGPRSOAVARARVSLLRSSLRFSISYRR
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IRHRALAAETFSAILTLEGPPQQGVGGITLLTLSDTEDSLHFLLLFRGLLEPRSGGLTQVPLRLQI
QGQLLRELQANVSAQEPGFAEVLPNLTVQEMDWLVLGELQMALEWAGRPGLRISGHIAARKSCDVL
VLCGADALIPVQTGAAGSASLTLLGNGSLIYQVQWGTSSEWAMTLETKPQRRDQRTVLCHMAGL
GGHTAVGICPGLGARGAHMLLQNELFLNVGTKDFPDGELRGHVAALPYCGHSARHDTLPVPLAGAL
PALAPAKPGGPGRPRDPNTCFFEGQQRPHGARWAPNYDPLCSLCTCQRRTVICDPWC
APDOCCPVCPEKODVRDLPGLPRSRDPC~ECC~HwRC~AT,
1c, CG121992-04 SEQ >D NO: 5 2319 by
Sequence ORF Start: at 1 a ORF Ston: at end of
GGACAAGCAGTGAGGTGGTGGCCATGACACTGGAGACCAAGCCTCAGCGGAGGGATCAGCGCACTGTC
CACCTCCTTGGGCCTCCTGGAACGCCAGGGCCTCGGC
T
ACGACCCGCTCTGCTCACTCTGCACCTGCCAGAGACGAACGGTGA
1c, CG121992-04 ~SEQ >D NO: 6 X773 as BMW at 82722.9kD
VKUAE1G(:'1'FGGKVYALDETWHPDLGEPFGVMRCVLCACEAPQWGRRTRGPGRVSCKNIKPEC
QPRQLPGHCCQTCPQERSSSERQPSGLSFEYPRDPEHRSYSDRGEPGAEERARGDGHTDFVA
SQAVARARVSLLRSSLRFSISYRRLDRPTRIRFSDSNGSVLFEHPAAPTQDGLVCGVWRAVP
LRAEQLHVALVTLTHPSGEVWGPLIRHRALAAETFSAILTLEGPPQQGVGGITLLTLSDTED
LFRGLLEPRSGGKWDGGKTREKVRESTCLRKAHMCGLAGLTQVPLRLQILHQGQLLRELQAN
GFAEVLPNLTVQEMDWLVLGELQMALEWAGRPGLRISGHIAARKSCDVLQSVLCGADALIPV
SASLTLLGNGSLIYQVQWGTSSEWAMTLETKPQRRDQRTVLCHMAGLQPGGHTAVGICPG
H_~~LQNELFLNVGTKDFPDGELRGHVAALPYCGHSARHDTLPVPLAGALVLPPVKSQAAGHA
FICHLHYEVLLAGLGGSEQGTVTAHLLGPPGTPGPRRLLKGFYGSEAQGVVKDLEPELLRHLA
MITTKGSPRGELRGQVHIANQCEVGGLRLEAAGAEGVRALGAPDTASAAPPWPGLPALAPA
RPRDPNTCFFEGQQRPHGARWAPNYDPLCSLCTCQRRTVICDPWCPPPSCPHPVQAPDQCC
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 1B.
Table 1B. Comparison of the NOV1 protein sequences.
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NOVla MPSLPAPPAPLLLLGLLLLGSRPARGAGPEPPVLPIRSEKEPLPVRGAAGCTFGGKWAL
NOVlb MPSLPAPPAPLLLLGLLLLGSRPARGAGPEPPVLPIRSEKEPLPVRGAAGCTFGGKVYAL
NOVlc _____-_-____________________________________~G~GCTFGGKVYAL
NOVla DETWHPDLGEPFGVMRCVLCACE_APQWGRRTRGPGRVSCKNIKPECPTPACGQPRQLPGH
NOVlb DETWHPDLGEPFGVMRCVLCACEAPQWGRRTRGPGRVSCKNIKPECPTPACGQPRQLPGH',
NOVlc DETWHPDLGEPFGVMRCVLCACEAPQWGRRTRGPGRVSCKNIKPECPTPACGQPRQLPGH',
i
NOVI.a CCQTCPQERSSSERQPSGLSFEYPRDPEHRSYSDRGEPGAEERARGDGHTDFVALLTGPR
NOVlb CCQTCPQERSSSERQPSGLSFEYPRDPEHRSYSDRGEPGAEERARGDGHTDFVALLTGPR
NOVlc CCQTCPQERSSSERQPSGLSFEYPRDPEHRSYSDRGEPGAEERARGDGHTDFVALLTGPR
NOVla SQAVARARVSLLRSSLRFSISYRRLDRPTRIRFSDSNGSVLFEHPAAPTQDGLVCGVWRA
NOVlb SQAVARARVSLLRSSLRFSISYRRLDRPTRIRFSDSNGSVLFEHPAAPTQDGLVCGVWRA
NOVlc SQAVARARVSLLRSSLRFSISYRRLDRPTRIRFSDSNGSVLFEHPAAPTQDGLVCGVWRA
NOVla VPRLSLRLLRAEQLHVALVTLTHPSGEVWGPLIRHRALAAETFSAILTLEGPPQQGVGGI
NOVlb VPRLSLRLLRAEQLHVALVTLTfiPSGEVWGPLIRHRALAAETFSAILTLEGPPQQGVGGI
NOVlc VPRLSLRLLRAEQLHVALVTLTHPSGEVWGPLIRHRALAAETFSAILTLEGPPQQGVGGI
NOVla TLLTLSDTEDSLHFLLLFRGLLEPRSGGKWDGGKTREKVRESTCLRKAHMCGLAGLTQVP
NOVlb TLLTLSDTEDSLHFLLLFRGLLEPRSGG---------------------------LTQVP
NOVlc TLLTLSDTED$LHFLLLFRGLLEPRSGGKWDGGKTREKVRESTCLRKAHMCGLAGLTQVP
NOVla LRLQILHQGQLLRELQANVSAQEPGFAEVLPNLTVQEMDWLVLGELQMALEWAGRPGLRI
NOVlb LRLQILHQGQLLRELQANVSAQEPGFAEVLPNLTVQEMDWLVLGELQMALEWAGRPGLRI
NOVlc LRLQILHQGQLLRELQANVSAQEPGFAEVLPNLTVQEMDWLVLGELQMALEWAGRPGLRI
NOVla SGHIAARKSCDVLQSVLCGADALIPVQTGAAGSASLTLLGNGSLIYQVQWGTSSEWAM
NOVlb SGHIAARKSCDVLQSVLCGADALIPVQTGAAGSASLTLLGNGSLIYQVQWGTSSEWAM
NOVlc SGHIAARKSCDVLQSVLCGADALIPVQTGAAGSASLTLLGNGSLIYQVQWGTSSEWAM
NOVla TLETKPQRRDQRTVLCHMAGLQPGGHTAVGICPGLGARGAHMLLQNELFLNVGTKDFPDG
NOVlb TLETKPQRRDQRTVLCHMAGLQPGGHTAVGICPGLGARGAHMLLQNELFLNVGTKDFPDG
NOVlc TLETKPQRRDQRTVLCHMAGLQPGGHTAVGICPGLGARGAHMLLQNELFLNVGTKDFPDG
NOVla ELRGHVAALPYCGHSARHDTLPVPLAGALVLPPVKSQAAGHAWLSLDTHCHLHYEVLLAG
NOVlb ELRGHVAALPYCGHSARHDTLPVPLAGALVLPPVKSQAAGHAWLSLDTHCHLHYEVLLAG
NOV1C ELRGHVAALPYCGHSARHDTLPVPLAGALVLPPVKSQAAGHAWLSLDTHCHLHYEVLLAG
NOVla LGGSEQGTVTAHLLGPPGTPGPRRLLKGFYGSEAQGVVKDLEPELLRHLAKGMASLMITT
NOVlb LGGSEQGTVTAHLLGPPGTPGPRRLLKGFYGSEAQGVVKDLEPELLRHLAKGMASLLITT
NOVlc LGGSEQGTVTAHLLGPPGTPGPRRLLKGFYGSEAQGVVKDLEPELLRHLAKGMASLMITT
NOVla KGSPRGELRGQVHIANQCEVGGLRLEAAGAEGVRALGAPDTASAAPPWPGLPALAPAK
NOVlb KGSPRGELRGQVHIANQCEVGGLRLEAAGAEGVRALGAPDTASAAPPWPGLPALAPAK
NOVlc KGSPRGELRGQVHIANQCEVGGLRLEAAGAEGVRALGAPDTASAAPPWPGLPALAPAK
NOVla PGGPGRPRDPNTCFFEGQQRPHGARWAPNYDPLCSLCTCQRRTVICDPW
NOVlb PGGPGRPRDPNTCFFEGQQRPHGARWAPNYDPLCSLCTCQRRTVICDPW
NOVlc PGGPGRPRDPNTCFFEGQQRPHGARWAPNYDPLCSLCTCQRRTVICDPW
NOVla CPPPSCPHPVQAPDQCCPVCPEKQDVRDLPGLPRSRDPGEGCYFDGDRSWRAAGTRW
NOVlb CPPPSCPHPVQAPDQCCPVCPEKQDVRDLPGLPRSRDPGEG------GHWRGAL---
NOVlc CPPPSCPHPVQAPDQCCPVCPEKQDVRDLPGLPRSRDPGEG------GHWRGAL---
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NOVla HPVVPPFGLIKCAVCTCKGGTGEVHCEKVQCPRLACAQPVRVNPTDCCKQCPVGSGAHPQ
NOVlb _________________ ________________________'__________________
~NOVlc ________________-___________________________________________
NOVla LGDPMQADGPRGCRFAG,QWFPESQSWHPSVPPFGEMSCITCRCGAGVPHCERDDCSLPLS
___________________________________________
NOVlb _______________ .
NOVlc ____________________________________________________________
NOVla CGSGKESRCCSRCTAHRRPAPETRTDPELEKEAEGS
NOVlb ____________________________________
NOVlc ____________________________________
NOVla (SEQ ID NO: 2)
NOVlb (SEQ ID N0: 4)
NOVlc (SEQ ID NO: 6)
NOV 1a, lb have a cleavable signal peptide corresponding to amino acid
residues 1
to 23 of SEQ ID N0:2 and 4.respectively. NOVla mature protein corresponds to
amino
acid residues 24-982 of SEQ ID N0:2. NOVlb mature protein corresponds to amino
acid
residues 24-790 of SEQ ID N0:4. NOV 1 sequences contain von Willebrand factor
type C
domains corresponding to amino acid residues 51-125 and 705-762 of NOVIb, SEQ
ID
NO:4; amino acid residues 51-125, 732-789, 811-877 and 899-959 of NOVla SEQ )D
N0:2; and amino acid residues 7-81 and 688-745 of NOVlc SEQ >D N0:6. NOVla and
NOVlc have a novel insertion at amino acid residues 329-355 of SEQ ID N0:2 and
residues 285-311 of SEQ >D N0:6 respectively.
A search of the NOVla 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.
Table 1D. Geneseq Results for NOVla
NOVIa Identities/
Geneseq Protein/Organism/Length Residues/Similarities for
Expect
Identifier [Patent #, Date] Match the Matched Value
Residues Region
ABG31265 Human chordin (CHRD) protein 1..982 954/982 (97%) 0.0
-
Homo sapiens, 955 aa. 1..955 955/982 (97%)
[W0200254940-A2, 18-JUL-
2002]
AAE12889 Human chordin protein - Homo 1..982 954/982 (97%) 0.0
sapiens, 955 aa. [W0200164885- 1..955 955/982 (97%)
A1, 07-SEP-2001]
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AAW48978 Mature human chordin protein - 1..982 954/982 (97%) 0.0
Homo Sapiens, 954 aa. 1..954 954/982 (97%)
[W09821335-A1, 22-MAY-1998]
In a BLAST search of public sequence databases, the NOVla protein was found to
have homology to the proteins shown in the BLASTP data in Table 1E.
Table 1E. Public BLASTP Results for NOVla
NOVla Identities/
Protein Residues/ Similarities Expect
for
AccessionProtein/Organism/LengthMatch the Matched Value
Number Residues Portion
Q9H2X0 Chordin precursor - 1..982 954/982 (97%)0.0
Homo
sapiens (Human), 955 1..955 955/982 (97%)
aa.
Q9ZOE2 Chordin precursor - 1..982 824/985 (83%)0.0
Mus ,
~
musculus (Mouse), 948 1..948 863/985 (86%)
aa.
057465 Chordin - Gallus gallus11..966 523/985 (53%)0.0
(Chicken), 940 aa. 5..927 651/985 (65%)
Q8N2W7 Hypothetical protein 570..982 413/413 (100%)0.0
- Homo
Sapiens (Human), 413 1..413 413/413 (100%)
as
(fragment).
Q8TEH7 FLJ00220 protein - Homo382..815 430/434 (99%)0.0
sapiens (Human), 503 68..501 432/434 (99%)
as
(fragment).
Chordin
is a
bone
morphogenetic
protein
(BMP)
antagonist.
BMPs
were
originally
identified by an ability of demineralized bone extract to induce endochondral
osteogenesis
in vivo in an extraskeletal site. To date, 15 BMPs have been identified and
all are members
of the transforming growth factor-beta superfamily of secreted signaling
molecules and
regulate tissue differentiation and maintenance. They play roles in
embryogenesis by
binding to specific serine/threonine kinase receptors, which transduce the
signal to the
nucleus. In contrast, there are proteins that antagonize the BMP functions by
specifically
binding to BMPs and preventing their binding to specific receptors or their
signaling.
Chordin can interfere with normal embryogenesis by binding to TGF-beta-
likeBMPs and sequestering them in latent complexes. It has been shown that
BMP1 and
TLL1 counteracted the effects of chordin upon overexpression in Xe'nopus
embryos (Scott
et al. "Mammalian BMP-l/Tolloid-related metalloproteinases, including novel
family
member mammalian Tolloid-like 2, have differential enzymatic activities and
distributions
of expression relevant to patterning and skeletogenesis." Dev. Biol. 213: 283-
300, 1999).
They suggested that BMP1 is the major chordin antagonist in early mammalian
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embryogenesis and in pre- and postnatal skeletogenesis. It also directly binds
BMP-4 and
BMP-2, and interferes with the binding of these proteins to their receptors.
c
Bone metastases are a frequent clinical problem in patients with breast,
prostate,
and other cancers. Formation of these lesions is a site-specific process
determined by
multiple cellular and molecular interactions between the cancer cells and the
bone
microenvironment. BMP has been shown to be one of the significant factors in
the
prognosis of bone tumors. The overexpression of BMP2, BMP4, and BMP6 were
found in
most osteosarcomas or prostate cancers with metastases (Hamdy, F., Autzen, P.,
Robinson,
MC., Wilson Horne, CH., Neal, DE. and Robson CN. "Immunolocalization and
messenger
RNA expression of bone morphogenetic protein-6 in human bening and malignant
prostatic
tissue." Cancer Research 57: 4427-4431, 1997; Guo, W., Gorlick, R., Ladanyi,
M.,
Meyers, PA., Huvos, AG.~ Bertino, JR., and Henley, JH.
"Expression of bone morphogenetic proteins and receptors in sarcomas."
Clinical
Orthopaedics and Related Research 365: 175-183, 1999:) suggesting a close
association
~ between BMPs and skeletal metastases. BMP-2, -4, -6 may be responsible, in
part, for
osteoblastic changes in metastatic lesions secondary to prostate cancer. NOV1
has a role in
the regulation of morphogenesis and cancer development. It is an important
antibody or
protein therapeutic target for the related diseases. -
NOVla has n nucleic acid of 3628 nucleotides (designated CuraGen Acc. No.
CG121992-03) encoding a novel CHORDIN-like splice variant with deletion of
exon 19
causing a frameshift staring from 784 aa. An open reading frame was identified
beginning
at nucleotides 247-249 and ending at nucleotides 3193-3195. This sequence
represents a
splice form of CHORDIN as indicated with 1 amino acid change L630M and
insertion in
frame of 27 amino acids KWDGGKTREKVRESTCLRKAHMCGLAG (SEQ ID 1V0:77).
The encoded protein having 982 amino acid residues contains 2 of 4 repeated
von
Willebrand factor type C domains compared to full length chordin. The von
Willebrand
factor (VWF) type C domain is found in multidomain protein/multifunctional
proteins
involved in maintaining homeostasis. The duplicated VWFC domain participates
in
oligomerization, but not in the initial dimerization step. The presence of
this region in a
number of other complex-forming proteins points to involvment of the VWFC
domain in
complex formation.
The CHORDIN-like genes disclosed in this invention map to chromosome 3. The
PSORT, SignalP results for the CHORDIN-like protein NOVla predict that this
sequence
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has a signal peptide and is likely to be localized extracellularly with a
certainty of 0.5469.
The signal peptide is predicted by SignalP to be cleaved at amino acid between
position 26
and 27: ARG-AG.
Example 2. NOV2, CG186275, ADAM 22
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are shown in Table 2A.
Table 2A. NOV2 Sequence Analysis
OV2a, CG186275-03 SEQ >D NO: 7 2847 by
NA Sequence ORF Start: ATG at 47 ORF Stop: TAA at 2795
TTCA
ATGTTTAAAAAACATCGGCTTTCCGTTGT
AGATTTAATATATAAAGACCAACTTAAGA
ATCTTCCTAAA.AAGTTCACCCAGTGT
TCGTGAAACGTGCTCAGGAAATTCAAGCCAGTGTGCCCCTAAT
.TATTGCTATGAGAAACTGAA
TATTGGCAATATCCCAAGGCTTGGAGAACTCGATGGTGAAA
TATGTGGAAGATGGGACACCTTGTGGTCCCCAAATGATGTGCTTAGAACACAGGTGT
TTCTTTCAACTTTAGTACTTGCTTGAGCAGTAAAGAAGGCACTATTTGCTCAGGAAA
AGACCTCGGTCTAATTCAACTGAGTATTT
TCTCCTGCCAAGTCTCCTTCTTCATCAACTGGGTCTATTGCCTCCAGCAGAAAATACC
OV2a, CG186275-03 ~SEQ ID NO: 8 ,916 as ~MW at 102480.1kD
MQAAVAVSVPFLLLCVLGTCPPARCGQAGDASLMELEKRKENRFVERQSIVPLRLIYRSGGEDES
ALDTRVRGDLGGRQIQMFLKSESQKTIYQIQLTHVDQASFQVDAFGTSFILDVVLNHDLLSSEYI
IEHGGKTVEVKGGEHCYYQGHIRGNPDSFVALSTCHGLHGMFYDGNHTYLIEPEENDTTQEDFHF
YKSRLFEFSLDDLPSEFOOINITPSKFILKPRPKRSKROLRRYPRNVEEETKYIELMIVNDHLMF
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RLSUVIiTNTYAKSWNMADLIYKDQLKTRIVLVAMETWATDNKFAISENPLITLREFMKYRRDFIKEK
SDAVHLFSGSQFESSRSGAAYIGGICSLLKGGGVNEFGKTDLMAVTLAQSLAHNIGIISDKRKLASGE
CKCEDTWSGCIMGDTGYYLPKKFTQCNIEEYHDFLNSGGGACLFNKPSKLLDPPECGNGFIETGEECD
CGTPAECVLEGAECCKKCTLTQDSQCSDGLCCKKCKFQPMGTVCREAVNDCDIRETCSGNSS,QCAPNI
HKMDGYSCDGVQGICFGGRCKTRDRQCKYIWGQKVTASDKYCYEKLNIEGTEKGNCGKDKDTWIQCNK
RDVLCGYLLCTNIGNIPRLGELDGEITSTLWQQGRTLNCSGGHVKLEEDVDLGYVEDGTPCGPQMMC
LEHRCLPVASFNFSTCLSSKEGTICSGNGVCSNELKCVCNRHWIGSDCNTYFPHNDDAKTGITLSGNG
VAGTNIIIGIIAGTILVLALILGITAWGYKNYREQRSNGLSHSWSERIPDTKHISDICENG~tPRSNSW
n~rrr.r_r_,.TUUU-rRnur. FRpR
cnl.~mFVT,WPWFKRDYNVAKWVEDVNKNTEEPYFRTLSPAKSPSSSTGSI
Further analysis of the NOV2a protein yielded the following properties. NOV2a
has a cleavable signal peptide corresponding to amino acid residues 1-25 of
SEQ ID N0:8.
NOV2a has a novel insertions at amino acid residues 81-98 and 841-871 as well
as a
deletion of 36 amino acids between residues 784-784 of SEQ 1D NO: 8.
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 2B.
Table 2B. Geneseq Results
for NOV2a
NOV2a Identities/
Geneseq Protein/Organism/L.ength Residues/Similarities Expect
[Patent for
Identifier#, Date] Match the Matched Value
ResiduesRegion
AAY25119 Human N1DC2-beta protein 1..840 821/840 (97%)0.0
- Homo
sapiens, 823 aa. [JP11155574-A,1..823 822!840 (97%)
15-JUN-1999]
AAY30208 Amino acid sequence of 40..916 830/913 (90%)0.0
3 the human
SVPH3-13 protein - Homo 1..867 831/913 (90%)
sapiens,
867 aa. [W09941388-A2,
19-AUG-
1999]
AAY25118 Human MDC2-alpha protein 1..840 821/876 (93%)0.0
-
Homo sapiens, 859 aa. 1..859 822/876 (93%)
[JP11155574-A, 15-JUN-1999]
AAR75352 Human fetal brain MDC 52..787 407/742 (54%)0.0
protein -
Homo sapiens, 769 aa. 50..768 518/742 (68%)
[EP633268-
A2, 11-JAN-1995]
AAR67759 Human fetal brain MDC 123..787382/670 (57%)0.0
protein -
Homo sapiens, 670 aa. 4..669 485/670 (72%)
[EP633268-
A2, 11-JAN-1995]
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 2C.
Table 2C. Public BLASTP Results for NOV2a
Protein ~ Proteill/Orgariism/Lerigth ~ N()V2a ~ TrlentitiPC/ ~ Fxnect-
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Accession ~ Residues/ Similarities for
Value
Number Match the Matched
Residues Portion
Q9POK1 ADAM 22 precursor (A disintegrin1..916 8681952 (91%) 0.0
and metalloproteinase domain1..906 8691952 (91%)
22)
(Metalloproteinase-like,
disintegrin-
like, and cysteine-rich
protein 2)
(Metalloproteinase-disintegrin
ADAM22-3) = Homo Sapiens
(Human), 906 aa.
Q9R1V6 ADAM 22 precursor (A disintegrin1..840 ~ 751/876 (85%) 0.0
and metalloproteinase domain1..857 783/876 (88%)
22) - ~
Mus musculus (Mouse), 857
aa. E
3
042596 ADAM 22 precursor (A disintegrin13..916 613/970 (63%) .
0.0
and metalloproteinase domain12..935 709/9?0 (72%)
22)
(Metalloprotease-disintegrin
MDCllb) (MDC11.2) - Xenopus
laevis (African clawed frog),
935 aa.
075078 ADAM 11 precursor (A disintegrin52..787 408/742 (54%) i
~ 0.0
and metalloproteinase domain50..768 518/742 (68%)
11)
(Metalloproteinase-like,
disintegrin-
Iike, and cysteine-rich
protein) (MDC)
- Homo sapiens (Human),
769 aa.
Q9R1V4 ; ADAM 11 precursor (A disintegrin52..787 409/743 (55%) 0.0
and metalloproteinase domain54..772 517/743 (69%)
11) t
(Metalloproteinase-like,
disintegrin-
Iike, and cysteine-rich
protein) (MDC) .
- Mus musculus (Mouse),
773 aa.
PFam analysis
predicts that
the NOV2a
protein contains
the domains
shown in the
Table 3F.
Table 2D. Domain Analysis of NOV2a
Identities/
Pfam Domain NOV2a Match Region AminoSimilarities Expect
Acid residues of SEQ for the MatchedValue
ID NO: 8
Region
Pep_M12B_propep120..228 31/119 (26%) 6.3e-17
;
76/119 (64%)
Reprolysin 256..455 69/206 (33%) 1.4e-89
172/206 (83%)
disintegrin 470..546 35/79 (44%) 4.2e-17
52/79 (66%)
EGF 696..728 1 n/4R l21 %nl 0.4
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21/48 (44%)
The cellular disintegrins, also known as ADAM (a disintegrin and
metalloproteinase) and MDC (metalloproteinase-like, disintegrin-like, and
cysteine-rich)
proteins, are regulators of cell-cell and cell-matrix interactions. They
contain multiple
regions, including pro-, metalloproteinase-like, disintegrin-like, cysteine-
rich, epidermal
growth factor-like, transmembrane, and cytoplasmic domains.
NOV 2a has a nucleic acid of 2847 nucleotides (designated CuraGe~ Acc. No.
CG186275-03) encoding a novel ADAM 22-like protein. An open reading frame was
identified beginning at nucleotides 47-49 and ending at nucleotides 2795-2797.
The
encoded protein has 916 amino acid residues and is a splice form of ADAM 22 as
indicated
in position 81 with one exon insertion of 18 amino acids RQIQMFLKSESQKTIYQI
(SEQ
ID N0:79). NOV3 genes disclosed in this invention map to chromosome 7q21
The presence of identifiable domains in the protein was determined by searches
of
domain databases such as Pfam, PROSITE, ProDom, Blocks or Prints and then
identified
by the Interpro domain accession number. Significant domains include
reprolysin,
disintegrin and metalloendopeptidase domains.
Reprolysin, found in CD156 (also called ADAMS (EC 3.4.24.-) or MS2 human) has
been implicated in extxavasation of leukocytes. The members of this family are
enzymes
that cleave peptides. These proteases require zinc for catalysis. Members of
this family are
also known as adamalysins. Most members of this family are snake venom
endopeptidases,
but there are also some mammalian proteins such as P78325, and fertilin
Q28472. Fertilin
and closely related proteins appear to not have some active site residues and
may not be
active enzymes.
Metalloendopeptidase M12B contains a sequence motif similar to the'cysteine
switch' of the matrixins. Many of the proteins with this domain are zinc
proteases that may
mediate cell-cell or cell-matrix interactions. The adhesion of platelets to
the extracellular
matrix, and platelet-platelet interactions, are essential in thrombosis and
haemostasis.
Platelets adhere to damaged blood vessels, release biologically active
chemicals, and
aggregate, a function that is inhibited in normal blood. The binding of
fibrinogen to the
glycoprotein IIb/Illa complex of activated platelets is essential to platelet
aggregation and
is induced by many agonists, including ADP, collagen, thrombin, epinephrine
and
prostaglandin endoperoxide analogue. Snake venoms affect blood coagulation and
platelet
function in a complex manner: some induce aggregation and release reactions,
and some
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inhibit them. Disintegrin, a component of some snake venoms, rather than
inhibiting the
release reactions, operates by inhibiting platelet aggregation, blocking the
binding of
fibrinogen to the receptor-glyco-protein complex of activated platelets. They
act by binding
to the integrin glycoprotein IIb-IIIa.receptor on the platelet surface and
inhibit aggregation
induced by ADP, thrombin, platelet-activating factor and collagen. The role of
disintegrin
in preventing blood coagulation renders it of medical interest, particularly
with regard to its
use as an anti-coagulant.
Disintegrins are peptides of about 70 amino acid residues that contain many
cysteines all involved in disulfide bonds. Disintegrins contain an Arg-Gly-Asp
(RGD)
sequence, a recognition site of many adhesion proteins. The RGD sequence of
disintegrins
interacts with the glycoprotein IIb-IIIa complex.
Example 3. NOV3 CG50586, Beta-secretase
The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide
114
sequences are shown in Table 3A:
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OV3b, 260368280 SEQ m NO: 11 786 by
NA Sequence _
OV3b. 260368280 ORF Start: at 1 ORF Ston: end of
TCCAGCTCAACAGACTCTGTACTTTGACGACAAGAAAGCTTTAAGGGACAA
TGTGGACAAAGGATGGCGGAGAATTACCAGATCCTGACCGAATGGTTGTGAGTGGTAGGGAGCTAAAC
ATTCTTTTCCTGAACAAAACGGATAATGGTACATATCGATGTGAAGCC
NOV3b, 260368280 SEQ m NO: 12 262 as MW at 29748.3kD
GGTAILTCRVDQNBNTSLQWSNPAQQTLYFDDKKALRDNRIELVRASWHELSISVSDVSLSDEGQ
YTCSLFTMPVKTSKAYLTVLGVPEKPQISGFSSPVMEGDLMQLTCKTSGSKPAADIRWFKNDKEIKI
KYLKEEDANRKTFTVSSTLDFRVDRSDDGVAVICRVDHESLNATPQVAMQVLEIHYTPSVKIIPSTI
PQEGQPLILTCESKGKPLPEPVLWTKDGGELPDPDRMWSGRELNILFLNKTDNGTYRCE
NOV3c, 267441066 ~SEQ m NO: 13 1074 by
DNA Sequence ' O~ Start: at I ORF Stop: end of sequence
ATGATTTGGAAACGCAGCGCCGTTCTCCGCTTCTACAGTGTCTGCGGGCTCCTGG
TCAAAATGAT
CTGGCATGAATTGAGT
TGATGGAGTGGCGGTCATCTGCAGAGTAGATCACGAATCCCTCAATGCC
AGGTGCTAGAAATACACTATACACCATCAGTTAAGATTATACCATCGAC
CAGCCTTTAATTTTGACTTGTGAATCCAAAGGAAAACCACTGCCAGAAC
ITGCGGAATATGTTCTCATTGTGCATGATCCTAATGCTTTGGCTGGCCAGAATGGCCCTGACCATG
'TCATAGGAGGAATAGTGGCTGTAGTTGTATTTGTCACGCTGTGTTCTATCTTTCTGCTTGGTCGA
'CTGGCAAGGCATAAAGGAACGTATTTAACAAATGAAGCTAAAGGAGCTGAAGATGCACCAGATGC
.TACAGCC_ATTATCAATGCTGAAGGCAGCCAAGTCAATGC_TGAAGAGAAAAAAGAGTATTTCATT
V3c, 267441066 SEQ m NO: 14 358 as MW at 40019.9kD~
tein Sequence
'KRSAVLRFYSVCGLLVQAAASKNKVKGSQGQFPLTQNVTVVEGGTAILTCRVDQNDNTSLQ
fPAQQTLYFDDKKALRDNRIELVRASWHELSISVSDVSLSDEGQYTCSLFTMPVKTSKAYLTVLDV
WVFVTLCSI
SEQ m NO: 15 X918 bp_
ORF Start: a 1 ORF Stop: end of sequence
AGTTAAAGGCAGCCAAGGGCAGTTTCCACTAACACAGAATGTAACCGTTGTTGAAGGTGG
TTTTGACCTGCAGGGTTGATCAAAATGATAACACCTCCQTCCAGTGGTCAAATCCAGCTC
CTGTACTTTGACGACAAGAAAGCTTTAAGGGACAATAGGATCGAGCTGGTTCGCGCTTCC
ATTGAGTATTAGTGTCAGTGATGTGTCTCTCTCTGATGAAGGACAGTACACCTGTTCTTT
TGCCTGTCAAAACTTCCAAGGCATATCTCACCGTTCTGGGTGTTCCTGAAAAGCCTCAGA
TTCTCATCACCAGTTATGGAGGGTGACTTGATGCAGCTGACTTGCAAAACATCTGGTAGT
AGCTGATATAAGATGGTTCAAAAATGACAAAGAGATTAAAGATGTAAAATATTTAAAAGA
TACACCATCAGTTAAGATTA
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A ClustalW comparison of the above protein sequences yields the following
sequence alignment shown in Table 3B.
Table 3B. Comparison of the NOV3 protein sequences.
NOV3d1 32
___________________________
~
NOV3b~ 11.
1
_______________________________
______________
NOV3a1 ~ 33
__________________________
NOV3c1 60
TGSTMIWKRSAVLRFYSVCGLLVQAAA
NOV3d33 92
NOV3b12 71
NOV3a34 93
NOV3c61 120
NOV3d93 152
NOV3b72 131
NOV3a94 153
NOV3c121 135
NOV3d 153 21.2
NOV3b 132 191
NOV3a 154 21;3
NOV3c 136 194
NOV3d 213 272
NOV3b 192 2~~
NOV3a 214 273
NOV3c 195 25'4
NOV3d 273 ~ _____________________ __ 306
NOV3b 252 _______________________________________________.___ 261,
NOV3a 274 ~ ~ 333
NOV3c 255 ~ ' ' ~ 314
NOV3d '~''''
NOV3b "~"
NOV3a334 ~ 384
NOV3c315 I~ ~ 365
NOV3a(SEQ ID 10)
NO:
NOV3b(SEQ ID 12)
N0:
NOV3c(SEQ ID 14)
NO:
NOV3d(SEQ ID 16)
N0:
Further analysis of the NOV3c protein yielded the following properties shown
in Table 3C.
Table 3C. Protein Sequence Properties NOV3a
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SignalP analysis: ~ Cleavage site between pos. 28 and 29
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 9; pos.chg 2; neg.chg 0
H-region: length 4; peak value -0.57
PSG score: -4.98
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -4.54
possible cleavage site: between 27 and 28
»> 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: 2
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood =-11_94 Transmembrane 299 - 315
PERIPHERAL Likelihood = 6.26 (at 183)
ALOM score: -11.94 (number of TMSs: 1)
MTOP: Prediction of membrane topology (Hartmann et a1.)
Center position for calculation: 306 .
Charge difference: 3.5 C( 2.5) - N(-1.0)
C > N: C-terminal side will be inside
»> Single TMS is located near the C-terminus
»> membrane topology: type Nt (cytoplasmic tail 1 to 298)
MITDISC: discrimination of mitochondrial targeting seq
R content: 2 Hyd Moment(75): 7.03
Hyd Moment(.95): 5.62 G content: 4
DlE content: 1 S/T content: 9
Score: -2.29
Gavel: prediction of cleavage 'sites for mitochondrial preseq
R-3 motif at 17 LRFY~S
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 9.6~
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
SKL2: 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: too long tail
Dileucine motif in the tail: found
LL at 21 '
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 Cytplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 89
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
A search of the NOV3c protein against the Geneseq database, a proprietary
database that contains sequences published in patents and patent publication,
yielded
several homologous proteins shown in Table 3D.
Table 3D. Geneseq Results for NOV3a
NOV3a Identities/
Geneseq Protein/Organism/Length Residues/ SimilaritiesExpect
for
Identifier[Patent #, Date] Match the Matched Value
Residues Region
AAB61142 Human NOV 12 protein 105...362 236/262 (90%)0.0
- Homo
sapiens, 404 aa. [W0200075321-143...404 240/262 (91%)
A2, 14-DEC-2000]
ABG66677 Human novel polypeptide 14...444 236/262 (90%)0.0
#12 -
Homo Sapiens, 404 aa. 6...437 240/262 (91%)
AAY33741 Beta-secretase - Homo 105...286 159/187 (85%)0.0
sapiens,
444 aa. , 143...329 163/187 (87%)
_ _. _~_ _~ -__.~_-._ ~_ ,
_~
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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 3E.
Table 3E. Public BLASTP Results for NOV3a
NOV3a Identities/
Protein Residues/Similarities Expect
for
Accession Protein/Organism/Length Match the Matched Value
Number Residues Portion
CAC22523 Sequence 23 from Patent 105...362236/262 (90%)0.0
W00075321- Homo sapiens 143...404240/262 (91
%)
(Human), 404 aa.
Q8BLQ9 Weakly similar to BK134P22.1-105...362232/262 (88%)0.0
Mus musculus (Mouse), 143...404237/262 (90%)
404 aa.
Q8BYP1 Weakly similar to BK134P22.1105...362232/262 (88%)0.0
-
Mus musculus (Mouse), 143...404237/262 (90%)
404 aa.
PFam analysis Table
predicts
that the
NOV3c protein
contains
the domains
shown in
the
3F.
Table 3F. Domain Analysis of NOV3a
NOV3a Match Region
Pfam Domain A~no acid residues of SEQ ID NO:10 Score E-Value
ig 50...119 26.1 0 . oooss
ig' 208...265 ~, 38.3 1.7e-b7
Adeno_E3_CRl 211...280 -20.2 3.3
Example 4. NOV4, CG50637, T-CELL SURFACE GLYCOPROTEIN CD1B
PRECURSOR
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are shown in Table 4A.
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TTCGATGACATGGC
CTATACCACCCTAGTGGGCTGTATCCTTAGTGTGGTCCTGGTCCTCATATACCTATACCTCACCC
OV4a, CG50637-01 SEQ m NO: 18 493 as MW at 55238.6kD
DPRGLWLLLPSLSLLLFEVARAGRAWSCPAACLCASNILSCSKQQLPNVPHSLPSY
~SRLRAEWTPTRLTQLHSLLLSHNHLNFISSEAFSPVPNLRYLDLSSNQLRTLDEFLF
rYNNHIMAVDRCAFDDMAQLQKLYLSQNQISRFPLELVKEGAKLPKLTLLDLSSNKLK
~AWIKNGLYLHNNPLNCDCELYQLFSHWQYRQLSSVMDFQEDLYCMNSKKLHNVFNLS
..WEAHLGDTLIIKCDTKQQGMTKVWVTPSNERVLDEVTNGTVSVSKDGSLLFQQVQVE
~ETFNETLSVELKVHNFTLHGHHDTLNTAYTTLVGCILSVVLVLIYLYLTPCRCWCRG
SVSSVFSDTPIW
V4b, 277577082 SEQ m NO: 19i 1476 by J~.
A Sequence ORF.Start: at 1 ORF Stop: end of
ACCCAACCTGCGCTACCTGGACCTCTCCTCCAACCAGCTGCGTACACTGGATGAGTTCC
a rr'~mt_r'~ n ArmArm(,C,AGGTGCTGCTGCTCTACAATAACCACATCATGGCGGTGGACCGG
TTAGTGTGGTCCTGGTCCTCATATACCTATACCTCACCCCTTGCCGCTGCTGGTGCC
AAGCCTTCCAGCCATCAAGGAGACAGCCTCAGCTCTTCCATGCTTAGTACCACACCC
TATGGCTGGTGGGGACAAAGATGATGGTTTTGACCGGCGGGTGGCTTTCCTGGAACC
4b, 277577082 SEQ m NO: 20 492 as MW at 56294.8kD
SLSLLLFEVARAGRAWSCPAACLCANILSCSKQQLPNVPHSLPS
SDLQVLEVLLLYNNHIMAVDRCAFDDMAQLQKLYLSQNQISRFPLELVKEGAKLPKLTLLDLS
NLPLPDLQKLPAWIKNGLYLHNNPLNCDCELYQLFSHWQYRQLSSVMDFQEDLYCMNSKKLHN
FNETLSVELKVHNFTLHGHHDTLNTAYTTLVGCILSVVLVLIYL
SSMLSTTPNHDPMAGGDKDDGFDRRVAFLEPAGPGQGQNGKLKP
SVFSDTPIW _._____.._~__
m NO: 21 ~ 1398
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V4c, 277577094 SEQ ID NO: 21 1398 by
A Sequence _
~V4c, 277577094 ORF Start: at 1 ORF Stop: end of sequence
GGCCGAGCCGTGGTTAGCTGTCCTGCCGCCTGCTTGTGCGCCAGCAACATCCTCAGCT
GCTCCAAGCAGCAGCTGCCCAATGTGCCCCATTCCTTGCCCAGTTACACAGCACTACT
TGC
AACCACATCATGGCGGTGGACCGGTGCGCCTTCGATGACA
GCAGTATCGGCAGCTGAGCTCCGTGATGGACTTTCAAGAGGATC
TGCACAATGTCTTCAACCTGAGTTTCCTCAACTGTGGCGAGTAC
ATACCTGC
GATCCAGAATCAGTCAGCTCGGTCTTCTCTGATACGCCCATTGTGGTG
NOV4c, 277577094 SEQ >D NO: 22 466 as MW at 52731.6kD
RAWSCPAACLCASNILSCSKQQLPNVPHSLPSYTALLDLSHNNLSRLRAEWTPTRLTQLHSLL
SHNHLNFISSEAFSPVPNLRYLDLSSNQLRTLDEFLFSDLQVLEVLLLYNNHIMAVDRCAFDDM
KLYLSQNQISRFPLELVKEGAKLPKLTLLDLSSNKLKNLPLPDLQKLPAWIKNGLYLHNNPLNC
YQLFSHWQYRQLSSVMDFQEDLYCMNSKKLHNVFNLSFLNCGEYKERAWEAHLGDTLIIKCDTK
TKVWVTPSNERVLDEVTNGTVSVSKDGSLLFQQVQVEDGGVYTCYAMGETFNETLSVELKVHNF
HHDTLNTAYTTLVGCILSVVLVLIYLYLTPCRCWCRGVEKPSSHQGDSLSSSMLSTTPNHDPMA
ID NO: 23 a 717 by
Start: at 1 ORF Stop: end of
TCTGAGGCCTTTTCCCCGGT
CACATCA
CGCTCC
TCGGCAGCTGAGCTCCGTGATGGACTTTCAAGAGGA
4d,~277577141 ~SEQ >D NO: 24 239 as ~MW at 28046.9kD
PAACLCASNILSCSKQQLPNVPHSLPSYTALLDLSHNNLSRLRAEWTPTRLTQLHSLLLSHNH
FISSEAFSPVPNLRYLDLSSNQLRTLDEFLFSDLQVLEVLLLYNNHIMAVDRCAFDDMAQLQKL
NQISRFPLELVKEGAKLPKLTLLDLSSNKLKNLPLPDLQKLPAWIKNGLYLHNNPLNCDCELYQ
WOYROLSSVMDFOEDLYCMNSKKLHNVFNLSFLNCG
A ClustalW comparison of the above protein sequences yields the following
sequence alignment shown in Table 4B.
Table 4B. Comparison of the NOV4 protein sequences.
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NOV4a MHPHRDPRGLWLLLPSLSLLLFEVARAGRAVVSCPAACLCASNILSCSKQQL
'NOV4b MHPHRDPRGLWLLLPSLSLLLFEVARAGRAVVSCPAACLCA-NILSCSKQQL
'NOV4c ----------------------'----G~wSCPAACLCASNILSCSKQQL
NOV4d --------------------------------SCPAACLCASNILSCSKQQL
NOV4a PNVPHSLPSYTALLDLSHNNLSRLRAEWTPTRLTQLHSLLLSHNHLNFISSEAFSPVPNL
NOV4b PNVPHSLPSYTALLDLSHNNLSRLRAEWTPTRLTøLHSLLLSHNHLNFISSEAFSPVPNL
NOV4C PNVPHSLPSYTALLDLSHNNLSRLRAEWTPTRLTQLHSLLLSHNHLNFISSEAFSPVPNL
NOV4d PNVPHSLPSYTALLDLSHNNLSRLRAEWTPTRLTQLHSLLLSHNHLNFISSEAFSPVPNL
NOV4a RYLDLSSNQLRTLDEFLFSDLQVLEVLLLYNNHIMAVDRCAFDDMAQLQKLYLSQNQISR
NOV4b RYLDLSSNQLRTLDEFLFSDLQVLEVLLLYNNHIMAVDRCAFDDMAQLQKLYLSQNQISR
NOV4c RYLDLSSNQLRTLDEFLFSDLQVLEVLLLYNNHIMAVDRCAFDDMAQLQKLYLSQNQISR
NOV4d RYLDLSSNQLRTLDEFLFSDLQVLEVLLLYNNHIMAVDRCAFDDMAQLQKLYLSQNQISR
NOV4a FPLELVKEGAKLPKLTLLDLSSNKLKNLPLPDLQKLPAWIKNGLYLHNNPLNCDCELYQL
NOV4b FPLELVKEGAKLPKLTLLDLSSNKLKNLPLPDLQKLPAWIKNGLYLHNNPLNCDCELYQL
NOV4C FPLELVKEGAKLPKLTLLDLSSNKLKNLPLPDLQKLPAWIKNGLYLHNNPLNCDCELYQL
NOV4d FPLELVKEGAKLPKLTLLDLSSNKLKNLPLPDLQKLPAWIKNGLYLHNNPLNCDCELYQL
NOV4a FSHWQYRQLSSVMDFQEDLYCMNSKKLHNVFNLSFLNCGEYKERAWEAHLGDTLIIKCDT
NOV4b FSHWQYRQLSSVMDFQEDLYCMNSKKLHNVFNLSFLNCGEYKERAWEAHLGDTLIIKCDT
NOV4C FSHWQYRQLSSVMDFQEDLYCMNSKKLHNVFNLSFLNCGEYKERAWEAHLGDTLIIKCDT
NOV4d FSHWQYRQLSSVMDFQEDLYCMNSKKLHNVFNLSFLNCG--------------------
NOV4a KQQGMTKVWVTPSNERVLDEVTNGTVSVSKDGSLLFQQVQVEDGGVYTCYAMGETFNETL
NOV4b KQQGMTKVWVTPSNERVLDEVTNGTVSVSKDGSLLFQQVQVEDGGVYTCYAMGETFNETL',
NOV4c KQQGMTKVWVTPSNERVLDEVTNGTVSVSKDGSLLFQQVQVEDGGVYTCYAMGETFNETL'
NOV4d ____________________________________________________________
NOV4a SVELKVHNFTLHGHHDTLNTAYTTLVGCILSVVLVLIYLYLTPCRCWCRGVEKPSSHQGD
NOV4b SVELKVHNFTLHGHHDTLNTAYTTLVGCILSVVLVLIYLYLTPCRCWCRGVEKPSSHQGD
NOV4c SVELKVHNFTLHGHHDTLNTAYTTLVGCILSVVLVLIYLYLTPCRCWCRGVEKPSSHQGD
NOV4d ________________________-___________________________________
NOV4a SLSSSMLSTTPNHDPMAGGDKDDGFDRRVAFLEPAGPGQGQNGKLKPGNTLPVPEATGKG
NOV4b SLSSSMLSTTPNHDPMAGGDKDDGFDRRVAFLEPAGPGQGQNGKLKPGNTLPVPEATGKG
NOV4C SLSSSMLSTTPNHDPMAGGDKDDGFDRRVAFLEPAGPGQGQNGKLKPGNTLPVPEATGKG
NOV4d _______-____________________________________________________
NOV4a QRRMSDPESVSSVFSDTPIW
NOV4b QRRMSDPESVSSVFSDTPIVV
NOV4c QRRMSDPESVSSVFSDTPIVV
NOV4d _-_____-_____________
NOV4a (SEQ ID NO: 18)
NOV4b (SEQ ID NO: 20)
NOV4c (SEQ ID NO: 22)
NOV4d (SEQ ID NO: 24)
Further analysis of the NOV4a protein yielded the following properties shown
in Table 4C.
Table 4C. Protein Sequence Properties NOV4a
SignalP analysis: ~ Cleavage site between residues 35'and 36.
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PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 5; pos.chg 1; neg.chg 0
H-region: length 7; peak value -5.92
PSG score: -10.32
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 0.90
possible cleavage site: between 35 and 36
»> 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
INTEGRAL Likelihood = -2.28 Transmembrane 17 - 33
INTEGRAL Likelihood = -2.18 Transmembrane 38 - 54
INTEGRAL Likelihood =-10.83 Transmembrane 384 - 400
PERIPHERAL Likelihood = 2.44 (at 132)
ALOM score: -10.83 (number of TMSs: 3)
MTOP: Prediction of membrane topology (Hartmann et~al.)
Center position for calculation: 24'
Charge difference: -2.0 C( 1.0) - N( 3.0)
N >= C: N-terminal side will be inside
»> membrane topology: type 3a
MTTDISC: discrimination of mitochondrial targeting seq
R content: 2 Hyd Moment(75): 5.88
Hyd Moment(95): 6.17 C~ content: 1
D/E content: 2 S/T content: 5
Score: -4.21
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 47 GRAIVV
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 8.5~
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
SKL2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: found
KLPK at 190
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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:
Leucine zipper pattern (PS00029): *** found ***
LSCSKQQLPNVPHSLPSYTALL at 52
LDLSSNQLRTLDEFLFSDLQVL at 122
LKVHNFTLHGHHDTLNTAYTTL at 363
none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytplasmic/Nuclear discrimination
Prediction: nuclear
Reliability: 55.5
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
A search of the NOV4a protein against the Ueneseq 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 [Patent Residues/ Similarities for Expect
Identifier #, Date] Match the Matched Value
Residues Region
AAB49650 Human SEC2 protein sequence SEQ 9..500 492/493 (99%) 0.0
ID 4 - Homo sapiens, 493 aa. 1..493 492/493 (99%)
[W0200070046-A2, 23-NOV-2000]
ABJ10921 Human secreted protein (SECP) #17 9..500 492/493 (99%) 0.0
- Homo sapiens, 493 aa. 1..493 492/493 (99%)
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ABB17119 Human nervous system related 158..335 177/178 (99%) ~ 0.0
polypeptide SEQ ID NO 5776 - 27..204 177/178 (99%)
Homo Sapiens, 212 aa.
[W0200159063-A2, 16-AUG-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.
Table 4E. Public BLASTP Results for NOV4a
NOV4a Identities/
Protein Residues/Similarities Expect
for
Accession Protein/Organism/Length Match the Matched Value
Number Residues Portion
Q86WK6 Transmembrane protein 9...500 492/493 (99%)0.0
AMIGO
- Homo sapiens (Human), 1...493 492/493 (99%)
493 aa.
Q8IW71 Hypothetical protein 9..500 491/493 (99%)0.0
- Homo
sapiens (Human), 493 1..493 493/493 (99%)
aa.
Q80ZD8 Transmembrane protein 9..500 440/492 (89%)0.0
AMIGO
- Mus musculus (Mouse), 1..492 463/492 (94%)
492 aa.
PFam analysis s the domainsn in
predicts show the
that the
NOV4a
protein
contain
Table 4F.
Table 4F. Domain Analysis of NOV4a
Pfam DomainNOV4a Match Region ScoreE-Value
~
Amino acid residues of SEQ
ID N0:18
L~ 41..67 11.6 1.2
I,~ ~~~69..91 4.2 3e+02
94..117 15.4 1.3
Leg 118..141 23.4 0.0053
L~ 142..165 8.3 78
Lgg 166..185 11.2 24
LRR 193..216 ~ 0.093
19.3
LRRCT 228..278 30.0 5.6e-05
Ig 290..350 ' 20.7 0.036
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Example 5. NOVS, CG51117-09, Nephronectin
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide
sequences are shown in Table 5A.
Table SA. N_OVS Sequence A_nalysi_s
NOV5a, 306433917 SEQ m NO: 25 1413 by _
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
TGCCAACCACGATGCAAACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCATC
CTGGTTATGCTGGAAA.AACCTGTAATCAAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAAG
CACAGGTGCATGAACACTTACGGCAGCTACAAGTGCTACTGTCTCAACGGATATATGCTCATGCCGGA
TGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAAG
GACAAATACGGTGCCAGTGCCCATCCCCTGGCCTGCAGCTGGCTCCTGATGGGAGGACCTGTGTAGAT
GTTGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGAG
CTACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGACATAGACGAATGCTCACTTG
GTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAACGTACGTGGGTCCTACAAGTGCAAATGTAAA
GAAGGATACCAGGGTGATGGACTGACTTGTGTGTATATCCCAAAAGTTATGATTGAACCTTCAGGTCC
AATTCATGTACCAAAGGGAAATGGTACCATTTTAAAGGGTGACACAGGAAATAATAATTGGATTCCTG
ATGTTGGAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTATCATTACCAACAGGCCTACT
TCTAAGCCAACAACAAGACCTACACCAAAGCCAACACCAATTCCTACTCCACCACCACCACCACCCCT
GCCAACAGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCAACCACCGGACTGACAACTA
TAGCACCAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAGGGTACAGACAGACCCTCAG
AAACCCAGAGGAGATGTGTTCAGTGTTCTGGTACACAGTTGTAATTTTGACCATGGACTTTGTGGATG
GATCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCCAGCAGGTGGACAATATCTGA
CAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCTACCTCTCGGCCGCCTCATG
CATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGCACTCTGGCACACTCCAGGT
GTTTGTGAGAAAACACGGTGCCCACGGAGCAGCCCTGTGGGGAAGAAATGGTGGCCATGGCTGGAGGC
AAACACAGATCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAAAGGTGAAAAAAGGCGTGGT
CACACTGGGGAGATTGGATTAGATGATGTGAGCTTGAA:~AAAGGCCACTGCTCTGAAGAACGC
NOVSa, 306433917 SEQ ID NO: 26 471 as MW at 51775.8kD
Protein Sequence
CQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPD
GSCSSALTCSMANCQYGCDVVKGQIRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRFRQCVNTFGS
YICKCHKGFDLMYIGDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGP
IHVPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPL
PTELRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGW
IREKDNDLHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQV
FVRKHGAHGAALWGRNGGHGWRQTQITLRGADIKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEER
NOVSb, 306447063 SEQ ID NO: 27 1743 by
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
ATGGATTTTCTCCTGGCGCTGGTGCTGGTATCCTCGCTC
TACCTGCAGGCGGCCGCCGAGTTCGACGGGAGTAGGTGGCCCAGGCAAATAGTGTCATCGATTGGCCT
ATGTCGTTATGGTGGGAGGATTGACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGTGTCAGC
CTTTCTACGTCTTAAGGCAGAGAATAGCCAGGATAAGGTGCCAGCTCAAAGCTGTGTGCCAACCACGA
TGCAAACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCATCCTGGTTATGCTGGAAAAACCTG
TAATCAAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAAGCACAGGTGCATGAACACTTACG
GCAGCTACAAGTGCTACTGTCTCAACGGATATATGCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTG
ACCTGCTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAAGGACAAATACGGTGCCAGTGCCC
ATCCCCTGGCCTGCAGCTGGCTCCTGATGGGAGGACCTGTGTAGATGTTGATGAATGTGCTACAGGAA
GAGCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGAGCTACATCTGCAAGTGTCATAAA
GGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGACATAGACGAATGCTCACTTGGTCA
GTATCAGTGCAGCAGCTTTGCTCGATGTTATAACGTACGTGGGTCCTACAAGTGCAAATGTAAAGAAG
GATACCAGGGTGATGGACTGACTTGTGTGTATATCCCAAAAGTTATGATTGAACCTTCAGGTCCAATT
CATGTACCAAAGGGAAATGGTACCATTTTAAAGGGTGACACAGGAAATAATAATTGGATTCCTGATGT
TGGAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTATCATTACCAACAGGCCTACTTCTA
AGCCAACAACAAGACCTACACCAAAGCCAACACCAATTCCTACTCCACCACCACCACCACCCCTGCCA
ACAGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCAACCACCGGACTGACAACTATAGC
ACCAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAGGGTACAGACAGACCCTCAGAAAC
CCAGAGGAGATGTGTTCATTCCACGGCAACCTTCAAATGACTTGTTTGAAATATTTGAAATAGAAAGA
GGAGTCAGTGCAGACGATGAAGCAAAGGATGATCCAGGTGTTCTGGTACACAGTTGTAATTTTGACCA
TGGACTTTGTGGATGGATCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCCAGCAG'
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G TGGACAATATCTGACAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCr~c:c~c:'1"1'~~'1'~~-
1H~.~
1-
GGCCGCCTCATGCATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGCACTC
C TC
T GGCACACTCCAGGTGTTTGTGAGAAAACACGGTGCCCACGGAGCAGCCCTGTGGGGAAGAAATGGTG
G CCATGGCTGGAGGCAAACACAGATCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAAAGGT
G Ap,AAAAGGCGTGGTCACACTGGGGAGATTGGATTAGATGATGTGAGCTTGAAAAI~AGGCCACTGCTC
T GAA
NOVSb, 306447063 SEQ m NO: 28 581 as MW at 65022.9kD
P rotein Sequence ~ ~__
M DFLLALVLVSSLYLQAA.AEFDGSRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQP
F YVLRQRIARIRCQLKAVCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYG
S YKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLQLAPDGRTCVDVDECATGR
A SCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEG
Y QGDGLTCWIPKVMIEPSGPIHVPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSK
PTTRPTPKPTPIPTPPPPPPLPTELRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKP
RGDVFIPRQPSNDLFEIFEIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAG
GQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGG
HGWRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKG_HCSE
NOVSc, 306447071 ' SEQ m NO: 29 '1689 by
.__
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
ATGGATTTTCTCCTGGCGCTGGTGCTGGTATCCTCGCTCTACCTGCAGGCGGCCGCCG
AGTTCGACGGGAGGTGGCCCAGGCAAATAGTGTCATCGATTGGCCTATGTCGTTATGGTGGGAGGATT
GACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGTGTCAGCCTGTGTGCCAACCACGATGCAA
ACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCATCCTGGTTATGCTGGAAAAACCTGTAATC
AAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAAGCACAGGTGCATGAACACTTACGGCAGC
' TACAAGTGCTACTGTCTCAACGGATATATGCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTGACCTG
CTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAAGGACAAATACGGTGCCAGTGCCCATCCC
CTGGCCTGCACCTGGCTCCTGATGGGAGGACCTGTGTAGATGTTGATGAATGTGCTACAGGAAGAGCC
TCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGAGCTACATCTGCAAGTGTCATAAAGGCTT
CGATCTCATGTATATTGGAGGCAAATATCAATGTCATGACATAGACGAATGCTCACTTGGTCAGTATC
AGTGCAGCAGCTTTGCTCGATGTTATAACGTACGTGGGTCCTACAAGTGCAA.ATGTAAAGAAGGATAC
CAGGGTGATGGACTGACTTGTGTGTATATCCCAAAAGTTATGATTGAACCTTCAGGTCCAATTCATGT
ACCAAAGGGAAATGGTACCATTTTAA.AGGGTGACACAGGAAATAATAATTGGATTCCTGATGTTGGAA
GTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTATCATTACCAACAGGCCTACTTCTAAGCCA
ACAACAAGACCTACACCAAAGCCAACACCAATTCCTACTCCACCACCACCACCACCCCTGCCAACAGA
GCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCAACCACCGGACTGACAACTATAGCACCAG
CTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAGGGTACAGACAGACCCTCAGAAACCCAGA
_ GGAGATGTGTTCATTCCACGGCAACCTTCAAATGACTTGTTTGAAATATTTGAAATAGAAAGAGGAGT
CAGTGCAGACGATGAAGCAAAGGATGATCCAGGTGTTCTGGTACACAGTTGTAATTTTGACCATGGAC
TTTGTGGATGGATCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCCAGCAGGTGGA
CAATATCTGACAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCTACCTCTCGG
CCGCCTCATGCATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGCACTCTGGCA
CACTCCAGGTGTTTGTGAGAA.AACACGGTGCCCACGGAGCAGCCCTGTGGGGAAGAAATGGTGGCCAT
GGCTGGAGGCAAACACAGATCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAAAGGTGAAAA
AAGGCGTGGTCACACTGGGGAGATTGGATTAGATGATGTGAGCTTGAAAAAAGGCCACTGCTCTGAA
NOVSc, 306447071 SEQ m NO: 30 563 as MW at 62132.5kD
Protein Sequence
MDFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCK
HGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTC
SMANCQYGCDWKGQIRCQCPSPGLHLAPDGRTCVDWECATGRASCPRFRQCVNTFGSYICKCHKGF
DLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHV
PKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTE
LRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFIPRQPSNDLFEIFEIERGV
SADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLG
RLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHGWRQTQITLRGADIKSWFKGEK
RRGHTGEIGLDDVSLKKGHCSE
NOVSd, 306447075 SEQ m NO: 31 1740 by
.
ORF Start: at 1 ORF Stop: end of sequence
DNA Sequence
ATGGATTTTCTCCTGGCGCTGGTGCTGGTATCCTCGCTCTACCTGCAGGCGGCCGCCG
AGTTCGACGGGAGGTGGCCCAGGCAAATAGTGTCATCGATTGGCCTATGTCGTTATGGTGGGAGGATT
GACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGTGTCAGCCTTTCTACGTCTTAAGGCAGAG
AATAGCCAGGATAAGGTGCCAGCTCAAAGCTGTGTGCCAACCACGATGCAAACATGGTGAATGTATCG
GGCCAAACAAGTGCAAGTGTCATCCTGGTTATGCTGGAAAAACCTGTAATCAAGATCTAAATGAGTGT
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CCGGCCCTGTAAGCACAGGTGCATGAACACTTACGGCAGCTACAAGTGCT
TGCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCA
TGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTT
ACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATAT
TGTTATAACGTACGTGGGTCCTACAAGTGCAAATGTAAAGA
TGTGTATATCCCAAAAGTTATGATTGAACCTTCAGGTCCAA
TTTTAAAGGGTGACACAGGAAATAATAATTGGATTCCTGAT
ACACAGTTGT
GGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCT
306447075 ~SEQ m NO: 32 X586 as BMW at 64240.OkD
C~'mFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPFYVLRQR
IARIRCQLKAVCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYC
L~TGYMLMPDGSCSSALTCSMANCQYGCDVVKGQIRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRF
QCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGL
CVYIPKVMIEPSGPIHVPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPT
KPTPIPTPPPPPPLPTELRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFI
RQPSNDLFEIFEIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTV
AD_IKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEVDG
ie, CG51117-09 f SEQ m NO: 33 ~ 1839 by
Sequence . ORF Start: ATG at 1 t ORF Stop: at 1850
CAGGCAAATAGTGTCATCGA
ATGC
CCTGT
CTGCAGCTGGCTCCTGATGGGAGGACCTGTGTAGA
TCATGTATATTGGAGGCAAATATCAATGTCATGACAT
TACCAGGGTGATGGACTGACTTGTGTGTATA
AA
GGCAACCTTCAAATGACTTGTTTGAAATA
TATCTGACAGTGTCGGCAGCCAAAGCCC
CG51117-09 ~SEQ ll~ NO: 34 X613 as ~N1W at 67402.4kD
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MDFLLALVLVSSLYLQAAAEFDGSRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPFYVLRQRIA
RIRCQLKAVCQPRCKHGECIGPNKCKCHPGYAGKTCNQDEHIPAPLDQGSEQPLFQPLDHQATSLPSR
DLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDVVKGQTRCQCPSP
GLQLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECSLGQYQ
CSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKGNGTILKGDTGNNNWIPDVGS
TWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELRTPLPPTTPERPTTGLTTIAPA
ASTPPGGITVDNRVQTDPQKPRGDVFIPRQPSNDLFEIFEIERGVSADDEAKDDPGVLVHSCNFDHGL .
CGWIREKDNDLHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGT
LOVFVRKHGAHGAALWGRNGGHGWRQTQITLRGADIKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEER:
NOVSf, CG51117-14 SEQ m NO: 35 _ 933, bp-~__
DNA Sequence ORF Start: at 1 ~w ORF Stop: at 944
AC
TGCACGCTTGGTGCTACCTCTCGGCC
OVSf, CG51117-14 ~SEQ m NO: 36 311 as ~MW at 33658.1kD
LTCVYIPKVMIEPSGPIHVPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPT
TPKPTPIPTPPPPPPLPTELRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQ
IPRQPSNDLFEIFEIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDP
~5g, SNP13382208 of SEQ m..NO:.37_. ~2112_bp __ ~ w,~~~~
1117-03, DNA Sequence p~ Start: ATG~at 203 ORF Stop: TAA~at 1949
Pos: 1794 SNP Change: G to A
GGATTTTCTCCTGGCGCTGGTGCTGGTATCCTCGCTCTACCTGCAGGCGGCCGCCGAGTTCGACGGGA
GGTGGCCCAGGCAAATAGTGTCATCGATTGGCCTATGTCGTTATGGTGGGAGGATTGACTGCTGCTGG
GGC'_TGGGCTCGCCAGTCTTGGGGACAGTGTCAGCCTTTCTACGTCTTAAGGCAGAGAATAGCCAGGAT
TGTCATCCTGGTTATGCTGGAAAAACCTGTATTCAAGTTTTAAATGAGTGTGGCCTGAAGCCC
CTGTAAGCACAGGTGCATGAACACTTACGGCAGCTACAAGTGCTACTGTCTCAACGGATATAT
TGCCGGATGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGTG
..mmT T T r~nT rT a amar~r_m~rrn.rmrrrrAmrrrc~TGGC~CTGCAGCTGGCTCCTGATGGGAGG
TACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATATC
AGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAAC
ACAAGTGCAAATGTAAAGAAGGATACCAGGGTGATGGACTGACTTGTGTGTATAT
ATTGAACCTTCAGGTCCAATTCATGTACCAAAGGGAAATGGTACCATTTTAAAGG
mAAmAATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCCGAAGACACCATAT
ACTCCACCACCACCACCACCCCTGCCAACAGAGCTCAGAACACCTCTACCACCTACAACCC
GCCAACCACCGGACTGACAACTATAGCACCAGCTGCCAGTACACCTCCAGGAGGGATTACA
ACAGGGTACAGACAGACCCTCAGAAACCCAGAGGAGATGTGTTCATTCCACGGCAACCTTC
TTGTTTGAAATATTTGAAATAGAAAGAGGAGTCAGTGCAGACGATGAAGCAAAGGATGATC
TCGGCCGCCTTATGCATTCAGGGGACCTGTGCC
TGGAGGCAAACACAGATCACCTTGCG
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t~TOVSg, SNP13382208 of SEQ >D NO: 38 582 aa_ MW at 63963.9kD
CG51117-03, Protein Sequence SNp pos: 531 SNP Change: Arg to Lys
MDFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPFYVLRQRIAR
IRCQLKAVCQPRCKHGECIGPNKCKCHPGYAGKTCIQVLNECGLKPRPCKHRCMNTYGSYKCYCLNGY
MLMPDGSCSSALTCSMANCQYGCDVVKGQIRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRF12QCV
NTFGSYICKCHKGFDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVY
IPKVMIEPSGPIHVPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPT
PIPTPPPPPPLPTELRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFIPRQP
SNDLFEIFEIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAK
APGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGKNGGHGWRQTQITL
RGADIKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEER- _,~.~._-
Further analysis of the NOVSa protein yielded the following properties shown
in Table 5B.
Table SC. Protein Sequence Properties NOVSe
SignalP analysis: Cleavage site between residues 19 and 20
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 Q.00
PSG score: -4.40
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -0.34
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: 1
INTEGRAL Likelihood = -4.19 Transmembrane 3 - 19
PERIPHERAL Likelihood = 5.67 (at 516)
ALOM score: -4.19 (number of TMSs: 1)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 10
Charge difference: 0.0 C( 0.0) - N( 0.0)
N >= C: N-terminal side will be inside
»> membrane topology: type 2 (cytoplasmic tail 1 to 3)
MITDISC: discrimination of mitochondria) targeting seq
R content: 0 Hyd Moment(75): 4.41
Hyd Moment(95): 7.23 G content: 0
D/E content: 2 S/T content: 2
Score: -6.55
Gavel: prediction of cleavage sites for mitochondria) preseq
cleavage site motif not found
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NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none '
bipartite: none
content of basic residues.: 11.9
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
SKL2: 2nd peroxisomal targeting signal: found
RIARIRCQL at 66
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 Cytplasmic/Nuclear discrimination
Prediction: nuclear
Reliability: 89
COIL: Lupas's algorithm to detect coiled=coil regions
total: 0 residues
A search of the NOVSe protein against the Cieneseq database, a propneiary
database that contains sequences published in patents and patent publication,
yielded
several homologous proteins shown in Table 5D.
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Table SC. Geneseq Results for NOVSe
NOVSe Identities/
Geneseq Protein/Organism/Length Residues/Similarities Expect
[Patent for
Identifier#, Date] Match the Matched Value
ResiduesRegion
ABR47621 Breast cancer associated 138..613476/476 (100%)0.0
protein
sequence SEQ ll~ N0:484 107..582476/476 (100%)
- Homo
Sapiens, 582 aa. [W02003004989-
A2, 16-JAN-2003]
AAB70547 Human PR017 protein sequence138..613476/476 (100%)0.4
SEQ ID N0:34 - Homo Sapiens,107..582476/476 (100%)
582 aa. [W0200110902-A2,15-'
FEB-2001]
ABG69659 Human secreted.protein 135..613477/479 (99%)0.0
SCEP-39 -
Homo Sapiens, 566 aa. 88..566 479/479 (100%)
[WO200248337-A2, 20-JUN-2002]
'
ABJ37055 breast cancer / ovarian ~ 103..613506/511 (99%)0.0
; Human
cancer related protein 51..560 509/511(99%) i
#31 - Homo
sapiens, 560 aa. [W02003000012-
A2, 03-JAN-2003]
In a BLAST OVSe protein
search was found
of public to
sequence
databases,
the N
have homology to the proteins shown in the BLASTP data in Table 5D.
Table SD. Public BLASTP Results for NOVSe
NOVSe Identities/
Protein Residues/ Similarities Expect
for
Accession Protein/Organism/LengthMatch the Matched Value
Number Residues Portion
CAC33425 Sequence 33 from.Patent138..613 476/476 (100%)0.0
W00110902 - Homo Sapiens107..582 476/476 (100%)
(Human), 582 aa.
Q923T5 Nephronectin - Mus musculus1..578 536/609 (88%)0.0
~~ (Mouse), 609 aa. 1..609 566/609 (93%)
Q91XL5 Nephronectin - Mus musculus25..609 503/585 (85%)0.0
(Mouse), 592 aa. 24..592 534/585 (91
%)
PFam analysis
predicts
that the
NOVSe
protein
contains
the domains
shown
in the
Table 5E.
Table 5E.
Domain
Analysis
of NOVSe
Pfam DomainNOVSe Match Region ScoreE-Value
Amino acid residues of SEQ
ID NO: 34
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EG F ___.~-_.- 78..104 _._~.~8.9 0.12
_
EGF 141..175 15.6 0.26
EGF 181..215 20.7 0.035
EGF 221..260 2.7 6.1
Mp,M 470..611 102.4 8.6e-27
The epithelial-mesenchymal interactions required for kidney organogenesis are
disrupted in mice lacking the integrin alpha8betal. None of this integrin's
known ligands,
however, appears to account for this phenotype. To identify a more relevant
ligand, . .
Brandenberger et al. (2001) used a soluble integrin alpha8betal heterodimer
fused to
alkaline phosphatase (AP) to probe blots and cDNA libraries. In newborn mouse
kidney
extracts, alpha8betal-AP detects a novel ligand of 70-90 kD. This protein,
named
nephronectin, is an extracellular matrix protein with five EGF-like repeats, a
mucin region
containing a RGD sequence, and a COON-terminal MAM domain. Integrin
alpha8betal
and several additional RGD-binding integrins bind nephronectin. Nephronectin
mRNA is
expressed in the ureteric bud epithelium, whereas alpha8betal is expressed in
the
rnetanephric mesenchyme. Nephronectin is localized in the extracellular matrix
in the same
distribution as the ligand detected by alpha8betal-AP and forms a complex with
alpha8betal .in vivo. Thus, these results strongly suggest that nephronectin
is a relevant
ligand mediating alpha8betal function in the kidney. Nephronectin is expressed
at
numerous sites outside the kidney, so it may also have wider roles in
development.
(Brandenberger et al. J Cell Biol 2001 Jul 23;154(2):447-58)
NOVSe is a novel nucleic acid of 613 nucleotides (designated CuraGen Acc. No.
CG51117-09) encoding a novel Nephronectin-like protein. This sequence
represents a
splice form of Nephronectin as indicated in positions with one exon insertion
30 amino
acids and one amino acid S insertion at position 24 and maps to chromosome 6
'The presence of identifiable domains in the protein disclosed herein was
determined
by searches versus domain databases such as Pfam, PROSTTE, ProDom, Blocks or
Prints
and then identified by the Interpro domain accession number. A 170 amino acid
domain,
the so-called MAM domain, has been recognised in the extracellular .region of
functionally
diverse proteins. These proteins have a modular, receptor-like architecture
comprising a
signal peptide, an N-terminal extracellular domain, a single transmembrane
domain and an
intracellular domain. Such proteins include meprin (a cell surface
glycoprotein); A5 antigen
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(a developmentally-regulated cell .surface protein); and receptor-like
tyrosine protein
phosphatase. The MAM domain is thought to have an adhesive function. It
contains 4
conserved cysteine residues, which probably form disulphide bridges.
A sequence of about thirty to forty amino-acid residues long found in the
sequence
of epidermal growth factor (EGF) has been shown to be present, in a more or
less
conserved form, in a large number of other, mostly animal proteins. The list
of proteins
currently known to contain one or more copies of an EGF-like pattern is large
and varied..
The functional significance of EGF domains in what appear to be unrelated
proteins is not
yet clear. However, a common feature is that these repeats are found in the
extracellular . ~:
domain of membrane-bond proteins or in proteins known to be secreted
(exception:
prostaglandin G/H synthase). The EGF domain includes six cysteine residues
which have .
been shown (in EGF) to be involved in disulfide bonds. The main structure is a
two-
stranded beta-sheet followed by a loop to a C-terminal short two-stranded
sheet.
Subdomains between the conserved cysteines vary in length.
NOVSa, clone 306433917 is a splice variant with deletion of amino acid
sequences
GKYQCH , EHIPAPLDQGSEQPLFQPLDHQATSLPSR (SEQ ID N0:79) and
PRQPSNDLFEIFEIERGVSADDEAKDDPG (SEQ ID N0:80) one deleted exon 29 amino
acids, plus 1 amino acid changes I322S compared to NOVSe. NOVSb, 5c, 5d,
assemblies
306447063, 306447071, 306447075 respectively.were found to encode an open
reading .
frame between residues 1 to 611 of NOVSe, CG51117-09. The cloned insert NOVSc
306447071 is a splice variant of parent with one exon deletion 17 amino acids
FYVLRQRIARIRCQLKA (SEQ ID N0:81), deletion of amino acid sequence
EHIPAPLDQGSEQPLFQPLDHQATSLPSR (SEQ ID NO:82) plus 1 amino acid S
deletion at position 24, amino acid changes Q159H compare to NOVSe. The cloned
insert
NOVSd 306447075 has a deletion of amino acid sequence
EHIPAPLDQGSEQPLFQPLDHQATSLPSR (SEQ ID N0:83) plus 1 amino acid S
deletion at position 24 compared to NOVSe NOVSb, 306447063 has has a deletion
of
amino acid sequence EHIPAPLDQGSEQPLFQPLDHQATSLPSR (SEQ ID N0:84)
compared to NOVSe
Example 6. NOV6, CG51923, Protocadherin Fat 2 precursor
The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are shown in Table 6A.
- Table 6A. NOV6 Sequence Analysis
134
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ATACCATACATGGCAGCCAAGACCCAGGJ--
,~ ~LL~"~~,~,r,~",~c~~,~~..~"n~~".....,....~~~.........._____________________.
AATGCCCTGCTAGGCATCATTACTCTAGCTCAAAAGCTTGATCAGGCAAATCATGCCCCACATACT
GACAGTGAAGGCAGAAGATCAAGGCTCCCCACAATGGCATGACCTGGCTACAGTGATCATTCATGT
ATCCCTCAGATAGGAGTGCCCCCATCTTTTCAAAATCTGAGTACTTTGTAGAGATCCCTGAATCAA
CCTGTTGGTTCCCCAATCCTCCTTGTCTCTGCTATGAGCCCCTCTGAAGTTACCTATGAGTTAAGA
GGGAAATAAGGATGGAGTCTTCTCTATGAACTCATATTCTGGCCTTATTTCCACCCAGAAGAAATT
ACCATGAGAAAATCTCGTCTTACCAGCTGAAAATCCGAGGCAGCAATATGGCAGGTGCATTTACTG
. GTCATGGTGGTGGTTGACATAATTGATGAAAATGACAATGCTCCTATGTTCTTAAAGTCAACTTTT
_. __._-________,-...-..,-.,-.w..,.-~-....,r.,-.a..,r"aran,.,.nTTnnnnmmmnm
~CATGCCCTCTTTCCAATTCTGTGTCTATGTCCATGACCAAGGAAGCCC
CTGCCCAAGTCATCATTCATGTCAGAGATGTGAATGATTCCCCTCCCA
nmnTmn
TGCTGATGAAGCTGTT
~GTAGCATATCTGTGCTGAATCCTGCTTTCCTGGGACmmwm:u~~~~~r~H~~Hwy~~~~.1l~.lvHl
mml,mmmn-n nnnT w nmnnmmna na a a a!~_I~TTf_!''af_'TT~
TTTCCTACTTTCTCTTGAATGGCACAGATATGTTTCA
CTATTGAGGATGTCAATGACAA
TTACACATATTTCCGAA
TCTACAACATTGTGGAGGAAGAACC
GTTCACAGTCAGAGCCACGGA
TCGGGAATAATTTCTATGTTCAAC
AGCAGAGAATGCAATGGTTGGAACCAAGGTGATTGATTTGCTAGCCATAGACAAAGATAGTGGTCC
ATGGCACTATAGATTATACTATCATCAATAAACTAGCAAGTGAGAAGTTCTCCATAAACCCCAATG
CAGATTGCCACTCTGCAGAAACTGGATCGGGAAAATTCAACAGAGAGAGTCATTGCTATTAAGGTC
GGCTCGGGATGGAGGAGGAAGAGTAGCCTTCTGCACGGTGAAGATCATCCTCACAGATGAAAATGA
ACCCCCCACAGTTCAAAGCATCTGAGTACACAGTATCCATTCAATCCAATGTCAGTAAAGACTCTC
!GTTATCCAGGTGTTGGCCTATGATGCAGATGAAGGTCAGAACGCAGATGTCACCTACTCAGTGAAC
CCTTTGTATACTTTCTCTGCACCTGAAGACCTTCCAGAGGGGTCTGAAATTGGGATTGTTAAA
GGCAGCTCAAGATCCAGTCATCTACAGTCTAGTGCGGGGCACTACACCTGAGAGCAACAAGGA
TCTTCTCCCTAGACCCAGACACAGGGGTCATAAAGGTGAGGAAGCCCATGGACCACGAATCCA
TTGTACCAGATTGATGTGATGGCACATTGCCTTCAGAACACTGATGTGGTGTCCTTGGTCTCT
CATCCAAGTGGGAGACGTCAATGACAATAGGCGTGTATTTGAGGCTGATCCATATAAGGCTGT
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TCTGCCACAGACTTGGACACTGATACCAATGCTCAGATCACATATTCTCTGCATGGCCCTGGGGCGCA
TGAATTCAAGCTGGATCCTCATACAGGGGAGCTGACCACACTCACTGCCCTAGACCGAGAAAGGAAGG
AT(~TGTTC'.AAC'_GTTGTTGCCAAGGCGACGGATGGAGGTGGCCGATCGTGCCAGGCAGACATCACCCTC
TTTGCCCGGGATCCCGACCAAGGCGCCAATGCCC
TACCGCTGTCCACGCTGGGCACCGTCACAGTCTCGGTGGTGGGCCTAGAAGACTACC
TGAACACCGGCCCCAATTCCCCCAAGATCCAT
AGGAGGGAACCAGCTTGGGCACTTCACCATTCACCCC
GTCAC
AAGCTGGACATCAAACGGGCTAACATTCAC
TAGCATCCA'
ACCACTGTCTGGGTGGTTTCTATGGAAACCTTTCCTCCCA
TC"C'ATC'('TGGTGGAGGAGATGGACGCTTCCATTCGCCTGA
GCCTCATTCTGTTGCATTCTTCCTCGAATGTCTCCCAGGGC
CCTGCCTGGAAGGTGGAACTTGCATC
CCGAGATCCAAAGGGGGGACTGGGGGCAGCAGGAGTTACTGATCA
ATCATAAGCACTGTCGGGCTTCTCTTCTACTGCCGCCGTTGCAAG
'TC:AC'TGTGTC~TTTGTC'AAGTGT('.T(CC:TCC'AC'GTCAGGCAC:TGTC'('TTTGC
137
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GGAGAAAATGGTGATGGAGGGCAACAAGGACTCCGAGGAGCACCACCAGGCCTCGGGCCCCAGAGGTC
CCGCTCCTCAGCCTACACGCAGAGGAACGGGCCCACCTCAGAGTCACACCACTGGCTGCCAGTCAGGG
CCTGCCAGGAGTCTACACAGCTCTGAACCTTCTTTGTTAAAGAATTCAGACCTCATGGAACTCTGGGT
TCTTCATCCCAAGTTTCCCAGGCACTTTTGGCCAAAGGAAGGAAGGAACTAATTCTTCATTTTAAAAA
TTCTTAGGCACTTTTTGACCTTGCTGTCTGGATGAGTTTCCTCAATGGGATTTTTCTTCCCTAGACAC
AAGGAAGTCTGAACTCCTATTTAGGGCCGGTTGGAAGCAGGGAGCTGGACCGCAGTGTCCAGGCTGGA
CACCTGCCATTGCCTCCTCTCCACTGCAGACGCCTGCCCATCAAGTATTACCTGCAGCGACTCAACCC
TATGCATGGAGGGTCAATGTGGGCACATGTCTACACATGTGGGTGCCCATGGATAGTACGTGTGTACA
CATGTGTAGAGTGTATGTAGCCAGGAGTGGTGGGGACCAGAAGCCTCTGTGGCCTTTGGTGACCTCAC
CACTCCCTCCCACCCAGTCCCTCCCTCTGGTCCACTGCCTTTTCATATGTGTTGTTTCTGGAGACAGA
AGTCAAAAGGAAGAGCAGTGGAGCCTTGCCCACAGGGCTGCTGCTTCATGCGAGAGGGAGATGTGTGG
GCGAGAGCCAATTTGTGTGAGTGGTTTGTGGCTGTGTGTGTGACTGTGAGTGTGAGTGACAGATACAT
AGTTTCATTGGTCATTTTTTTTTTTAACAATAAAGTATCTTTTTTTACTGTT
~~~
MW at 479387.3kD
NOV6a, CG51923-O1 SEQ DJ NO: 404349 as
Protein Sequence
MTIALLGFAIFLLHCATCEKPLEGILSSSAWHFTHSHYNATIYENSSPKTWESFEKMGIYLAEPQWA
VRYRIISGDVANVFKTEEYWGNFCFLRIRTKSSNTALLNREVRDSYTLIIQATEKTLELEALTRWV
HILDQNDLKPLFSPPSYRVTISEDMPLKSPICKVTATDADLGQNAEFYYAFNTRSEMFAIHPTSGVVT
VAGKLNVTWRGKHELQVLAVDRMRKISEGNGFGSLAALWHVEPALRKPPAIASVWTPPDSNDGTTY
ATVLVDANSSGAEVESVEWGGDPGKHFKAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARSGSG
PYFYSQIRGFHLPPSKLSSLKFEKAWRVQLSEFSPPGSRWMVRVTPAFPNLQYVLKPSSENVGFKL
NARTGLITTTKLMDFHDRAHYQLHIRTSPGQASTVWIDIWCNNHAPLFNRSSYDGTLDENIPPGTS
VLAVTATDRDHGENGYVTYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGSPFRR
EKEVSIFLQLRNLNDNQPMFEEVNCTGSIRQDWPVGKSIMTMSAIDVDELQNLKYEIVSGNELEYFDL
NHFSGVISLKRPFINLTAGQPTSYSLKITASDGKNYASPTTLNITWKDPHFEVPVTCDKTGVLTQFT
KTILHFIGLQNQESSDEEFTSLSTYQINHYTPQFEDHFPQSIDVLESVPINTPLARLAATDPDAGFNG
KLVWIADGNEEGCFDIELETGLLTVAAPLDYEATNFYILNVTVYDLGTPQKSSWKLLTVNVKDWNDN
APRFPPGGYQLTISEDTEVGTTIAELTTKDADSEDNGRVRYTLLSPTEKFSLHPLTGELWTGHLDRE
SEPRYILKVEARDQPSKGHQLFSVTDLIITLEDVNDNSPQCITEHNRLKVPEDLPPGTVLTFLDASDP
DLGPAGEVRYVLMDGAHGTFRWLMTGALILERELDFERRAGYNLSLWASDGGRPLARRTLCHVEVIV
LDVNENLHPPHFASFVHQGQVQENSPSGTQVIWAAQDDDSGLDGELQYFLRAGTGLAAFSINQDTGM
IQTLAPLDREFASYYWLTVLAVDRGSVPLSSWEVYIEVTDANDNPPQMSQAVFYPSIQEDAPVGTSV
LQLDAWDPDSSSKGKLTFNITSGNYMGFFMIHPVTGLLSTAQQLDRENKDEHILEVTVLDNGEPSLKS
TSRVWGILDVNDNPPIFSHKLFNVRLPERLSPVSPGPWRLVASDLDEGLNGRVTYSIEDSYEEAFS
IDLVTGWSSNSTFTAGEYNILTIKATDSGQPPLSASVRLHIEWIPWPRPSSIPLAFDETYYSFTVME
TDPVNHMVGVISVEGRPGLFWFNISGGDKDMDFDIEKTTGSIVIARPLDTRRRSNYNLTVEVTDGSRT
IATQVHIFMIANINHHRPQFLETRYEVRVPQDTVPGVELLRVQAIDQDKGKSLIYTIHGSQDPGSASL
FQLDPSSGVLVTVGKLDLGSGPSQHTLTVMVRDQEIPIKRNFVWV'I'IHVEDGNLHPPRFTQLHYEAS
PDTIAPGTELLQVRAMDADRGVNAEVHYSLLKGNSEGFFNINALLGIITLAQKLDQANHAPHTLTVKA
EDQGSPQWHDLAWIIHWPSDRSAPIFSKSEYFVEIPESIPVGSPILLVSAMSPSEVTYELREGNKD
GVFSMNSYSGLISTQKKLDHEKISSYQLKIRGSNMAGAFTDVMVWDIIDENDNAPMFLKSTFVGQIS
EAAPLYSMIMDKNNNPFVIHASDSDKEANSLLWKILEPEALKFFKIDPSMGTLTIVSEMDYESMPSF
QFCVYVHDQGSPVLFAPRPAQVIIHVRDVNDSPPRFSEQIYEVAIVGPIHPGMELLMVRASDEDSEVN
YSIKTGNADEAVTIHPVTGSISVLNPAFLGLSRKLTIR.ASDGLYQDTALVKISLTQVLDKSLQFDQDV
YWAAVKENLQDRKALVILGAQGNHLNDTLSYFLLNGTDMFHMVQSAGVLQTRGVAFDREQQDTHELAV
EVRDNRTPQRVAQGLVRVSIEDVNDNPPKFKHLPYYTIIQDGTEPGDVLFQVSATDEDLGTNGAVTYE
FAEDYTYFRIDPYLGDISLKKPFDYQALNKYHLKVIARDGGTPSLQSEEEVLVTVR'NFCSNPLFQSPYY
KVRVPENITLYTPILHTQARSPEGLRLIYNIVEEEPLMLFTTDFKTGVLTVTGPLDYESKTKHVFTVR
ATDTALGSFSEATVEVLVEDVNDNPPTFSQLWTTSISEGLPAQTPVIQLLASDQDSGRNRDVSYQIV
EDGSDVSKFFQINGSTGEMSTVQELDYEAQQHFHVKVFtAMDKGDPPLTGETLVWNVSDINDNPPEFR
QPQYEANVSELATCGHLVLKVQAIDPDSRDTSRLEYLILSGNQDRHFFINSSSGIISMFNLCKKHLDS
SYNLRVGASDGVFRATVPWINTTNANKYSPEFQQHLYEAELAENAMVGTKVIDLLAIDKDSGPYGTI
DYTIINKLASEKFSINPNGQIATLQKLDRENSTERVIAIKVMARDGGGRVAFCTVKIILTDENDNPPQ
FKASEYTVSIQSNVSKDSPVIQVLAYDADEGQNADVTYSVNPEDLVKDVIEINPVTGVVKVKDSLVGL
ENQTLDFFIKAQDGGPPHWNSLVPVRLQWPKKVSLPKFSEPLYTFSAPEDLPEGSEIGIVKAVAAQD
PVIYSLVRGTTPESNKDGVFSLDPDTGVIKVRKPMDHESTKLYQIDVMAHCLQNTDWSLVSVNIQVG
DVNDNRPVFEADPYKAVLTENMPVGTSVIQVTAIDKDTGRDGQVSYRLSADPGSNVHELFAIDSESGW
ITTLQELDCETCQTYHFHWAYDHGQTIQLSSQALVQVSITDENDNAPRFASEEYRGSWENSEPGEL
VATLKTLDADISEQNRQVTCYITEGDPLGQFGISQVGDEWRISSRKTLDREHTAKYLLRVTASDGKFQ
ASVTVEIFVLDVNDNSPQCSQLLYTGKVHEDVFPGHFILKVSATDLDTDTNAQITYSLHGPGAHEFKL
DPHTGELTTLTALDRERKDVFNLVAKATDGGGRSCQADITLHVEDVNDNAPRFFPSHCAVAVFDNTTV
KTPVAWFARDPDQGANAQVVYSLPDSAEGHFSIDATTGVIRLEKPLQVRPQAPLELTVRASDLGTPI
PLSTLGTVTVSWGLEDYLPVFLNTEHSVQVPEDAPPGTEVLQLATLTRPGAEKTGYRWSGNEQGRF
RLDARTGILYVNASLDFETSPKYFLSIECSRKSSSSLSDVTTVMVNITDVNEHRPQFPQDPYSTRVLE
NALVGDVILTVSATDEDGPLNSDITYSLIGGNQLGHFTIHPKKGELQVAKALDREQASSYSLKLRATD
SGQPPLHEDTDIAIQVADVNDNPPRFFQLNYSTTVQENSPIGSKVLQLILSDPDSPENGPPYSFRITK
GNNGSAFRVTPDGWLVTAEGLSRR.AOEWYOLOIOASDSGIPPLSSLTSVRVHVTEOSHYAPSALPLEI
138
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
~iFITVGEDEFQGGMVGKIHATDRDPQDTLTYSLAEEETLGRHFSVGAPDGKIIAAQGLPRGHYSFNVTV
SDGTFTTTAGL'Fi~WWHVGQEALQC2AMWMGFYQLTPEELVSDHWRNLQRFLSHKLDIKRANIHLASLQ
pAEAVAGVDVLLVFEGHSGTFYEFQELASIITHSAKEMEHSVGVQMRSAMPMVPCQGPTCQGQICHNT
VHLDPKVGPTYSTARLSILTPRHHLQRSCSCNGTATRFSGQSYVRYRAPAARNWHIHFYLKTLQPQAI
LLFTNETASVSLKLASGVPQLEYHCLGGFYGNLSSQRHVNDHEWHSILVEEMDASIRLMVDSMGNTSL
WPENCRGLRPERHLLLGGLILLHSSSNVSQGFEGCLDAWVNEEALDLLAPGKTVAGLLETQALTQC
CLHSDYCSQNTCLNGGKCSWTHGAGWCKCPPQFSGKHCEQGRENCTFAPCLEGGTCILSPKGASCNC
PHPYTGDRCEMEARGCSEGHCLVTPEIQRGDWGQQELLIITVAVAFIIISWGLLFYCRRCKSHKPVA
MEDPDLLARSVGVDTQAMPAIELNPLSASSCNNLNQPEPSKASVPNELVTFGPNSKQRPWCSVPPRL
PPAAVPSHSDNEPVIKRTWSSEEMVYPGGAMVWPPTYSRNERWEYPHSEVTQGPLPPSAHRHSTPWM
PEPNGLYGGFPFPLEMENKRAPLPPRYSNQNLEDLMPSRPPSPRERLVAPCLNEYTAISYYHSQFRQG
GGGPCLADGGYKGVGMRLSRAGPSYAVCEVEGAPLAGQGQPRVPPNYEGSDMVESDYGSCEEVMF
_ --
NOV6b, 305869563 SEQ m NO: 41 2_019 by _ _
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
GATGGAGGAGGAAGAGTAGCCTTCTGCACGGTGAAGATCATCCTCACAGATGAAAATGA
CAACCCCCCACAGTTCAAAGCATCTGAGTACACAGTATCCATTCAATCCAATGTCAGTAAAGACTCTC
CGGTTATCCAGGTGTTGGCCTATGATGCAGATGAAGGTCAGAACGCAGATGTCACCTACTCAGTGAAC
CCAGAGGACCTAGTTAAAGATGTCATTGAAATTAACCCAGTCACTGGTGTGGTCAAGGTGAAAGACAG
CCTGGTGGGATTGGAAAATCAGACCCTTGACTTCTTCATCAAAGCCCAAGATGGAGGCCCTCCTCACT
GGAACTCTCTGGTGCCAGTACGACTTCAGGTGGTTCCTAAAAAAGTATCCTTACCGAAATTTTCTGAA
CCTTTGTATACTTTCTCTGCACCTGAAGACCTTCCAGAGGGGTCTGAAATTGGGATTGTTAAAGCAGT
GGCAGCTCAAGATCCAGTCATCTACAGTCTAGTGCGGGGCACTACACCTGAGAGCAACAAGGATGGTG
TCTTCTCCCTAGACCCAGACACAGGGGTCATAAAGGTGAGGAAGCCCATGGACCACGAATCCACCAAA
TTGTACCAGATTGATGTGATGGCACATTGCCTTCAGAACACTGATGTGGTGTCCTTGGTCTCTGTCAA
CATCCAAGTGGGAGACGTCAATGACAATAGGCCTGTATTTGAGGCTGATCCATATAAGGCTGTCCTCA
CTGAGAATATGCCAGTGGGGACCTCAGTCATTCAAGTGACTGCCATTGACAAGGACACTGGGAGAGAT
GGCCAGGTGAGCTACAGGCTGTCTGCAGACCCTGGTAGCAATGTCCATGAGCTCTTTGCCATTGACAG
TGAGAGTGGTTGGATCACCACACTCCAGGAACTTGACTGTGAGACCTGCCAGACTTATCATTTTCATG
TGGTGGCCTATGACCACGGACAGACCATCCAGCTATCCTCTCAGGCCCTGGTTCAGGTCTCCATTACA
GATGAGAATGACAATGCTCCCCGATTTGCTTCTGAAGAGTACAGAGGATCTGTGGTTGAGAACAGTGA
GCCTGGCGAACTGGTGGCGACTCTAAAGACCCTGGATGCTGACATTTCTGAGCAGAACAGGCAGGTCA
CCTGCTACATCACAGAGGGAGACCCCCTGGGCCAGTTTGGCATCAGCCAAGTTGGAGATGAGTGGAGG
ATTTCCTCAAGGAAGACCCTGGACCGCGAGCATACAGCCAAGTACTTGCTCAGAGTCACAGCATCTGA
TGGCAAGTTCCATGCTTCGGTCACTGTGGAGATCTTTGTCCTGGACGTCAATGATAACAGCCCACAGT
GTTCACAGCTTCTCTATACTGGCAAGGTTCATGAAGATGTATTTCCAGGACACTTCATTTTGAAGGTT
TCTGCCACAGACTTGGACACTGATACCAATGCTCAGATCACATATTCTCTGCATGGCCCTGGGGCGCA
TGAATTCAAGCTGGATCCTCATACAGGGGAGCTGACCACACTCACAGCCCTAGACCGAGAAAGGAAGG
ATGTGTTCAACCTTGTTGCCAAGGCGACGGATGGAGGTGGCCGATCGTGCCAGGCAGACATCACCCTC
CATGTGGAGGATGTGAATGACAATGCCCCGCGGTTCTTCCCCAGCCACTGTGCTGTGGCTGTCTTCGA
CAACACCACAGTGAAGACCCCTGTGGCTGTAGTATTTGCCCGGGATCCCGACCAAGGCGCCAATGCCC
AGGTGGTTTACTCTCTGCCGGATTCAGCCGAAGGCCACTTTTCCATCGACGCCACCACGGGGGTGATC
CGCCTGGAAAAGCCGCTGCAGGTCAGGCCCCAGGCACCACTGGAGCTCACGGTCCGTGCCTCTGACCT
GGGCACCCCAATACCGCTGTCCACGCTGGGCACCGTCACAGTCTCGGTGGTGGGCCTAGAAGAC'ACC
TGCCCGTGTTCCTGAACACCGAGCACAGCGTGCAGGTGCCCGAGGACGCCCCACCT
NOV6b, 305869563 SEQ ll~ NO: 42 ~ 679 as MW at ~73948kD
Protein Sequence
DGGGRVAFCTVKIILTDENDNPPQFKASEYTVSIQSNVSKDSPVIQVLAYDADEGQNADVTYSVN
PEDLVKDVIEINPVTGWKVKDSLVGLENQTLDFFIKAQDGGPPHWNSLVPVRLQWPKKVSLPKFSE
PLYTFSAPEDLPEGSEIGIVKAVAAQDPVIYSLVRGTTPESNKDGVFSLDPDTGVIKVRKPMDHESTK
LYQIDVMAHCLQNTDWSLVSVNIQVGDVNDNRPVFEADPYKAVLTENMPVGTSVIQVTAIDKDTGRD
GQVSYRLSADPGSNVHELFAIDSESGWITTLQELDCETCQTYHFHWAYDHGQTIQLSSQALVQVSIT
DENDNAPRFASEEYRGSWENSEPGELVATLKTLDADISEQNRQVTCYITEGDPLGQFGISQVGDEWR
ISSRKTLDREHTAKYLLRVTASDGKFHASVTVEIFVLDVNDNSPQCSQLLYTGKVHEDVFPGHFILKV
SATDLDTDTNAQITYSLHGPGAHEFKLDPHTGELTTLTALDRERKDWNLVAKATDGGGRSCQADITL
HVEDVNDNAPRFFPSHCAVAVFDNTTVKTPVAWFARDPDQGANAQWYSLPDSAEGHFSIDATTGVI
RLEKPLQVRPQAPLELTVRASDLGTPIPLSTLGTVTVSWGLEDYLPVFLNTEHSVQVPEDAPP
NOV6c, 305869567 SEQ m NO: 43 _ 2037 by
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
GATGGAGGAGGAAGAGTAGCCTTCTGCACGGTGAAGATCATCCTCACAGATGAAAATGA
CAACCCCCCACAGTTCAAAGCATCTGAGTACACAGTATCCATTCAATCCAATGTCAGTAAAGACTCTC
CGGTTATCCAGGTGTTGGCCTATGATGCAGATGAAGGTCAGAACGCAGATGTCACCTACTCAGTGAAC
CCAGAGGACCTAGTTAAAGATGTCATTGAAATTAACCCAGTCACTGGTGTGGTCAAGGTGAAAGACAG
CCTGGTGGGATTGGAAAATCAGACCCTTGACTTCTTCATCAAAGCCCAAGATGGAGGCCCTCCTCACT
GGAACTCTCTGGTGCCAGTACGACTTCAGGTGGTTCCTAAAAAAGTATCCTTACCGAAATTTTCTGAA
139
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
:CTTTGTATACTTTCTCTGCACCTGAAGACCTTCCAGAGGGGTCTGAAATTGGGATTGTTAAAGC:ACi'1'
iGCAGCTCAAGATCCAGTCATCTACAGTCTAGTGCGGGGCACTACACCTGAGAGCAACAAGGATGGTG
CCTTCTCCCTAGACCCAGACACAGGGGTCATAAAGGTGAGGAAGCCCATGGACCACGAATCCACCAAA
CTGTACCAGATTGATGTGATGGCACATTGCCTTCAGAACACTGATGTGGTGTCCTTGGTCTCTGTCAA
:ATCCAAGTGGGAGACGTCAATGACAATAGGCCTGTATTTGAGGCTGATCCATATAAGGCTGTCCTCA
:TGAGAATATGCCAGTGGGGACCTCAGTCATTCAAGTGACTGCCATTGACAAGGACACTGGGAGAGAT
3GCCAGGTGAGCTACAGGCTGTCTGCAGACCCTGGTAGCAATGTCCATGAGCTCTTTGCCATTGACAG
CGAGAGTGGTTGGATCACCACACTCCAGGAACTTGACTGTGAGACCTGCCAGACTTATCATTTTCATG
CGGTGGCCTATGACCACGGACAGACCATCCAGCTATCCTCTCAGGCCCTGGTTCAGGTCTCCATTACA
3ATGAGAATGACAATGCTCCCCGATTTGCTTCTGAAGAGTACAGAGGATCTGTGGTTGAGAACAG,TGA
~CCTGGCGAACTGGTGGCGACTCTAAAGACCCTGGATGCTGACATTTCTGAGCAGAACAGGCAGGTCA
~CTGCTACATCACAGAGGGAGACCCCCTGGGCCAGTTTGGCATCAGCCAAGTTGGAGATGAGTGGAGG
~1TTTCCTCAAGGAAGACCCTGGACCGCGAGCATACAGCCAAGTACTTGCTCAGAGTCACAGCATCTGA
rGGCAAGTTCCAGGCTTCGGTCACTGTGGAGATCTTTGTCCTGGACGTCAATGATAACAGCCCACAGT
3TTCACAGCTTCTCTATACTGGCAAGGTTCATGAAGATGTATTTCCAGGACACTTCATTTTGAAGGTT
rCTGCCACAGACTTGGACACTGATACCAATGCTCAGATCACATATTCTCTGCATGGCCCTGGGGCGCA
rGAATTCAAGCTGGATCCTCATACAGGGGAGCTGACCACACTCACTGCCCTAGACCGAGAAAGGAAGG
ATGTGTTCAACCTTGTTGCCAAGGCGACGGATGGAGGTGGCCGATCGTGCCAGGCAGACATCACCCTC
CATGTGGAGGATGTGAATGACAATGCCCCGCGGTTCTTCCCCAGCCACTGTGCTGTGGCTGTCTTCGA
CAACACCACAGTGAAGACCCCTGTGGCTGTAGTATTTGCCCGGGATCCCGACCAAGGCGCCAATGCCC
AGGTGGTTTACTCTCTGCCGGATTCAGCCGAAGGCCACTTTTCCATCGACGCCACCACGGGGGTGATC
CGCCTGGAAAAGCCGCTGCAGGTCAGGCCCCAGGCACCACTGGAGCTCACGGTCCGTGCCTCTGACCT
GGGCACCCCAATACCGCTGTCCACGCTGGGCACCGTCACAGTCTCGGTGGTGGGCCTAGAAGACTACC
TGCCCGTGTTCCTGAACACCGAGCACAGCGTGCAGGTGCCCGAGGACGCCCCACCT
NOV6c, 305869567 SEQ m NO: 44 679 as MW at 73939 kD
Protein Sequence _ _
DGGGRVAFCTVKIILTDENDNPPQFKASEYWSIQSNVSKDSPVIQVLAYDADEGQNADVTYSVN
PEDLVKDVIEINPVTGWKVKDSLVGLENQTLDFFIKAQDGGPPHWNSLVPVRLQWPKKVSLPKF'SE
PLYTFSAPEDLPEGSEIGIVKAVAAQDPVIYSLVRGTTPESNKDGVFSLDPDTGVIKVRKPMDHESTK
LYQIDVMAHCLQNTDWSLVSVNIQVGDVNDNRPVFEADPYKAVLTENMPVGTSVIQVTAIDKDTGRD
'GQVSYRLSADPGSNVHELFAIDSESGWITTLQELDCETCQTYHFHWAYDHGQTIQLSSQALVQVSIT
DENDNAPRFASEEYRGSWENSEPGELVATLKTLDADISEQNRQVTCYITEGDPLGQFGISQVGDEWR
ISSRKTLDREHTAKYLLRVTASDGKFQASVWEIFVLDVNDNSPQCSQLLYTGKVHEDVFPGHFILKV
SATDLDTDTNAQITYSLHGPGAHEFKLDPHTGELTTLTALDRERKDVFNLVAKATDGGGRSCQADITL
HVEDVNDNAPRFFPSHCAVAVFDNTTVKTPVAWFARDPDQGANAQVVYSLPDSAEGHFSIDATTWI
RLEKPLQVRPQAPLELTVRASDLGTPIPLSTLGWWSWGLEDYLPVFLNTEHSVQVPEDAPP
NOV6d, 306076041 SEQ m NO: 45 1455 by
DNA Sequence ORF Start: at 1 ORF Stop: end of sequenca
GACCCCCAGGACACGCTGACCTATAGCCTGGCAGAAGAGGAGACCCTGGGCAGGCACTT
CTCAGTGGGTGCGCCTGATGGCAAGATTATCGCCGCCCAGGGCCTGCCTCGTGGCCACTACTCGTTCA
ACGTCACGGTCAGCGATGGGACCTTCACCACGACTGCTGGGGTCCATGTGTATGTGTGGCATGTGGGG
CAGGAGGCTCTGCAGCAGGCCATATGGATGGGCTTCTACCAGCTCACCCCCGAGGAGCTGGTGAGTGA
CCACTGGCGGAACCTGCAGAGGTTCCTCAGCCATAAGCTGGACATCAAACGGGCTAACATTCACTTGG
CCAGCCTCCAGCCTGCAGAGGCCGTGGCTGGTGTGGATGTGCTCCTGGTCTTTGAGGGGCATTCTGGA
ACCTTCTACGAGTTTCAGGAGCTAGCATCCATCATCACTCACTCAGCCAAGGAGATGGAGCATTCAGT
GGGGGTTCAGATGCGGTCAGCTATGCCCATGGTGCCCTGCCAGGGGCCAACCTGCCAGGGTCAAATCT
GCCATAACACAGTGCATCTGGACCCCAAGGTTGGGCCCACGTACAGCACCGCCAGGCTCAGCATCCTA
ACCCCGCGGCACCACCTGCAGAGGAGCTGCTCCTGCAATGGTACTGCTACAAGGTTCAGTGGTCAGAG
CTATGTGCGGTACAGGGCCCCAGCGGCTCGGAACTGGCACATCCATTTCTATCTGAAAACACTCCAGC
CACAGGCCATTCTTCTATTCACCAATGAAACAGCGTCCGTCTCCCTGAAGCTGGCCAGTGGAGTGCCC
CAGCTGGAATACCACTGTCTGGGTGGTTTCTATGGAAACCTTTCCTCCCAGCGCCATGTGAATGACCA
CGAGTGGCACTCCATCCTGGTGGAGGAGATGGACGCTTCCATTCGCCTGATGGTTGACAGCATGGGCA
ACACCTCCCTTGTGGTCCCAGAGAACTGCCGTGGTCTGAGGCCCGAAAGGCACCTCTTGCTGGGCGGC
CTCATTCTGTTGCATTCTTCCTCGAATGTCTCCCAGGGCTTTGAAGGCTGCCTGGATGCTGTCGTGGT
CAACGAAGAGGCTCTAGATCTGCTGGCCCCTGGCAAGACGGTGGCAGGCTTGCTGGAGACACAAGCCC
TCACCCAGTGCTGCCTCCACAGTGACTACTGCAGCCAGAACACATGCCTCAATGGTGGGAAGTGCTCA
TGGACCCATGGGGCAGGCTATGTCTGCAAATGTCCCCCACAGTTCTCTGGGAAGCACTGTGAACAAGG
AAGGGAGAACTGTACTTTTGCACCCTGCCTGGAAGGTGGAACTTGCATCCTCTCCCCCAAAGGAGCTT
CCTGTAACTGCCCTCATCCTTACACAGGAGACAGGTGTGAAATGGAGGCGAGGGGTTGTTCAGAAGGA
CACTGCCTAGTCACTCCCGAGATCCAAAGGGGGGAC
NOV6d, 306076041 SEQ m NO: 46 485 as MW at ~53871kD
Protein Sequence
DPQDTLTYSLAEEETLGRHFSVGAPDGKIIAAQGLPRGHYSFNVWSDGTFTTTAGVHVYVWHVG
OEALOOAIWMGFYOLTPEELVSDHWRNLORFLSHKLDIKRANIHLASLOPAEAVAGVDVLLVFEGHSG
140
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
TFYEFQELASIITHSAKEMEHSVGVQMRSAMPMVPCQGPTCQGQICHNTVHLDPKVGPTYSTARLSIL
TPRHHLQRSCSCNGTATRFSGQSYVR.YRAPAARNWHIHFYLKTLQPQAILLFTNETASVSLKLASGVP
QLEYHCLGGFYGNLSSQRHVNDHEWHSILVEEMDASIRLMVDSMGNTSLWPENCRGLRPERHLLLGG
LILLHSSSNVSQGFEGCLDAVVVNEEALDLLAPGKTVAGLLETQALTQCCLHSDYCSQNTCLNGGKCS
WTHGAGWCKCPPQFSGKHCEQGRENCTFAPCLEGGTCILSPKGASCNCPHPYTGDRCEMEARGCSEG
HCLVTPEIQRGD
NOV6e, 317868343 . S_EQ_mNO: 47 1_977 by
DNA Sequence _~ QR_F Start: at 1 ~ ORF Stop: end of sequence
ATGACTATTGCCCTGCTGGGTTTTGCCATATTCTTGCTCCATTGTGCGACCTGTGAGAA
GCCTCTAGAAGGGATTCTCTCCTCCTCTGCTTGGCACTTCACACACTCCCATTACAATGCCACCATCT
ATGAAAATTCTTCTCCCAAGACCTATGTGGAGAGCTTCGAGAAAATGGGCATCTACCTCGCGGAGCCA
CAGTGGGCAGTGAGGTACCGGATCATCTCTGGGGATGTGGCCAATGTATTTAAAACTGAGGAGTATGT
GGTGGGCAACTTCTGCTTCCTAAGAATAAGGACAAAGAGCAGCAACACAGCTCTTCTGAACAGAGAGG
TGCGAGACAGCTACACCCTCATCATCCAAGCCACAGAGAAGACCTTGGAGTTGGAAGCTTTGACCCGT
GTGGTGGTCCACATCCTGGACCAGAATGACCTGAAG6CTCTCTTCTCTCCACCTTCGTACAGAGTCAC
CATCTCTGAGGACATGCCCCTGAAGAGCCCCATCTGCAAGGTGACTGCCACAGATGCTGATCTAGGCC
AGAATGCTGAGTTCTATTATGCCTTTAACACAAGGTCAGAGATGTTTGCCATCCATCCCACCAGCGGT
GTGGTCACTGTGGCTGGGAAGCTTAACGTCACCTGGCGAGGAAAGCATGAGCTCCAGGTGCTAGCTGT
GGACCGCATGCGGAAAATCTCTGAGGGCAATGGGTTTGGCAGCCTGGCTGCACTTGTGGTTCATGTGG
AGCCTGCCCTCAGGAAGCCCCCAGCCATTGCTTCAGTGGTGGTGACTCCACCAGACAGCAATGATGGT
ACCACCTATGCCACTGTACTGGTCGATGCAAATAGCTCAGGAGCTGAAGTGGAGTCAGTGGAAGTTGT
TGGTGGTGACCCTGGAAAGCACTTCAAAGCCATCAAGTCTTATGCCCGGAGCAATGAGTTCAGTTTGG
TGTCTGTCAAAGACATCAACTGGATGGAGTACCTTCATGGGTTCAACCTCAGCCTCCAGGCCAGGAGT
GGGAGCGGCCCTTATTTTTATTCCCAGATCAGGGGCTTTCACCTACCACCTTCCAAACTGTCTTCCCT
CAAATTCGAGAAGGCTGTTTACAGAGTGCAGCTTAGTGAGTTTTCCCCTCCTGGCAGCCGCGTGGTGA
TGGTGAGAGTCACCCCAGCCTTCCCCAACCTGCAGTATGTTCTAAAGCCATCTTCAGAGAATGTAGGA
TTTAAACTTAATGCTCGAACTGGGTTGATCACCACCACAA.AGCTCATGGACTTCCACGACAGAGCCCA
CTATCAGCTACACATCAGAACCTCACCGGGCCAGGCCTCCACCGTGGTGGTCATTGACATTGTGGACT
GCAACAACCATGCCCCCCTCTTCAACAGGTCTTCCTATGATGGTACCTTGGATGAGAACATCCCTCCA
GGCACCAGTGTTTTGGCTGTGACTGCCACTGACCGGGATCATGGGGAAAATGGATATGTCACCTATTC
CATTGCTGGACCAAAAGCTTTGCCATTTTCTATTGACCCTTACCTGGGGATCATCTCCACCTCCAAAC
CCATGGACTATGAACTCATGAAAAGAATTTATACCTTCCGGGTAAGAGCATCAGACTGGGGATCCCCT
TTTCGCCGGGAGAAGGAAGTGTCCATTTTTCTTCAGCTCAGGAACTTGAATGACAACCAGCCTATGTT
TGAAGAAGTCAACTGTACAGGTTCTATCTGCCAAGACTGGCCAGTAGGGAAATCGATAATGACTATGT
CAGCCATAGATGTGGATGAGCTTCAGAACCTAAAATACGAGATTGTATCAGGCAATGAACTAGAGTAT
TTTGATCTAAATCATTTCTCCGGAGTGATATCCCTCAAACGCCCTTTTATCAATCTTACTGCTGGTCA
ACCCACCAGTTATTCCCTGAAGATTACAGCCTCAGATGGCAAAAACTATGCCTCACCCACAACTTTGA
ATATTACTGTGGTG _
---.-
~
,
..
:..
NOV6e, 317868343 ' SEQ DJ NO: 48 606 as MW at ~73978kD
Protein Sequence ..._._.~..._.....:~ ~I___.~._~..._,____
MTIALLGFAIFLLHCATCEKPLEGILSSSAWHFTHSHYNATIYENSSPKTYVESFEKMGIYLAEP,
QWAVRYRIISGDVANVFKTEEYWGNFCFLRIRTKSSNTALLNREVRDSYTLIIQATEKTLELEALTR
VVVHILDQNDLKPLFSPPSYRVTISEDMPLKSPICKVTATDADLGQNAEFWAFNTRSEMFAIHPTSG
WTVAGKLNVTWRGKHELQVLAVDRMRKISEGNGFGSLAALWHVEPALRKPPAIASWVTPPDSNDG
TTYATVLVDANSSGAEVESVEWGGDPGKHFKAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARS
GSGPYFYSQIRGFHLPPSKLSSLKFEKAVYRVQLSEFSPPGSRWMVRVTPAFPNLQYVLKPSSENVG
FKLNARTGLITTTKLMDFHDRAHYQLHIRTSPGQASTWVIDIVDCNNHAPLFNRSSYDGTLDENIPP
GTSVLAVTATDRDHGENGWTYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGSP
FRREKEVSIFLQLRNLNDNQPMFEEVNCTGSICQDWPVGKSIMTMSAIDVDELQNLKYEIVSGNE
FDLNHFSGVISLKRPFINLTAGQPTSYSLKITASDGKNYASPTTLNITWLEA
NOV6f, 317868367 S_EQ_m NO: 49 1977 bp_ ___ _
~
~
~ORF Stop: end of sequence
l._,__
DNA Sequence ORF 5tt
ATGACTATTGCCCTGCTGGGTTTTGCCATATTCTTGCTCCATTGTGCGACCTGTGAGAA
GCCTCTAGAAGGGATTCTCTCCTCCTCTGCTTGGCACTTCACACACTCCCATTACAATGCCACCATCT
ATGAAAATTCTTCTCCCAAGACCTATGTGGAGAGCTTCGAGAAAATGGGCATCTACCTCGCGGAGCCA
CAGTGGGCAGTGAGGTACCGGATCATCTCTGGGGATGTGGCCAATGTATTTAAAACTGAGGAGTATGT
GGTGGGCAACTTCTGCTTCCTAAGAATAAGGACAAAGAGCAGCAACACAGCTCTTCTGAACAGAGAGG
TGCGAGACAGCTACACCCTCATCATCCAAGCCACAGAGAAGACCTTGGAGTTGGAAGCTTTGACCCGT
GTGGTGGTCCACATCCTGGACCAGAATGACCTGAAGCCTCTCTTCTCTCCACCTTCGTACAGAGTCAC
CATCTCTGAGGACATGCCCCTGAAGAGCCCCATCTGCAAGGTGACTGCCACAGATGCTGATCTAGGCC
AGAATGCTGAGTTCTATTATGCCTTTAACACAAGGTCAGAGATGTTTGCCATCCATCCCACCAGCGGT
GTGGTCACTGTGGCTGGGAAGCTTAACGTCACCTGGCGAGGAAAGCATGAGCTCCAGGTGCTAGCTGT
GGACCGCATGCGGAAAATCTCTGAGGGCAATGGGTTTGGCAGCCTGGCTGCACTTGTGGTTCATGTGG
AGCCTGCCCTCAGGAAGCCCCCAGCCATTGCTTCGGTGGTGGTGACTCCACCAGACAGCAATGATGGT
141
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
ACCACCTATGCCACTGTACTGGTCGATGCAAATAGCTCAGGAGCTGAAGTGGAGTCAGTGGAAGTTGT
TGGTGGTGACCCTGGAAAGCACTTCAAAGCCATCAAGTCTTATGCCCGGAGCAATGAGTTCAGTTTGG
TGTCTGTCAAAGACATCAACTGGATGGAGTACCTTCATGGGTTCAACCTCAGCCTCCAGGCCAGGAGT
GGGAGCGGCCCTTATTTTTATTCCCAGATCAGGGGCTTTCACCTACCACCTTCCAAACTGTCTTCCCT
CAAATTCGAGAAGGCTGTTTACAGAGTGCAGCTTAGTGAGTTTTCCCCTCCTGGCAGCCGCGTGGTGA
TGGTGAGAGTCACCCCAGCCTTCCCCAACCTGCAGTATGTTCTAAAGCCATCTTCAGAGAATGTAGG
TTTAAACTTAATGCTCGAACTGGGTTGATCACCACCACAAAGCTCATGGACTTCCACGACAGAGCCCA
CTATCAGCTACACATCAGAACCTCACCGGGCCAGGCCTCCACCGTGGTGGTCATTGACATTGTGGACT
GCAACAACCATGCCCCCCTCTTCAACAGGTCTTCCTATGATGGTACCTTGGATGAGAACATCCCTCCA
GGCACCAGTGTTTTGGCTGTGACTGCCACTGACCGGGATCATGGGGAAAATGGATATGTCACCTATTC
CATTGCTGGACCAAAAGCTTTGCCATTTTCTATTGACCCTTACCTGGGGATCATCTCCACCTCCAAAC
CCATGGACTATGAACTCATGAAAAGAATTTATACCTTCCGGGTAAGAGCATCAGACTGGGGATCCCCT
TTTCGCCGGGAGAAGGAAGTGTCCATTTTTCTTCAGCTCAGGAACTTGAATGACAACCAGCCTATGTT
TGAAGAAGTCAACTGTACAGGGTCTATCCGCCAAGACTGGCCAGTAGGGAAATCGATAATGACTATGT
CAGCCATAGATGTGGATGAGCTTCAGAACCTAAAATACGAGATTGTATCAGGCAATGAACTAGAGTAT
TTTGATCTAAATCATTTCTCCGGAGTGATATCCCTCAAACGCCCTTTTATCAATCTTACTGCTGGTCA
ACCCACCAGTTATTCCCTGAAGATTACAGCCTCAGATGGCAAAAACTATGCCTCACCCACAACTTTGA
ATATTACTGTGGTG '_ _
NOV6f, 317868367 ~~ SEQ m NO: 50 ~ 659 as ~ MW at 74031.4kD
3
Protein Sequence
MTIALLGFAIFLLHCATCEKPLEGILSSSAWHFTHSHYNATIYENSSPKTYVESFEKMGIYLAEP
QWAVRYRIISGDVANVFKTEEYVVGNFCFLRIRTKSSNTALLNREVRDSYTLIIQATEKTLELEALTR
VVVHILDQNDLKPLFSPPSYRVTISEDMPLKSPICKVTATDADLGQNAEFYYAFNTRSEMFAIHPTSG
WTVAGKLNVTWRGKHELQVLAVDRMRKISEGNGFGSLAALVVHVEPALRKPPAIASVWTPPDSNDG
TTYATVLVDANSSGAEVESVEWGGDPGKHFKAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARS
GSGPYFYSQIRGFHLPPSKLSSLKFEKAVYRVQLSEFSPPGSRWMVRVTPAFPNLQYVLKPSSENVG
FKLNARTGLITTTKLMDFHDRAHYQLHIRTSPGQASTVWIDIVDCNNHAPLFNRSSYDGTLDENIPP
GTSVLAVTATDRDHGENGYVTYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGSP
FRREKEVSIFLQLRNLNDNQPMFEEVNCTGSIRQDWPVGKSIMTMSAIDVDELQNLKYEIVSGNELEY
FDLNHFSGVISLKRPFINLTAGQPTSYSLKITASDGKNYASPTTLNITW
NOV6g, 317871203 SEQ m NO: 51 1923 by
DNA Sequence ORF Start: at 1 ORF Stop: end of, sequence
GAGAAGCCTCTAGAAGGGATTCTCTCCTCCTCTGCTTGGCACTTCACACACTCCCATTA
CAATGCCACCATCTATGAAAATTCTTCTCCCAAGACCTATGTGGAGAGCTTCGAGAAAATGGGCATCT
ACCTCGCGGAGCCACAGTGGGCAGTGAGGTACCGGATCATCTCTGGGGATGTGGCCAATGTATTTAAA
ACTGAGGAGTATGTGGTGGGCAACTTCTGCTTCCTAAGAATAAGGACAAAGAGCAGCAACACAGCTCT
TCTGAACAGAGAGGTGCGAGACAGCTACACCCTCATCATCCAAGCCACAGAGAAGACCTTGGAGTTGG
AAGCTTTGACCCGTGTGGTGGTCCACATCCTGGACCAGAATGACCTGAAGCCTCTCTTCTCTCCACCT
TCGTACAGAGTCACCATCTCTGAGGACATGCCCCTGAAGAGCCCCATCTGCAAGGTGACTGCCACAGA
TGCTGATCTAGGCCAGAATGCTGAGTTCTATTATGCCTTTAACACAAGGTCAGAGATGTTTGCCATCC
ATCCCACCAGCGGTGTGGTCACTGTGGCTGGGAAGCTTAACGTCACCTGGCGAGGAAAGCATGAGCTC
CAGGTGCTAGCTGTGGACCGCATGCGGAAAATCTCTGAGGGCAATGGGTTTGGCAGCCTGGCTGCACT
TGTGGTTCATGTGGAGCCTGCCCTCAGGAAGCCCCCAGCCATTGCTTCAGTGGTGGTGACTCCACCAG
ACAGCAATGATGGTACCACCTATGCCACTGTACTGGTCGATGCAAATAGCTCAGGAGCTGAAGTGGAG
TCAGTGGAAGTTGTTGGTGGTGACCCTGGAAAGCACTTCAAAGCCATCAAGTCTTATGCCCGGAGCAA
TGAGTTCAGTTTGGTGTCTGTCAAAGACATCAACTGGATGGAGTACCTTCATGGGTTCAACCTCAGCC
TCCAGGCCAGGAGTGGGAGCGGCCCTTATTTTTATTCCCAGATCAGGGGCTTTCACCTACCACCTTCC
AAACTGTCTTCCCTCAAATTCGAGAAGGCTGTTTACAGAGTGCAGCTTAGTGAGTTTTCCCCTCCTGG
CAGCCGCGTGGTGATGGTGAGAGTCACCCCAGCCTTCCCCAACCTGCAGTATGTTCTAAAGCCATCTT
CAGAGAATGTAGGATTTAAACTTAATGCTCGAACTGGGTTGATCACCACCACAAAGCTCATGGACTTC
CACGACAGAGCCCACTATCAGCTACACATCAGAACCTCACCGGGCCAGGCCTCCACCGTGGTGGTCAT
TGACATTGTGGACTGCAACAACCATGCCCCCCTCTTCAACAGGTCTTCCTATGATGGTACCTTGGATG
AGAACATCCCTCCAGGCACCAGTGTTTTGGCTGTGACTGCCACTGACCGGGATCATGGGGAAAATGGA
TATGTCACCTATTCCATTGCTGGACCAAAAGCTTTGCCATTTTCTATTGACCCTTACCTGGGGATCAT
CTCCACCTCCAAACCCATGGACTATGAACTCATGAAAAGAATTTATACCTTCCGGGTAAGAGCATCAG
ACTGGGGATCCCCTTTTCGCCGGGAGAAGGAAGTGTCCATTTTTCTTCAGCTCAGGAACTTGAATGAC
AACCAGCCTATGTTTGAAGAAGTCAACTGTACAGGGTCTATCTGCCAAGACTGGCCAGTAGGGAAATC
GATAATGACTATGTCAGCCATAGATGTGGATGAGCTTCAGAACCTAAAATACGAGATTGTATCAGGCA
ATGAACTAGAGTATTTTGATCTAAATCATTTCTCCGGAGTGATATCCCTCAAACGCCCTTTTATCAAT
CTTACTGCTGGTCAACCCACCAGTTATTCCCTGAAGATTACAGCCTCAGATGGCAAAAACTATGCCTC
ACCCACAACTTTGAATATTACTGTGGTG
NOV6g, 317871203 SEQ m NO: 52 641 as MW at 72058.OkD
Protein Sequence
EKPLEGILSSSAWHFTHSHYNATIYENSSPKTYVESFEKMGIYLAEPOWAVRYRIISGDVANVFK
142
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
YWGNFCFLRIRTKSSNTALLNREVRDSYTLIIQATEKTLELEALTRWVHILDQNDLKPLFSP
VTISEDMPLKSPICKVTATDADLGQNAEFWAFNTRSEMFAIHPTSGWTVAGKLNVTWRGKHE
AWRMRKISEGNGFGSLAALWHVEPALRKPPAIASVWTPPDSNDGTTYATVLVDANSSGAEV
WGGDPGKHFKAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARSGSGPYFYSQIRGFHLPP
SLKFEKAVYRVQLSEFSPPGSRWMVR.VTPAFPNLQYVLKPSSENVGFKLNARTGLITTTKLMD
_ _ x_.._.._. _ _
WTYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGSPFRREKEVSIFLQLRNLND
NQPMFEEVNCTGSICQDWPVGKSIMTMSAIDVDELQNLKYEIVSGNELEYFDLNHFSGVISLKRPFIN
LTAGQPTSYSLKITASDGKNYASPTTLNITW
NOV6h, 317871219 SEQ m NO: 53 1518 by
DNA Se uence ORF Start: at 1 ORF Stop: end of sequence
q
CAATGAGTTCAGTTTGGTGTCTGTCAAAGACATCAACTGGATGGAGTACCTTCA
GACAACCAGCCTATGTTTGAAGAAGTCAACTGTACAGGTTCTATCTGCCAAGA
ATCGATAATGACTATGTCAGCCATAGATGTGGATGAGCTTCAGAACCTAAAAT
GCAATGAACTAGAGTATTTTGATCTAAATCATTTCTCCGGAGTGATATCCCTC
ATTACTGTGGTG
6h, 317871219 SEQ m NO: 54 506 as MW at ~56527kD
RVTISEDMPLKSPICKVTATDADLGQNAEFWAFNTRSEMFAIHPTSGWTVAGKLNVTWRGKHE
LQVLAWRMRKISEGNGFGSLAALWHVEPALRKPPAIASWVTPPDSNDGTTYATVLVDANSSGAE
ESVEWGGDPGKHFKAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARSGSGPYFYSQIRGFHLP
SKLSSLKFEKAWRVQLSEFSPPGSRWMWVTPAFPNLQYVLKPSSENVGFKLNARTGLITTTKLM
FHDRAHYQLHIRTSPGQASTWVIDIVDCNNHAPLFNRSSYDGTLDENIPPGTSVLAVTATDRDHGE
GWTYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGSPFRREKEVSIFLQLRNL
DNQPMFEEVNCTGSICQDWPVGKSIMTMSAIDWELQNLKYEIVSGNELEYFDLNHFSGVISLKRPF
NLTAGOPTSYSLKITASDGKNYASPTTLNITW _.,.,-rw,_
317871243 ~SEQ m NO: 55 1518
NA Sequence ORF Start: at 1 ORF Stop: end of
ATT
TGTAGGATTTAAAC
CGGGCCAGGC
CAAAAGCTTTGCCATTTTCTATTGACCCTTACCTGGGGA
143
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
GACAACCAGCCTATGTTTGAAGAAGTCAACTGTACAGGGTCTATCCGCCAAGACTGGCCAGTAGGGAA
ATCGATAATGACTATGTCAGCCATAGATGTGGATGAGCTTCAGAACCTAAAATACGAGATTGTATCAG
GCAATGAACTAGAGTATTTTGATCTAAATCATTTCTCCGGAGTGATATCCCTCAAACGCCCTTTTATC
AATCTTACTGCTGGTCAACCCACCAGTTATTCCCTGAAGATTACAGCCTCAGATGGCAAAAACTATGC
CTCACCCACAACTTTGAATATTACTGTGGTG
NOV6i, 317871243 SEQ m NO: 56 506 as MW at ~56580kD
Protein Sequence _
RVTISEDMPLKSPICKVTATDADLGQNAEFYYAFNTRSEMFAIHPTSGWTVAGKLNVTWRGKHE
LQVLAWRMRKISEGNGFGSLAALWHVEPALRKPPAIASVWTPPDSNDGTTYATVLVDANSSGA
ESVEWGGDPGKHFKAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARSGSGPYFYSQIRGFHLPP
SKLSSLKFEKAWRVQLSEFSPPGSRWMVRVTPAFPNLQYVLKPSSENVGFKLNARTGLITTTKLMD
FHDRAHYQLHIRTSPGQASTVWIDIVD~NNHAPLFNRSSYDGTLDENIPPGTSVLAVTATDRDHGEN
GYWYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGSPFRREKEVSIELQLRNLN
DNQPMFEEVNCTGSIRQDWPVGKSIMTMSAIDWELQNLKYEIVSGNELEYFDLNHFSGVISLKRPFI
NLTAGQPTSYSLKITASDGKNYASPTTLNITW
NOV6j, 317871246 SEQ m NO: 57 ~ 1992 by
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
ACAGGGTCTATCCGCCAAGACTGGCCAGTAGGGA
AATCGATAATGACTATGTCAGCCATAGATGTGGATGAGCTTCAGAACCTAAAATACGAGATTGTATCA
GGCAATGAACTAGAGTATTTTGATCTAAATCATTTCT,CCGGAGTGATATCCCTCAAACGCCCTTTTATw
CAATCTTACTGCTGGTCAACCCACCAGTTATTCCCTGAAGATTACAGCCTCAGATGGCAAAAACTATG
CCTCACCCACAACTTTGAATATTACTGT.GGTGAAGGACCCTCATTTTGAAGTTCCTGTAACATGTGAT
AAAACAGGGGTATTGACACAATTCACAAAGACTATCCTCCACTTTATTGGGCTTCAGAACCAGGAGTC
CAGTGATGAGGAATTCACTTCTTTAAGCACATATCAGATTAATCATTACACCCCACAGTTTGAGGACC
ACTTCCCCCAATCCATTGATGTCCTTGAGAGTGTCCCTATCAACACCCCCTTGGCCCGCCTAGCAGCC'
ACTGACCCTGATGCTGGTTTTAATGGCAAACTGGTCTATGTGATTGCAGATGGCAATGAGGAGGGCTG
CTTTGACATAGAGCTGGAGACAGGGCTGCTCACTGTAGCTGCTCCCTTGGACTATGAAGCCACCAATT
TCTACATCCTCAATGTAACAGTATATGACCTGGGCACACCCCAGAAGTCCTCCTGGAAGCTGCTGACA
GTGAATGTGAAAGACTGGAATGACAACGCACCCAGATTTCCTCCCGGTGGGTACCAGTTAACCATCTC
GGAGGACACAGAAGTTGGAACCACAATTGCAGAGCTGACAACCAAAGATGCTGACTCGGAAGACAATG
GCAGGGTTCGCTACACCCTGCTAAGTCCCACAGAGAAGTTCTCCCTCCACCCTCTCACTGGGGAACTG
GTTGTTACAGGACACCTGGACCGCGAATCAGAGCCTCGGTACATACTCAAGGTGGAGGCCAGGGATCA
GCCCAGCAAAGGCCACCAGCTCTTCTCTGTCACTGACCTGATAATCACATTGGAGGATGTCAACGACA
ACTCTCCCCAGTGCATCACAGAACACAACAGGCTGAAGGTTCCAGAGGACCTGCCCCCCGGGACTGTC
TTGACATTTCTGGATGCCTCTGATCCTGACCTGGGCCCCGCAGGTGAAGTGCGATATGTTCTGATGGA
TGGCGCCCATGGGACCTTCCGGGTGGACCTGATGACAGGGGCGCTCATTCTGGAGAGAGAGCTGGACT
TTGAGAGGCGAGCTGGGTACAATCTGAGCCTGTGGGCCAGTGATGGTGGGAGGCCCCTAGCCCGCAGG
ACTCTCTGCCATGTGGAGGTGATCGTCCTGGATGTGAATGAGAATCTCCACCCTCCCCACTTTGCCTC
CTTCGTGCACCAGGGCCAGGTGCAGGAGAACAGCCCCTCGGGAACTCAGGTGATTGTAGTGGCTGCCC
AGGACGATGACAGTGGCTTGGATGGGGAGCTCCAGTACTTCCTGCGTGCTGGCACTGGACTCGCAGCC
TTCAGCATCAACCAAGATACAGGAATGATTCAGACTCTGGCACCCCTGGACCGAGAATTTGTATCTTA
CTACTGGTTGACGGTATTAGCAGTGGACAGGGGTTCTGTGCCCCTCTCTTCTGTAACTGAAGTCTACA
TCGAGGTTACGGATGCCAATGACAACCCACCCCAGATGTCCCAAGCTGTGTTCTACCCCTCCATCCAG
GAGGATGCTCCCGTGGGCACCTCTGTGCTTCAACTGGATGCCTGGGACCCAGACTCCAGCTCCAAAGG
GAAGCTGACCTTCAACATCACCAGTGGGAACCACATGGGATTCTTTATGATTCACCCTGTTACAGGTC
TCCTATCTACAGCCCAGCAGCTGGACAGAGAGAACAAGGATGAACACATCCTGGAGGTGACTGTGCTG
GACAATGGGGAACCCTCACTGAAGTCCACCTCCAGGGTGGTGGTAGGCATCTTG
_
.. .,.,. ,.~~~~y _
~
SEQ m NO: 58 664 as ~ MW at ~74703kD
NOV6j, 317871246
Protein Sequence
TGSIRQDWPVGKSIMTMSAIDVDELQNLKYEIVSGNELEYFDLNHFSGVISLKRPFI
NLTAGQPTSYSLKITASDGKNYASPTTLNITVVKDPHFEVPVTCDKTGVLTQFTKTILHFIGLQNQES
SDEEFTSLSTYQINHYTPQFEDHFPQSIDVLESVPINTPLARLAATDPDAGFNGKLVYVIADGNEEGC
FDIELETGLLTVAAPLDYEATNFYILNVTVYDLGTPQKSSWKLLTVNVKDWNDNAPRFPPGGYQLTIS
EDTEVGTTIAELTTKDADSEDNGRVRYTLLSPTEKFSLHPLTGELWTGHLDRESEPRYILKVEARDQ
PSKGHQLFSWDLIITLEDVNDNSPQCITEHNRLKVPEDLPPGTVLTFLDASDPDLGPAGEVRYVLMD
GAHGTFRVDLMTGALILERELDFERRAGYNLSLWASDGGRPLARRTLCHVEVIVLDVNENLHPPHFAS
FVHQGQVQENSPSGTQVIWAAQDDDSGLDGELQYFLRAGTGLAAFSINQDTGMIQTLAPLDREFVSY
YWLTVLAWRGSVPLSSVTEVYIEVTDANDNPPQMSQAWYPSIQEDAPVGTSVLQLDAWDPDSSSKG
KLTFNITSGNHMGFFMIHPWGLLSTAQQLDRENKDEHILEVTVLDNGEPSLKSTSRVWGIL
NOV6k, 317999764 SEQ m NO: 59 1773 by
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
TACCCCTCCATCCAGGAGGATGCTCCCGTGGGCACCTCTGTGCTTCAACTGGATGCCTG
.
144
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
ACAGGTCTCCTATCT
ICATGGTGGGGGTCATCAGCGTAGAGGGCAGACCCGGACTCTTCTGGTTCAACATCTCAGGTGGGGAT
TGGCATGACCTGGCTACAGTGATCATTCA
V6k, 317999764 ~SEQ B? NO: 60 591 as MW at ~65888kD
PSIQEDAPVGTSVLQLDAWDPDSSSKGKLTFNITSGNHMGFFMIHPVTGLLSTAQQLDRENKDE
ILEVTVLDNGEPSLKSTSRVVVGILDVNDNPPIFSHKLFNVRLPERLSPVSPGPVYRLVASDLD
GRVTYSIEDSDEEAFSIDLVTGVVSSSSTFTAGEYNILTIKATDSGQPPLSASVRLHIEWIPWP
IPLAFDETYYSFTVMETDPVNHMVGVISVEGRPGLFWFNISGGDKDMDFDIEKTTGSIVIARPL
SLIYTIHGSQDPGSASLFQLDPS SGVLVTVGKLDLG~Gr~~H't'L'rvMV tc~ur;l rl~uvr-v w vwlrm
GNLHPPRFTQLHYEASVPDTIAPGTELLQVR.AMDADRGVNAEVHYSLLKGNSEGFFNINALLGIIT
QKLDQANHAPHTLTVKAEDQGSPQWHDLATVIIHVYPSDRSAPIFSKSEYFVEIPESIPVGSPILL
AMSPSEVTYELREGNKDGVFSMNSYSGLISTQKKLDHEKISSYQLKIRGS~,~ ~..m.. _
OV61, 318176301 SEQ m NO:61 2019 by
NA Sequence pRF Start: at 1 ~ ORF Stop: end of
TATGAGCCCCTCTGAAGTTACCTA
TGATGTCATGGTGGTGGTTGACATAATTGATGAAA.ATGACAATGCTCCT.
mmr~_mrrr_r_rr n a nmmn rmrA A~c ~ ArCTCCACTGTATAGCATGATCATGGA
ATGGGAACCCTAACCATTGTATCA
TGTCCATGACCAAGGAAGCCCTGT
TGAGGTAGCAATAGTCGGGCCTATCCATC:c:AGGC:
AwGG.aG~u°u~'twHw~~m~~~~~~~Hw
ACTCAGAAGTCAATTATAGCATCAAAACTGGCAATGCTGATGAAGCTGTT~CCATCCATCC~
GGTAGCATATCTGTGCTGAATCCTGCTTTCCTGGGACTCTCTCGGAAGCTCACCATCAGGG
GACACTCATGAGTTGGCAGTGGAAGTGAGGGACAATCGGACACCTCAGC
AGATGGCACAGAGCCAGGGGATGTCCTCTTTCAGG'1'
GGGCTGTTACATATGAATTTGCAGAAGATTACACAT
TCACTCAAGAAACCCTTTGATTATCAAGCTTTAAAT
145
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
CTGTTTCAGAGTCCTTATTACAAAGTCAGAGTACCTGAAAATATCACCCTCTATACCCCAATTCTCCA
_ _ _ __ ............-. ,-." r.r.r.T nmr~r~rnr~r~mramr-~mara
arAmmC,TCCtAGGAAGAACCCTTGATGC
NOV61, 318176301 ' SEQ m NO: 62 673 as MW at ~75571kD
Protein Sequence ~ __
EASVPDTIAPGTELLQVRAMDADRGVNAEVHYSLLKGNSEGFFNINALLGIITLAQKLDQANHAP
HTLTVKAEDQGSPQWHDLATVIIHVYPSDRSAPIFSKSEYFVEIPESIPVGSPILLVSAMSPSEVTYE
LREGNKDGVFSMNSYSGLISTQKKLDHEKISSYQLKIRGSNMAGAFTDVMWVDIIDENDNAPMFLKS
TFVGQISEAAPLYSMIMDKNNNPFVIHASDSDKEANSLLVYKILEPEALKFFKIDPSMGTLTIVSEMD
YESMPSFQFCVYVHDQGSPVLFAPRPAQVTIHVRDVNDSPPRFSEQIYEVAIVGPIHPGMELLMVRAS
DEDSEVNYSIKTGNADEAVTIHPVTGSISVLNPAFLGLSRKLTIRASDGLYQDTALVKISLTQVLDKS ..
LQFDQDVYWAAVKENLQDRKALVILGAQGNHLNDTLSYFLLNGTDMFHMVQSAGVLQTRGVAFDREQQ
DTHELAVEVRDNRTPQRVAQGLVRVSIEDVNDNPPKFKHLPYYTIIQDGTEPGDVLFQVSATDEDLGT
NGAVTYEFAEDYTYFRIDPYLGDISLKKPFDYQALNKYHLKVIARDGGTPSLQSEEEVLVTVRNKSNP
LFQSPYYKVRVPENITLYTPILHTQARSPEGLRLIYNIVEEEPLMLFTTDFKTGVLTVTGPLDY
NOV6rn, CG51923-02 SEQ m NO: 63 3666 by ''
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence '
TATAAGGCTGTCCTCACTGAGAATATGCCAGTGGGGACCTCAGTCATTCAAGTGACTGCCATTGACAA
__-___ _._ .........,.-.....,T r..-.,. TmnmnnTmr7vrr
.TGAGAATGACAATGCTCCC
ATAGCACAAGGGTCTTAGAGAATGCCCTTGTGGGTGACGTCATCCTCACGGTATCAUC:u
.GATGGACCCCTAAATAGTGACATTACCTATAGCCTCATAGGAGGGAACCAGCTTGGGCA ..
'TCACCCCAA.AAAGGGGGAGCTACAGGTGGCCAAGGCCCTGGACCGGGAACAGGCCTCTA
TGAAGCTCCGAGCCACAGACAGTGGGCAGCCTCCACTGCATGAGGACACAGACATCGCT
aGCTGATGTCAATGATAACCCACCGAGATTCTTCCAGCTCAACTACAGCACCACTGTCCA
_____ __......_....T r""m.,.-.mr.r.Tnnmr~amrrmr_ar~mGACC~ACCATTCTCCAGAGAATG .
.
GTGGCTGGTGTGGACGTGCTCCTGGTC~
CAGGGTCAAATC
146
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
V6m, CG51923-02 SEQ m NO: 641222 as MW at 133578.OkD
~YKAVLTENMPVGTSVIQVTAIDKDTGRDGQVSYRLSADPGSNVHELFAIDSESGWITTLC~ELUC~'u
TYHFHVVAYDHGQTIQLSSQALVQVSITDENDNAPRFASEEYRGSVVENSEPGELVATLKTLDADI
QNRQVTCYITEGDPLGQFGISQVGDEWRISSRKTLDREHTAKYLLRVTASDGKFQASVTVEIFVLD
__ --_ ______.. .....,-..,_...-.....a-."..,"~.rr rvrW rmnL~T mmr~.m
VAVFDNTTVKTPVAVVF
GLEDYLPVFLNTEHSVQVPEDAPPGTEVLQLATLTRPGAEKTGYRWSGNEQGRFRLDARTGILYV
SLDFETSPKYFLSIECSRKSSSSLSDVTTVMVNITDVNEHRPQFPQDPYSTRVLENALVGDVILTV
TDEDGPLNSDITYSLIGGNQLGHFTIHPKKGELQVAKALDREQASSYSLKLRATDSGQPPLHEDTD,
QASDSGIPPLSSST
GFYQLTPEELVSDHWRNLQRFLSHKLDIKRANIHLASLQPAEAVAGVDVLL
IITHSAKEMEHSVGVQMRSAMPMVPCQGPTCQGQICHNTVHLDPKVGPTYS
SCNGTATRFSGQSYVR'YRAPAARNWHIHFYLKTLQPQAILLFTNETAS,VSL
YGNLSSQRHVNDHEWFiSILVEEMDASIRLMVDSMGNTSLWPENCRGLI~PE
ILSPKGASCNCPHPYTGDRCEM
V6n, CG51923-03 SEQ m NO: 65 314279 by
A Sequence ORF Stmt: ATG at 14 ORF Stop: TAG at 12806
ATTGCCCTGCTGGGTTTTGCCATATTCTTGCTC
GGA
ATTATGCCTTTAACACAAGGTCAGAGATGTTTGCCATCCA
CTGGC
TTTTTA
TTTAAACTTAATGCTCGAACTGGGTTGATCACCACCACAAAGCTCA
GACTGCAACAACCATGCCCCCCTCTTCAACAGGTCTTCCTATGATGGTACCT'1'GCUA'1'U~c~~~~'r~~c
TCCAGGCACCAGTGTTTTGGCTGTGACTGCCACTGACCGGGATCATGGGGAAAATGGATATGTCACCT
ATTCCATTGCTGGACCAAAAGCTTTGCCATTTTCTATTGACCCCTACCTGGGGATCATCTCCACCTCC
AAAC~CCATGGACTATGAACTCATGAAAAGAATTTATACCTTCCGGGTAAGAGCATCAGACTGGGGATC
GTATTTTGATCTAAATCATTTCTCCGGAGTGATATCCCTCAAACGCCCTTTTATCAATCTT
AGAGC~
ATA
147
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
CCACAGAGAAGTTCTCCCTCCACCCTCTCACTGGGGAAC:'1'GCU'1"1'ci'1"nAl:~tzt~H~-H
TCAGAGCCTCGGTACATACTCAAGGTGGAGGCCAGGGATCAGCCCAGCAAAGGCC
CACATCTTCATGATTGCCAACATTAACCACCATCGGCCCC
ArmmrcrrAC~GACACCGTGCCAGGGGTAGAGCTCCTGCGA
GCTCGGGGCCCTCCCAGCACACACTGACAGTCATGGTCCGAGACCAGGAAATACCT
GACAGTGAAGGCAGAAGATCAAGGCTCCCCACAATGGCA
TAAGGATGGAGTCTTCTCTATGAACTCATATTCTGGCCTTA
ArAAAATCTCGTCTTACCAGCTGAAAATCCGAGGCAGCAAT
AATTGATGAAAATGACAATGCTCCTA
ATGCCCTCTTTCCAATTCTGTGTCTATGTCCATGACCAAGGAAGCCCTGTATTATTTGCACCCAGAC
TGCCCAAGTCATCATTCATGTCAGAGATGTGAATGATTCCCCTCCCAGATTCTCAGAACAGATATAT
_ __ ----__,-., .........,..-.r..-..-.nr",-n ~,1,.-mn mw ~n n-n
TCCCCCCAAATTTAAGCATCTGCCCT
GGAAC
TCAGA
TGGTGAAACCCTTGTGGTTGTCAATGTGTCTGATATCAATGACAACCC
148
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
AATCAGGACAGGCACTTCTTCATTAACAGCTCATCGGGAATAATTTCTATGTTCAACCTTTGCAAAAA
GCACCTGGACTCTTCTTACAATTTGAGGGTAGGTGCTTCTGATGGAGTCTTCCGAGCAACTGTGCCTG
TGTACATCAACACTACAAATGCCAACAAGTACAGCCCAGAGTTCCAGCAGCACCTTTATGAGGCAGAA
TTAGCAGAGAATGCAATGGTTGGAACCAAGGTGATTGATTTGCTAGCCATAGACAAAGATAGTGGTCC
CTATGGCACTATAGATTATACTATCATCAATAAACTAGCAAGTGAGAAGTTCTCCATAAACCCCAATG
ATCCAGGTGTTGGCCT
TT
GGCAGCTCAAGATCCAGTCATCTACAGT
TCTTCTCCCTAGACCCAGACACAGGGGT
TTGTACCAGATTGATGTGATGGCACATT
CATCCAAGTGGGAGACGTCAATGACAAT
' ~, TGAGAGTGGTTGGATCACCACA
TGGTGGCCTATGACCACGGACA
~GATGAGAATGACAATGCTCCCC
CC.TGGATGCTGACA
GTGGCTGTAGTATTTGCCCGGGATCC
TTCAGCCGAAGGCCACTTTTCCATCG
CCCTAAATAGTGACATTACCTATAGCCTCATAGGAGGGAACCAGCTTGGGCACTTCAC
AA.AAAGGGGGAGCTACAGGTGGCCAAGGCCCTGGACCGGGAACAGGCCTCTAGTTATT
CCGAGCCACAGACAGTGGGCAGCCTCCACTGCATGAGGACACAGACATCGCTATCCAA
TCAATGATAACCCACCGAGATTCTTCCAGCTCAACTACAGCACCACTGTCCAGGAGAA
GGC'AGCAAAGTCCTGCAGCTGATCCTGAGTGACCCAGATTCTCCAGAGAATGGCCCCC
GAACCTTCTACGAGTTTCAGGAGCTAGCATCCATCATCACTCACTCAGCCAAGGAGATGGAGCATTC.
GTGGGGGTTCAGATGCGGTCAGCTATGCCCATGGTGCCCTGCCAGGGGCCAACCTGCCAGGGTCAAA
CTGCCATAACACAGTGCATCTGGACCCCAAGGTTGGGCCCACGTACAGCACCGGCCAGGCNTTAACA
CCCTAACCCCGCGGCACCACCTGCAGAGGAGCTGCTCCTGCAATGGTACTGCTACAAGGTTCAGTGG
CAGAGCTATGTGCGGTACAGGGTCCCAGCGGCTCGGAACTGGCACATCCATTTCTATCTGAAAACAC
CCAGCCACAGGCCATTCTTCTATTCACCAATGAAACAGCGTCCGTCTCCCTGAAGGGCTTTGAAGGC
GCCTGGATGCTGTCGTGGTCAACGAAGAGGCTCTAGATCTGCTGGCCCCTGGCAAGACGGTGGCAGG
TTGCTGGAGACACAAGCCCTCACCCAGTGCTGCCTCCACAGTGACTACTGCAGCCAGAACACATGCC
CAATGGTGGGAAGTGCTCATGGACCCACGGGGCAGGCTATGTCTGCAAATGTCCCCCACAGTTCTCT
GGAAGCACTGTGAACAAGGAAGGGAGAACTGTACTTTTGCACCCTGCCTGGAAGGTGGAACTTGCAT
CT('_TC'CC:C"CAAAGGAG('.TTC'C('.TGTAAC:TGC'CC.TCATC'.C:TTAC".ACAC;GAGA('.AGGTGT
GAAATC;GAGG
149
CA 02495563 2005-02-O1
WO 2004/015079 PCT/US2003/024931
GAGGGGTTGTTCAGAAGGACACTGCCTAGTCACTCCCGAGATCCAAAGGGGGGACfiGGGGGCAGCAGG
P:GTTACTGATCATCACAGTGGCCGTGGCGTTCATTATCATAAGCACTGTCGGGCTTCTCTTCTACTGC~
.
CGCCGTTGCAAGTCTCACAAGCCTGTGGCCATGGAGGACCCAGACCTCCTGGCCAGGAGTGTTGGTGT
TGACACCCAAGCCATGCCTGCCATCGAGCTCAACCCATTGAGTGCCAGCTCCTGCAACAACCTCAACC
AACTGGAACCCAGCAAGGCCTCTGTTCCAAATGAACTCGTCACATTTGGACCCAATTCTAAGCAACGG
CCAGTGGTCTGCAGTGTGCCCCCCAGACTCCCGCCAGCTGCGGTCCCTTCCCACTCTGACAATGGGCC
TGTCATTAAGAGAACCTGGTCCAGTGAGGAGATGGTGTACCCTGGCGGAGCCATGGTCTGGCCCCCTA
CTTACTCCAGGAACGAACGCTGGGAATACCCCCACTCCGAAGTGACTCAGGGCCCTCTGCCGCCCTCG
GCTCACCGCCACTCAACCCCAGTCGTGATGCCAGAGCCTAATGGCCTCTATGGGGGCTTCCCCTTCCC
CCTGGAGATGGAAAACAAGCGGGCACCTCTCCCACCCCGTTACAGCAACCAGAACCTGGAAGATCTG
TGCCCTCTCGGCCCCCTAGTCCCCGGGAGCGCCTGGTTGCCCCCTGTCTCAATGAGTACACGGCCATC
AGCTACTACCACTCGCAGTTCCGGCAGGGAGGGGGAGGGCCCTGCCTGGCAGACGGGGGCTACAAGGG
GGTGGGTATGCGCCTCAGCCGAGCTGGGCCCTCTTATGCTGTCTGTGAGGTGGAGGGGGCACCTCTTG
CAGGCCAGGGCCAGCCCCGGGTGCCCCCCAACTATGAGGGCTCTGACATGGTGGAGAGTGATTATGGC
AGCTGTGAGGAGGTCATGTTCTAGCTTCCCATTCCCAGAGCAAGGCAGGCGGGAGGCCAAGGACTGGA
CTTGGCTTATTTCTTCCTGTCTCGTAGGGGGTGAGTTGAGTGTGGCTGGGAGAGTGGGAGGGAAGCCC'
.
TCAGCCCAGGCTGTTGTCCCTTGAAATGTGCTCTTCCAATCCCCCACCTAGTCCCTGAGGGTGGAGGG
AAGCTGAGGATAGAGCTCCAGAAACAGCACTAGGGTCCCAGGAGAGGGGCATTTCTAGAGCAGTGACC
CTGGAA.AACCAGGAACAATTGACTCCCGGGGTGGGCGAGAGACAGGAGGGCTCCCTGATCTGCCGGCT
CTCAGTCCCCGGGGCAGAGCCTGATTGACTGTGCTGGCTCAACTTCACCAAGATGCATTCTCATACCT
GCCCACAGCTCCATTTTGGAGGCAGGCAGGTTGGTGCCTGACAGACAACCACTACGCGGGCCGTACAG
AGGAGCTCTAGAGGGCTGCGTGGCATCCTCCTAGGGGCTGAGAGGTGAGCAGCAGGGGAGCGGGCACA
GTCCCCTCTGCCCCTGCCTCAGTCGAGCACTCACTGTGTCTTTGTCAAGTGTCTGCTCCACGTCAGGC
ACTGTGCTTTGCACCGGGGAGAAAATGGTGATGGAGGGCAACAAGGACTCCGAGGAGCACCACCAGGC
CTCGGGCCCCAGAGGTCCCACTCCTCAGCCTACACGCAGAGGAACGGGCCCACCTCAGAGTCACACCA
CTGGCTGCCAGTCAGGGCCTGCCAGGAGTCTACACAGCTCTGAACCTTCTTTGTTAAAGAATTCAGAC
CTCATGGAACTCTGGGTTCTTCATCCCAAGTTTCCCAGGCACTTTTGGCCAAAGGAAGGAAGGAACTA
ATTCTTCATTTTAAAAATTCTTAGGCACTTTTTGACCTTGCTGTCTGGATGAGTTTCCTCAATGGGAT
TTTTCTTCCCTAGACACAAGGAAGTCTGAACTCCTATTTAGGGCCGGTTGGAAGCAGGGAGCTGGACC
GCAGTGTCCAGGCTGGACACCTGCCATTGCCTCCTCTCCATTGCAGACGCCTGCCCATCAAGTATTAC
TGCGGCGACTCAACCCTATGCATGGAGGGTCAATGTGGGCACATGTCTACACATGTGGGTGCCCATGG
ATAGTACGTGTGTACACATGTGTAGAGTGTATGTAGCCAGGAGTGGTGGGGACCAGAAGCCTCTGTGG
CCTTTGGTGACCTCACCACTCCCTCCCACCCAGTCCCTCCCTCTGGTCCACTGCCTTTTCATATGTGT
TGTTTCTGGAGACAGAAGTCAAAAGGAAGAGCAGTGGAGCCTTGCCCACAGGGCTGCTGCTTCATGCG
AGAGGGAGATGTGTGGGCGAGAGCCAATTTGTGTGAGTGGTTTGTGGCTGTGTGTGTGACTGTGAGTG
TGAGTGACAGATACATAGTTTCATTGGTCATTTTTTTTTTAACAATAAAGTATCTTTTTTTACTGTT
NOV6n, CG51923-03 SEQ m NO: 66 4264 aa~ , MW at 469871.7kD
Protein Sequence a
,~_. _._
MTIALLGFAIFLLHCATCEKPLEGILSSSAWHFTHSHYNATIYENSSPKTWESFEKMGIYLAEPQWA
VRYRIISGDVANVFKTEEYWGNFCFLRIRTKSSNTALLNREVRDSYTLIIQATEKTLELEALTRVW
HILDQNDLKPLFSPPSYRVTISEDMPLKSPICKVTATDADLGQNAEFYYAFNTRSEMFAIHPTSGWT
VAGKLNVTWRGKHELQVLAVDRMRKISEGNGFGSLAALVVHVEPALRKPPAIASVWTPPDSNDGTTY
ATVLVDANSSGAEVESVEWGGDPGKHFKAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARSGSG
PYFYSQIRGFHLPPSKLSSLKFEKAWRVQLSEFSPPGSRVVMVRVTPAFPNLQYVLKPSSENVGFKL
NARTGLITTTKLMDFHDRAHYQLHIRTSPGQASTVWIDIVDCNNHAPLFNRSSYDGTLDENIPPGTS
VLAVTATDRDHGENGYWYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGSPFRR
EKEVSIFLQLRNLNDNQPMFEEVNCTGSIRQDWPVGKSIMTMSAIDVDELQNLKYEIVSGNELEYFDL
NHFSGVISLKRPFINLTAGQPTSYSLKITASDGKNYASPTTLNITWKDPHFEVPVTCDKTGVLTQFT
KTILHFIGLQNQESSDEEFTSLSTYQINHYTPQFEDHFPQSIDVLESVPINTPLARLAATDPDAGFNG
~LVYVIADGNEEGCFDIELETGLLTVAAPLDYEATNFYILNVTVYDLGTPQKSSWKLLTVNVKDWNDN
APRFPPGGYQLTISEDTEVGTTIAELTTKDADSEDNGRVRYTLLSPTEKFSLHPLTGELWTGHLDRE
SEPRYILKVEARDQPSKGHQLFSVTDLIITLEDVNDNSPQCITEHNRLKVPEDLPPGTVLTFLDASDP
DLGPAGEVRYVLMDGAHGTFRVDLMTGALILERELDFERRAGYNLSLWASDGGRPLARRTLCHVEVIV
LDVNENLHPPHFASFVHQGQVQENSPSGTQVIWAAQDDDSGLDGELQYFLRAGTGLAAFSINQDTGM
IQTLAPLDREFASYYWLTVLAVDRGSVPLSSVTEVYIEVTDANDNPPQMSQAVFYPSIQEDAPVGTSV
LQLDAWDPDSSSKGKLTFNITSGNYMGFFMIHPVTGLLSTAQQLDRENKDEHILEVTVLDNGEPSLKS
TSRVWGILDVNDNPPTFSHKLFNVRLPERLSPVSPGPWRLVASDLDEGLNGRVTYSIEDSYEEAFS
IDLVTGWSSNSTFTAGEYNILTIKATDSGQPPLSASVRLHIEWIPWPRPSSIPLAFDETYYSFTVME
TDPVNHMVGVISVEGRPGLFWFNISGGDKDMDFDIEKTTGSIVIARPLDTRRRSNYNLTVEVTDGSRT
IATQVHIFMIANINHHRPQFLETRYEVRVPQDTVPGVELLRVQAIDQDKGKSLIYTIHGSQDPGSASL
FQLDPSSGVLVWGKLDLGSGPSQHTLTVMVRDQEIPIKRNFVWVTIHVEDGNLHPPRFTQLHYEASV
PDTIAPGTELLQVRAMDADRGVNAEVHYSLLKGNSEGFFNINALLGIITLAQKLDQANHAPHTLTVKA
EDQGSPQWHDLAWIIHWPSDRSAPIFSKSEYFVEIPESIPVGSPILLVSAMSPSEVTYELREGNKD
GVFSMNSYSGLISTQKKLDHEKISSYQLKIRGSNMAGAFTDVNtWVDIIDENDNAPMFLKSTFVGQIS
EAAPLYSMIMDKNNNPFVIHASDSDKEANSLLWKILEPEALKFFKIDPSMGTLTIVSEMDYESMPSF
QFCVYVHDQGSPVLFAPRPAQVIIHVRDVNDSPPRFSEQIYEVAIVGPIHPGMELLMVRASDEDSEVN
YSIKTGNADEAVTIHPVTGSISVLNPAFLGLSRKLTIRASDGLYODTALVKISLTOVLDKSLOFDODV
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RVAQGLVRVSIEDVNDNPPKFKHLPYYTIIQDGTEPGDVLFQVSATDEDLGTNGAV
IDPYLGDISLKKPFDYQALNKYHLKVIARDGGTPSLQSEEEVLVTVRNKSNPLFQS
LYTPILHTQARSPEGLRLIYNIVEEEPLMLFTTDFKTGVLTVTGPLDYESKTKHVF
SEATVEVLVEDVNDNPPTFSQLWTTSISEGLPAQTPVIQLLASDQDSGRNRDVSY
FQINGSTGEMSTVQELDYEAQQHFHVKVRAMDKGDPPLTGETLVWNVSDINDNPP
EI~ATCGHLVLKVQAIDPDSRDTSRLEYLILSGNQDRHFFINSSSGIISMFNLCKKH
DGVFRATVPVYINTTNANKYSPEFQQHLYEAELAENAMVGTKVIDLLAIDKDSGPY
SEKFSINPNGQIATLQKLDRENSTERVIATKVMARDGGGRVAFCTVKIILTDENDN
IQSNVSKDSPVIQVLAYDADEGQNADVTYSVNPEDLVKDVIEINPVTGVVKVKDSL
KAQDGGPPHWNSLVPVRLQWPKKVSLPKFSEPLYTFSAPEDLPEGSEIGIVKAVA
TTPESNKDGVFSLDPDTGVIKVRKPMDHESTKLYQIDVMAHCLQNTDWSLVSVNI
ITTLQELDCETCQTYHFHWAYDHGQTIQLSSQALVQVSITDENDNAPRFASEEYRGSWENSEPGEL
VATLKTLDADISEQNRQVTCYITEGDPLGQFGISQVGDEWRISSRKTLDREHTAKYLLRVTASDGKFQ
ASVTVEIFVLDVNDNSPQCSQLLYTGKVHEDVFPGHFILKVSATDLDTDTNAQITYSLHGPGAHEFKL ,
DPHTGELTTLTALDRERKDVFNLVAKATDGGGRSCQADITLHVEDVNDNAPRFFPSHCAVAVFDNTTV
KTPVAVVFARDPDQGANAQVVYSLPDSAEGHFSIDATTGVIRLEKPLQVRPQAPLELTVRASDLGTPI
PLSTLGTVTVSWGLEDYLPVFLNTEHSVQVPEDAPPGTEVLQLATLTRPGAEKTGYRWSGNEQGRF
RLDARTGILYVNASLDFETSPKYFLSIECSRKSSSSLSDVTTVMVNITDVNEHRPQFPQDPYSTRVLE
NALVGDVILTVSA~'DEDGPLNSDITYSLIGGNQLGHFTIHPKKGELQVAKALDREQASSYSLKLRATD ,
SGQPPLHEDTDIAIQVADVNDNPPRFFQLNYSTTVQENSPIGSKVLQLILSDPDSPENGPPYSFRITK
GNNGSAFRVTPDGWLVTAEGLSRRAQEWYQLQIQASDSGIPPLSSLTSVRVHVTEQSHYAPSALPLEI
FITVGEDEFQGGMVGKIHATDRDPQDTLTYSLAEEETLGRHFSVGAPDGKIIAAQGLPRGHYSFNVTV
PAEAVAGVDVLLVFEGHSGTFYEFQELASIITHSAKEMEHSVGVQMRSAMPMVPCQGPTCQGQICHNT
VHLDPKVGPTYSTGQALTSLTPRHHLQRSCSCNGTATRFSGQSYVRYRVPAARNNIHIHFYLKTLQPQA
ILLFTNETASVSLKGFEGCLDAVVVNEEALDLLAPGKTVAGLLETQALTQCCLHSDYCSQNTCLNGGK
CSWTHGAGYVCKCPPQFSGKHCEQGRENCTFAPCLEGGTCILSPKGASCNCPHPYTGDRCEMEARGCS
EGHCLVTPEIQRGDWGQQELLIITVAVAFIIISTVGLLFYCRRCKSHKPVAMEDPDLLARSVGVDTQA
MPAIELNPLSASSCNNLNQLEPSKASVPNELVTFGPNSKQRPWCSVPPRLPPAAVPSHSDNGPVIKR
TWSSEEMWPGGAMVWPPTYSRNERWEYPHSEVTQGPLPPSAHRHSTPVVMPEPNGLYGGFPFPLEME
NKRAPLPPRYSNQNLEDLMPSRPPSPRERLVAPCLNEYTAISYYHSQFRQGGGGPCLADGGYKGVGMR
LSRAGPSYAVCEVEGAPLAGQGQPRVPPNYEGSDMVESDYGSCEEVMF
A ClustalW comparison of the above protein sequences yields the following
relationships between the NOV6 sequences. In comparison to NOV6a, CG51923-O1,
NOV6n is 4264 amino acid residues having the following sequence changes: amino
acids
3754 to 3759, -ARLSI becomes GQALTS; A3789V; amino acids 3900 to 3907,
HSSSNVSQ are deleted; P4117L; E4160G. NOV6m corresponds to amino acid residues
2802 to 4023 of NOV6a with the following sequence changes: V3033A; L3514S;
G3591D;
M3631I. NOV61 corresponds to amino acid residues1561 to 2233 of NOV6a. NOV6k
corresponds to amino acids 1143 to 1733 of NOV6a with the following sequence
changes:
Y1181H; Y1287D; N1303S. Both NOV6b and NOV6c correspond to amino acids 2561 to
3233 of NOV6a with NOV6b having a~ amino acid change Q2991H. NOV6e and NOV6f
correspond to amino acids 1 to 659 of NOV6a and NOV6e has an amino acid change
R574C. NOV6g corresponds to amino acid residues 19-659 of NOV6a with an amino
acid
change of R574C. NOV6h and NOV6i correspond to amino acids 154-659 of NOV6a
and
NOV6h has an amino acid change R574C. NOV6d corresponds to amino acids 3559 to
4043 of NOV6a with an amino acid change M3631I. NOV6j corresponds to NOV6a
amino acids 570 to 1233 with A1100V and Y1181H amino acid changes.
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Further analysis of the NOV6a protein yielded the following properties shown
in
Table 6B. ---
Table 6B. Protein Sequence Properties NOV6a
SignalP analysis: Cleavage site between'residues 19 and 20
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 0; pos.chg 0neg:chg 0
H-region: length 18; peak value 11.25
PSG score: 6.85
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -0.05
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: 1
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood =-10.40 Transmembrane 4049 -4065
PERIPHERAL Likelihood = 1.01 (at 3195)
ALOM score: -10.40 (number of ,TMSs: 1)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 9
Charge difference: -1.5 C(-0..5) - N( 1.0)
N >= C: N-terminal side will be inside
»> membrane topology: type 1a (cytoplasmic tail 4066 to 4349)
MITDISC: discrimination of mitochondrial targeting seq
R content: 0 Hyd Moment(75): 0.99
Hyd Moment(95): 1.82 G content:. 1
D/E content: 1 S/T content: 2
Score: -6.08
Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found
NUCDTSC: discrimination of nuclear localization signals
pat4: none
pat7: PFRREKE (4) at 541
pat7: PLDTRRR (3) at 1407
bipartite: none
content of basic residues: 8.0~
NLS Score: 0.13
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
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SKL: peroxisomal targeting signal in the C-terminus: none
'PTS2: 2nd peroxisomal targeting signal: found
KLASGVPQL at 3821
'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 4066
LL at 4086
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: 76.7
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9123):
44.4 ~: endoplasmic reticulum
22.2 ~: Golgi
22.2 ~: extracellular, including cell wall
11.1 ~: plasma membrane
» prediction for CG51923-01 is end (k=9)
A search of the NOV6a protein against the (ieneseq database, a proprietary
database that contains sequences published in patents and patent publication,
yielded
several homologous proteins shown in Table 6C.
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Table 6C. Geneseq Results
for NOV6a
NOV6a Identities/
Geneseq Protein/Organism/Length Residues/ Expect
Similarities
for the
Identifier [Patent #, Date] Match value
Matched Region
Residues
AA026792 Human cadherin (CAD) 1..4349 4349/4349 (100%)0.0
protein,
SEQ ID No 15 - Homo sapiens,1..4349 4349/4349 (100%)
4349 aa. [W0200299042-A2,
12-
DEC-2002]
AAU79940 Human protocadherin Fat 1..4349 4349/4349 (100%)0.0
2
(FAT2) protein NOV2 - 1..4349 4349/4349 (100%)
Homo
sapiens, 4349 aa.
[W0200229038-A2, 11-APR-
2002]
ABB97540 Novel human protein SEQ 1..4349 4346/4349 (99%)0.0
>D
NO: 808 - Homo Sapiens, 1..4349 4347/4349 (99%)
4349
aa. [W0200222660-A2,
21-
MAR-2002]
ABB97541 Novel human protein SEQ 1..3821 3819/3821 (99%)0.0
ID
NO: 809 - Homo Sapiens, 1..3821 3819/3821 (99%)
4263
aa. [W0200222660-A2,
21-
MAR-2002]
AAO26791 Human cadherin (CAD) ' 26..40331844/4089 (45%)0.0
protein,
I SEQ ID No 14 - Homo 27..41002647/4089 (64%)
sapiens,
4590 aa. [W0200299042-A2,
12-
DEC-2002]
In a BLAST NOV6a protein
search of was found to
public sequence
databases,
the
have homology to the proteins shown in the BLASTP data in Table 6D.
Table 6D. Public BLASTP Results for NOV6a
NOV6a
Protein Identities/
Residues/ Expect
Accession Protein/Organism/Length Similarities
for the
Match value
Number Matched Portion
Residues
Q9NYQ8 Protocadherin Fat 2 1..4349 4349/4349 (100%)0.0
precursor
(hFat2) (Multiple epidermal1..4349 4349/4349 (100%)
growth factor-like domains
1) -
Homo sapiens (Human),
4349 aa.
CAD35056 Sequence 364 from Patent1..4349 4346/4349 (99%)0.0
W00222660 - Homo sapiens1..4349 4347/4349 (99%)
(Human), 4349 aa.
CAD35057 Sequence 365 from Patent1..3821 3819/3821 (99%)0.0
W(~0222fi~,(1- Hnmn 1..3821 3819/3821 (99%)
sanien~
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(Human), 4263 aa..
088277 Protocadherin Fat 2 precursor1..4349 3557/4351 (81%) ' 0.0
(Multiple epidermal growth 1..4351 3915/4351 (89%)
factor-like domains 1) - Rattus
norvegicus (Rat), 4351 aa.
Q9QXA3 Mouse fat 1 cadherin - 33..4167 1890/4317 (43%) 0.0
Mus
musculus (Mouse), 4587 as 35..4315 270114317 (61%)
(fragment).
PFam analysis predicts that the
NOV6a protein contains the domains
shown in the
Table 6E.
Table 6E. Domain Analysis of NOV6a
Identities/
Pfam NOV6a Match Region Similarities Expect
Domain A~no Acid residues of SEQ for the Matched Valuey
ID
NO: 40 Region
cadherin 38..139 25/113 (22%) 0.05
671113 (59%)
cadherin 153..247 28/109 (26%) 2.1e-08
69/109 (63%)
cadherin 367..449 21/107 (20%) 0.94
54/107 (50%)
cadherin 463..553 40/107 (37%) 8.9e-20
69/107 (64%)
cadherin 569..659 27/110 (25%) 2.9e-06
64/110 (58%)
cadherin 720..811 35/107 (33%) 4.4e-23
70/107 (65%)
cadherin 825..916 35/107 (33%) 8.6e-24
75/107 (70%)
cadherin 930..1019 33/107 (31%) 2.8e-12
62/107 (58%)
cadherin 1037..1128 41/107 (38%) 7.6e-21
70/107 (65%)
cadherin 1142..1233 39/107 (36%) 3.7e-20
69/107 (64%)
cadherin 1247..1337 33/110 (30%) 2.2e-08
66/110 (60%)
cadherin 1354..1438 29/107 (27%) 1.4e-05
. 63/107 (59%)
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cadherin 1453..1546 29/107 (27%) 4.1e-08
67/107 (63%)
cadherin 1560..1651 40/107 (37%) 1.8e-21
69/107 (64%)
cadherin 1665..1749 26/107 (24%) 6e-13
63/107 (59%)
cadherin 1763..1861 23/116 (20%) 8.6e-09
,
73/116 (63%)
cadherin 1877..1959 30/111 (27%) 0.27
53/111 (48%)
cadherin 1973..2061 23/108 (21%) 7.1e-06
60/108 (56%)
cadherin 2075..2164 36/107 (34%) 4.6e-17
64/107 (60%)
cadherin 2176..2263 32/107 (30%), 1.5e-09
64/107 (60%)'
cadherin 2277..2370 31/109 (28%) 9.3e-27
73/109 (67%)
cadherin 2384..2472 34/111 (31%) 3e-09
65/111 (59%)
cadherin 2486..2576 34/107 (32%) 3.7e-15
_ _ 66/107 (62%)
__
cadherin 2590..2682 25/111 (23%) 1.9e-05
66/111 (59%)
cadherin 2696..2786 311112 (28%) 9.8e-07
68/112 (61%)
cadherin 2802..2897 36/110 (33%) 2.5e-22
76/110 (69%)
cadherin 2911..3002 37/107 (35%) 4.7e-12
63/107 (59%)
cadherin 3016..3104 33/107 (31%) 6e-21
69/107 (64%)
cadherin 3119..3209 35/107 (33%) 1.3e-13
66/107 (62%)
cadherin 3223..3312 301107 (28%) 1e-12
69/107 (64%)
cadherin 3326..3417 43/107 (40%) 7e-27
69/107 (64%)
cadherin 3431..3522 3f/1OR f:i3%1 6.9e-20
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Various open reading frames of CG51923-Ol were cloned as follows: assemblies
317868343 and 317868367, residues 1 to 659; assembly 317871203, residues 19 to
659;
assemblies 317871219 and 317871243, residues 154 to 659; assembly 317871246,
residues
570 to 1233; assembly 317999764, residues 1143 to 1733; assembly 318176301,
residues
1561 to 2233; assemblies 305869563 and 305869567 residues 2560 to 3233. The
cloned
inserts differ from the original sequence as follows: assembly 317868343 has
three silent
SNPs and one R574C amino acid change; assembly 317868367 has one silent SNP;
assembly 317871203 has two silent SNPs and one R574C amino acid change;
assembly
317871219 has three silent SNPs and one R574C amino acid change; assembly
317871243
has one silent, SNP; assembly 317871246 has two amino acid changes: A1100V and
Y1181H; assembly 317999764 has three amino acid changes: Y1181H, Y1287D and
N1303S; assembly 318176301 has no changes; assembly 305869563 differs from the
original sequence by a single amino acid change: Q2992H while the cloned
insert of
assembly 305869567 is 100% identical to the original sequence.
Example 7. NOV7, CG52919, SEZ-6
The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide
157
sequences are shown in Table 7A.
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CCCAGCAGAGCCTGGACAC
TCGTC
CCTACAACCGCATTACCATAGAGTCAGC
V7a, CG52919-06 SEQ m NO: 68 543 as MW at 58351.OkD
MRPVALLLLPSLLALLAHGLSLEAPTVUKU~Artilr:r;mt~~r~Lm~rwr~5~r ~n.tivrir-v
wwHrwLnL.mv
HHPLLEEFLQEGLEKGDEELRPALPFQPDPPAPFTPSPLPRLANQDSRPVFTSPTPAMAAVPTQPQSK '
EGPWSPESESPMLRITAPLPPGPSMAVPTLGPGEIASTTPPSRAWTPTQEGPGDMGRPWVAEVVSQGA
GIGIQGTITSSTASGDDEETTTTTTIITTTITTVQTPGPCSWNFSGPEGSLDSPTDLSSPTDVGLDCF
FYISVYPGYGVEIKVQNISLREGETVTVEGLGGPDPLPLANQSFLLRGQVIRSPTHQAALRFQSLPPP
~EAPPVYDSYEVEYLPIEGLLSSGKHFFVEPRPRPRPYNRITIESAFDNPTYETGSLSLAGDERI
V7b, 298521010 SEQ ~ NO: 69 444 by
A Sequence ORF Start: at 1 ORF Stop: end of sequence
GGCTGCTTGAGGCTCCTGAGGGCCAGCGGCTACACCTGCACTTTGAGAAGGTTTCCCTGGC
GATGACAGGCTCATCATTCGCAATGGGGACAACGTGGAGGCCCCACCAGTGTATGATTCCT
'GGAATACCTGCCCATTGAGGGCCTGCTCAGCTCTGGCAAACAC
V7b, 298521010 SEQ m NO: 70 148 as MW at ~16958kD
RPAYGDVTVTSLHPGGSARFHCATGYQLKGARHLTCLNVTQPFWDSKEPVCIAACGGVIR
RIVSPGFPGNYSNNLTCHWLLEAPEGQRLHLHFEKVSLAEDDDRLIIRNGDNVEAPPVYD
IEGLLSSGKH _ _ _ _
~c, CG52919-09 SEQ m NO: 71 ;1572_bp~_
Sequence ORF Start: at 1 ~ OR__F _St_op: at .1_583 ~
:TTTAGAGGCCCCAACCGTGGGGAAAGGACAAGCCCCAGGCATCGAGGAGACAG~
TGAGGAGCTGAGGCCAGCACTGCCCTTCCAGCCTGACCCACCTGCACCCTTCACCCCAAGTCCCC
TACCCTGGCTATGGCGTGGAAATCAA
GGAAGGCCTGGGGGGGCCTGACCCAC
TCCGCAGCCCCACCCACCAAGCGGCC
CCCGCTTCCATTGTGCCACTGGCTACCAGCTGAAGGGCGCCAGGCATCTCACCTGTC
CAGCCCTTCTGGGATTCAAAGGAGCCCGTCTGCATCGCTGCTTGCGGCGGAGTGATC
CACCGGCCGCATCGTCTCTCCAGGCTTCCCGGGCAACTACAGCAACAACCTCACCTG
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comparison. NOV7a is a 543 amino acid long protein sequence. NOV7b is the
mature
protein sequence corresponding to amino acid residues 20 to 543 of NOV7a.
NOV7c
corresponds to amino acid residues 356 to 504 of NOV7a, which includes the
sushi and
CUB domain as predicted by pfam, see below. .
Further analysis of the NOV7a protein yielded the following properties shown
in
Table 7B.
Table 7B. Protein Sequence Properties NOV7a
SignalP analysis: Cleavage site between residues 20 and 21
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 2; pos.chg 1; neg.chg 0
H-region: length 20; peak value 8.99
PSG score: 4.59
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 2.15
possible cleavage site: between 17 and 18
»> Seems to have a cleavable signal peptide (1 to 17)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 18
Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) .. fixed
PERIPHERAL Likelihood = 4.72 (at 267)
ALOM score: 4.72 (number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 8
Charge difference: -1.5 C( 0.5) - N( 2.0)
N >= C: N-terminal side will be inside
MITDISC: discrimination of mitochondrial targeting seq
159
A ClustalW comparison of the above protein sequences yields the following
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R content: 1 Hyd Moment(75): 5.75
Hyd Moment(95): 8.42 G content: 1
D/E content: 1 S/T content: 2
Score: -3.74
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 12 MRP~VA
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 6.4~
NLS Score: -0.47 '
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: RPVA
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: 55.5
COIL: Lupas's algorithm to detect coiled-coil regions
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total: 0 residues
4Fina1 Results (k = 9/23):
55.6 ~: extracellular, including cell wall
33.3 ~: mitochondrial ,
11.1 ~: vacuolar
» prediction for CG52919-06 is exc (k=9)
A search of the NOV7a protein against the Cieneseq
database, a proprietary
database that contains sequences published in d
patents and patent publication, yielde
several homologous proteins shown in Table 7C.
Table 7C. Geneseq Results for NOV7a
NOV7a Identities/
Geneseq Protein/Organism/Length [Patent - Residues/Similarities Expect
for
Identifier #, Date] Match the Matched Value
Residues Region
AAB70542 ~ Human PR012 protein sequence 1..543 518/543 (95%)0.0
,
SEQ ID NO:24 - Homo sapiens, 1..526 518/543 (95%)
526 aa. [W0200110902-A2, 15-
FEB-2001]
AAB70541 Human PR011 protein sequence 1..533 513/533 (96%)0.0
SEQ >D N0:22 - Homo sapiens, 1..516 513/533 (96%)
525 aa. [W0200110902-A2, 15-
FEB-2001]
AAB70540 Human PRO10 protein sequence 1..533 513/533 (96%)0.0
SEQ )D NO:20 - Homo Sapiens, 1..516 513/533 (96%)
525 aa. [W0200110902-A2, 15-
FEB-2001]
AAU81976 j Human secreted protein SECP2 - ' 1..508507/508 (99%)0.0
Homo sapiens, 994 aa. 1..508 507/508 (99010)
[W0200198353-A2, 27-DEC-2001]
ABP69306 Human polypeptide SEQ ID NO 1..508 507/508 (99%)0.0
1353 - Homo sapiens, 544 aa. 1..508 507/508 (99%)
[W0200270539-A2,12-SEP-2002]
In a BLAST search of public sequence databases,
the NOV7a protein was found to
have homology to the proteins shown in the BLASTP
data in Table 7D.
Table 7D. Public BLASTP Results for NOV7a
Protein protein/Organism/Length NOV7a Identities/ Expect
Arceccinn Recirlnec/ similarities Value
fnr
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Number Match the Matched
ResiduesPortion
CAC33420 Sequence 23 from Patent3 1..543 518/543 (95%) 0.0
~ ~
WO0110902 -Homo sapiens 1..526 518/543 (95%)
(Human), 526 aa.
CAC33418 Sequence 19 from Patent 1..533 513/533 (96%) 0.0
'
W00110902 - Homo sapiens 1..516 513/533 (96%)
(Human), 525 aa.
CAC33417 . Sequence 17 from Patent 1..533 509/533 (95%) 0.0
WO0110902 - Homo sapiens 1..516 509/533 (95%)
(Human), 525 aa.
CAC33416 Sequence 15 from Patent 1..508 503/508 (99%) 0.0
:
W00110902 - Homo sapiens ~ 1..508504/508 (99%)
(Human), 994 aa.
CAC33415 Sequence 13 from Patent 1..508 503/508 (99%) 0.0
;
WO0110902 - Homo sapiens 1..508 ~ 504/508 (99%)
(Human), 993 aa.
PFarn analysis s the domains n the
predicts shown i
that the
NOV7a
protein
contain
Table 7E.
Table 7E. Domain Analysis of NOV7a
Identities/
Pfam Domain NOV7a Match Region Similarities Expect Value
a for the Matched Region
sushi 357..412 15/65 (23%) f 1.9e-05
41/65 (63%)
CI~ 416..504 28/116 (24%) 1.3e-05
65/116 (56%)
Example 8. NOVB, CG94946, Agrin precursor.
The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide
162
sequences are shown in Table 8A.
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TGCTGTGCGGCTTCGGC
Z'GTGACCAGGCCCCGTCCCCATGCCTCGGGGTGCAGTGTGCATTTGGGGCGAC
TGGAGGCCACGGCCTGTACCCTC
TCTGAATGCGTGGCTTTGGCCCAGCCCGTGTGTGGCTCC
AC
GAGTGC
GTGTGTCTGTGACTTCAGCTGCC
CGCCCCGCTGCCGCCTGTGGCCCCCTT
GTCTGGCTCAGCCCACTGTGTCTGCCCGATGCTCACCTGTCCAGAGGCCAACGCTACCAAGGTC
GGTCAGATGGAGTCACATACGGCAACGAGTGTCAGCTGAAGACCATCGCCTGCCGCCAGGGCCT
ATCTCTATCCAGAGCCTGGGCCCGTGCCAGGAGGCTGTTGCTCCCAGCACTCACCCGACATCTG
CGTGACTGTGACCACCCCAGGGCTCCTCCTGAGCCAGGCACTGCCGGCCCCCCCCGGCGCCCTC
TGCCAGCGTCCCCAGGACCACCGTGTGGCCCGTGCTGACGGTGCCCCCCACGGCACCCTCCCCTGCAC
CCAGCCTGGTGGCGTCCGCCTTTGGTGAATCTGGCAGCACTGATGGAAGCAGCGATGAGGAACTGAGC
GGGGACCAGGAGGCCAGTGGGGGTGGCTCTGGGGGGCTCGAGCCCTTGGAGGGCAGCAGCGTGGCCAC
TTTATGGACTTTGACTGGTTTC
ACGTCAGGAGCCATTGCTGCGGGAGCCACGGCCAGAGCCAC;CAC;'1'GC:A
TGTGACCCCTCGGGCCCCGCACCCCAGTCACACAAGCCAGCCCGTTGC
CCCGGCCCCCCAGCAGCCTCCAAAGCCCTGTGACTCACAGCCCTGCTTCCACGGGGGGACCTGC
ACTGGGCATTGGGCGGGGGCTTCACCTGCAGCTGCCCGGCAGGCAGGGGAGGCGCCGTCTGTGA
GTGCTTGGCGCCCCTGTGCCGGCCTTCGAGGGCCGCTCCTTCCTGGCCTTCCCCACCCTCCGCG
GGCGGTGCTGACCAGTGCCGTGCCGGTAGAGCCGG
GGCGCCGGGGCACCCTCTCGGTGGATGGTGAGACC
TGGGGCGGCGCCCTGC
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CCGTGGTGCTGCGTTCCAC
NOVBa, CG94946-01 ~SEQ m NO: 74 2053 as ~MW at 215628.Ok1~
PAAGRPLLPLLWAACVLPGAGGTCPERALERREEEANVVLTGTVEE
SVVAPVCGSDASTYSNECELQRAQCSQQRRIRLLSRGPCGSRDPCSNVTCSFGSTCARSADGLT.
CPATCRGAPEGTVCGSDGADYPGECQLLRRACARQENVFKKFDGPCDPCQGALPDPSRSCRVNP:
PEMLLRPESCPARQAPVCGDDGVTYENDCVMGRSGAARGLLLQKVRSGQCQGRDQCPEPCRFNA
CAFGATCAVKNGQAACECLQACSSLYDPVCGSDGVTYGSACELEATACTLGREIQV
RFGALCEAETGRCVCPSECVALAQPVCGSDGHTYPSECMLHVHACTHQISLHVASA
FGAVCSAGQCVCPRCEHPPPGPVCGSDGVTYGSACELREAACLQQTQIEEARAGPC
GEDGDCEQELCRQRGGIWDEDSEDGPCVCDFSCQSVPGSPVCGSDGVTYSTECELK
AAQGACRGPAFAPLPPVAPLHCAQTPYGCCQDNITAARGVGLAGCPSACQCNPHGS
SCRPGVGGLRCDRCEPGFWNFRGIVTDGRSGCTPCSCDPQGAVRDDCEQMTGLCSC
QLKTIACRQGLQISIQSLGPCQEAVAPSTHPTSASVTVTTPGLLLSQALPAPPGALPLAPS
TPPPSSRPRTTASVPRTTVWPVLTVPPTAPSPAPSLVASAFGESGSTDGSSDEELSGDQEA
LEPLEGSSVATPGPPVERASCYNSALGCCSDGKTPSLDAEGSNCPATKVFQGVLELEGVEG
VSRRRSLGVRRPLQEHVRFMDFDWFPAFITGATSGAIAAGA'1'AttA'1"1'r~sttmr~~Hwrrtc
PVAKTTAAPTTRRPPTTAPSRVPGRRPPAPQQPPKPCDSQPCFHGGTCQDWALGGGFTC
VCEKVLGAPVPAFEGRSFLAFPTLRAYHTLRLALEFRALEPQGLLLYNGNARGKDFLAL
RFDTGSGPAVLTSAVPVEPGQWHRLELSRHWRRGTLSVDGETPVLGESPSGTDGLNLDT
DQAAVALERTFVGAGLRGCIRLLDVNNQRLELGIGPGAATRGSGVGECGDHPCLPNPCH
T r. owr~nr~anr_v~ Tr_~nmr an'~u cnr~nnnlpC~H~ A A PCRVLPEGGAOCEC PLGREGTFC
QTA
PFLADFNGFSHLELRGLHTIARDLGEKMALEAVFLARGPSGLLL
YVGGAPDFSKLARAAAVSSGFDGAIQLVSLGGRQLLTPEHVLRQVDVTSFAGHPCTRA
CVPREAAYVCLCPGGFSGPHCEKGLVEKSAGDVDTLAFDGRTFVEYLNAVTESEKALQ
VBb, 308909220 SEQ m NO: 75 1935 by
A Sequence ORF Start: at 1 ORF Stop: end of
TTCGTGGGCGCCGGCCTGAGGGGGTGCATCCGTTTGCTGGACGTCAACAACCAGC
r~T mmr~~r_~rr_rr~rrmaCrArrrr ArrCTC'TC'GCGTGGGCGAGTGCGGGGACCAC
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sequence comparisons. NOVBb corresponds to NOVBa protein sequence amino acid
residues 1404-2052 with the following changes: I1658F; A1670V; and deletion of
1756-
1759 of the NOV8a sequence and furthermore includes several laminin G and EGF
domains as predicted by pfam, below.
Further analysis of the NOVBa protein yielded the following properties shown
in
Table 8B. -
Table 8B. Protein Sequence Properties NOVBa
SignalP analysis: Cleavage site between residues 34 and 35
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 5; pos.chg 2; neg.chg 0
H-region: length 9; peak value 3.79
PSG score: -0.61
GvH: von Heijne'.s method for signal seq. recognition
GvH score (threshold: -2.1): 0.49
possible cleavage site: between 33 and 34
»> Seems to have no N-terminal signal peptide
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 1
165
A ClustalW comparison of the above protein sequences yields the following
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Tentative number of TMS(s) for the threshold 0.5: 1
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood = -4.35 Transmembrane 17 - 33
PERIPHERAL Likelihood = 0.53 (at 609)
ALOM score: -4.35 (number of TMSs: 1)
. MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 24
Charge difference: -6.5 C(-2.~) - N( 4.'S)
N >= C: N-terminal side will be inside '
»> membrane topology: type 2 (cytoplasmic tail 1 to 17)
MITDISC: discrimination of mitochondrial targeting seq
R content: 3 Hyd Moment(75): 2.17
Hyd Moment(95): 10.07 G content: 6
D/E cdntent: 1 S/T.content: 1
Score: -4.90
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 25 GRP~LL
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: PRTRRPE (5) at 339
pat7: PKSRKVP (5) at 1755
bipartite: none
content of basic residues: 9.5~
NLS Score: 0.39
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: RHGR
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
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Tyrosines in the tail: none
'Dileucine motif in the tail: none
'checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribpsomal protein motifs: none
i
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)
34.8 0: mitochondrial
34.8 0: nuclear
13.0 ~: cytoplasmic
4.3 ~: extracellular, including cell wall
4.3 0: vacuolar
4.3 ~: Golgi
4.3 ~: peroxisomal
» prediction for CG94946-01.i's mit (k=23)
_._ _........__._~-.-
A search of the NOVBa protein against
the Cieneseq database, a proprietary
database that contains sequences
published in patents and patent
publication, yielded
several homologous proteins shown
in Table 8C.
Table 8C. Geneseq Results for NOVBa
NOVBa Identities/
Geneseq Protein/Organism/Length Residues/Similarities for
Expect
Identifier [Patent #, Date] Match the Matched Value
Residues Region
ABIJ52400 Human GPCR related protein160..20531841/1894 (97%) 0.0
NOV40a - Homo Sapiens, 1931 51..1931 1853/1894 (97%)
aa. [W0200279398-A2, 10-OCT-
2002]
ABP43859 Human mRNA precursor - 137..16691530/1533 (99%) 0.0
Homo
Sapiens, 1741 aa. 1..1533 1530/1533 (99%)
[W0200231111-A2, 18-APR-
2002]
AAW26609 Human agrin - Homo Sapiens,1591..2053460/471 (97%) 0.0
492 aa_ fW(~9721 R11-A2_ 19- 22..492 461/471 (97%)
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JUN-1997]
AAB93754 Human protein sequence SEQ ID 583..968381/386 (98%) 0.0
N0:13424 - Homo sapiens, 413 1..386 384/386 (98%)
aa. [EP1074617-A2, 07-FEB-
2001]
AAY73993 Human prostate tumor EST 1634..2053414/420 (98%) 0.0
fragment derived protein #180 - 1..416 414/420 (98%)
Homo sapiens, 416 aa.
[DE19820190-Al, 04-NOV-
1999]
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 8D.
Table 8D. Public BLASTP Results for NOVBa
Protein NOVBa Identities/
AccessionProtein/Organism/LengthResidues/ Similarities Expect
for the
Number esi Matched PortionValue
dues
R
000468 AGRIN precursor - Homo 24..2053 2022/2030 (99%)0.0
~
sapiens (Human), 2026 1..2026 2022/2030 (99%)
as ;
(fragment).
P25304 Agrin precursor - Rattus160..2053 1558/1914 (81 0.0
' %)
norvegicus (Rat), 1959 51..1959 1663/1914 (86%)
aa.
P31696 Agrin precursor - Gallus128..2050 1234/1970 (62%)0.0
gallus
(Chicken), 1955 aa. 1..1952 1479/1970 (74%)
Q90404 Agrin - Discopyge ommata716..2051 ~ 733/1353 0.0
(54%)
(Electric ray), 1328 1..1325 932/1353 (68%)
as
(fragment).
Q96IC1 Hypothetical protein 1562..2053486/492 (98%) 0.0
- Homo
sapiens~(Human), 488 1..488 486/492 (98%)
as
(fragment).
PFam analysis protein
predicts contains
that the domains
the NOVBa shown
in the
Table 8E.
Table 8E. Domain Analysis of NOVBa
NOVBa Match Region Identities/
Pfam Amino Acid residues of SEQ ID Similarities Expect
Domain NO: 74 for the Matched Value
Region
NtA 34..161 125/135 (93%) 1.1e-111
128/135 (95%)
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k~~' 201..246 25/61 (41 %) 7.2e-18
36/61 (59%)
276..321 21/62 (34%) 5.1e-13
33/62 (53%)
kazal 346..393 18/61 (30%) 7.9e-12
33/61 (54%)
k~~ 420..465 21/61 (34%) 4.1e-16
~.. 38/61 (62%)
k~~ ~ 494..538 24/61 (39%) 3.6e-19
- 38/61 (62%)
k~~ 559..603 19/61 (31%) 1.4e-18
38/61 (62%)
k~~ ' 624..668 26/62 (42%) 1.5e-17
37/62 (60%)
k~~ 709..754 24/62 (39%) 1.2e-16
40/62 (65%)
laminin_EGF 797..848 28/61 (46%) l.le-20
46/61 (75%)
laminin_EGF 851..895 21/59 (36%) 3.6e-11
37/59 (63%)
kazal 927..973 25/62 (40%) 5.2e-18
41/62 (66%)
SEA 1134..1256 ~ 3 38/132 (29%) 4.6e-37
111/132 (84%)
EGF 1337..1370 16/47 (34%) 0.00055
24/47 (51 %)
laminin_G 1404..1535 70/154 (45%) 4.6e-55
120/154 (78%)
EGF 1557..1589 16/47 (34%) 5.2e-06
27/47 (57%)
EGF 1596..1628 16/47 (34%) 0.00021
25/47 (53%)
laminin_G 1672..1807 71/154 (46%) 5.8e-52
122/154 (79%)
EGF 1826..1860 14/47 (30%) 4.4e-07
25/47 (53%)
laminin_G 1905..2036 58/154 (38%) 1.2e-50
__ 125/154-(81%)
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Example B: Sequencing Methodology and Identification of NOVX Clones
1. GeneCalling~ Technology: This is a proprietary method of performing
differential gene expression profiling between two or more samples developed
at CuraGen
and described by Shimkets, et al., "Gene expression analysis by transcript
profiling coupled
to a gene database query" Nature Biotechnology 17:198-803 (1999). cDNA was
derived
from various human samples representing multiple tissue types, normal and
diseased states,
physiological states, and developmental states from different donors. Samples
were
obtained as whole tissue, primary cells or tissue cultured primary cells or
cell lines. Cells
and cell lines may have been treated with biological or chemical agents that
regulate gene
expression, for example, growth factors, chemokines or steroids. The cDNA thus
derived
was then digested with up to as many as 120 pairs of restriction enzymes and
pairs of
linker-adaptors specific for each pair of restriction enzymes were ligated to
the appropriate
end.~The restriction digestion generates a mixture of unique cDNA gene
fragments.
Limited PCR amplification is performed with primers homologous to the linker
adapter
sequence where one primer is biotinylated and the other is fluorescently
labeled. The
doubly labeled material is isolated and the fluorescently labeled single
strand is resolved by
capillary gel electrophoresis. A computer algorithm compares the
electropherograms from
an experimental and control group for each of the restriction digestions. This
and additional
sequence-derived information is used to predict the identity of each
differentially expressed
gene fragment using a variety of genetic databases. The identity of the gene
fragment is
confirmed by additionalr gene-specific competitive PCR or by isolation and
sequencing of
the gene fragment.
2. SeqCalling~ Technology: cDNA was derived from various human
samples representing multiple tissue types, normal and diseased states,
physiological states, .
and developmental states from different donors. Samples were obtained as whole
tissue,
primary cells or tissue cultured primary cells or cell lines. Cells and cell
lines may have
been treated with biological or chemical agents that regulate gene expression,
for example,
growth factors, chemokines or steroids. The cDNA thus derived was then
sequenced using
CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated
manually
and edited for corrections if appropriate. cDNA sequences from all samples
were
assembled together, sometimes including public human sequences, using
bioinformatic
programs to produce a consensus sequence for each assembly. Each assembly is
included in
CuraGen Corporation's database. Sequences were included as components for
assembly
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when the extent of identity with another component was at least 95% over 50
bp. Each
assembly represents a gene or portion thereof and includes information on
variants, such as
splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and
other
sequence variations.
3. PathCalling~ Technology: The NOVX nucleic acid sequences are
derived by laboratory screening of cDNA library by the two-hybrid approach.
cDNA
fragments covering either the full length of the DNA sequence, or part of the
sequence, or
both, are sequenced. In silico prediction was based on sequences available in
CuraGen
Corporation's proprietary sequence databases or in the public human sequence
databases,
and provided either the full length DNA sequence, or some portion thereof.
The laboratory screening was performed using the methods summarized below:
cDNA libraries were derived from various human samples representing multiple
tissue types, normal and diseased states, physiological states, and
developmental states
from different donors. Samples were obtained as whole tissue, primary cells or
tissue
cultured primary cells or cell lines. Cells and cell lines may have been
treated with
biological or chemical agents that regulate gene expression, for example,
growth factors,
chemokines or steroids. The cDNA thus derived was then directionally cloned
into the
appropriate two-hybrid vector (Gal4-activation domain (Gal4-AD) fusion). Such
cDNA
libraries as well as commercially available cDNA libraries from Clontech (Palo
Alto, CA)
were then transferred from E.coli into a CuraGen Corporation proprietary yeast
strain
(disclosed in U. S. Patents 6,057,101 and 6,083,693, incorporated herein by
reference in
their entireties).
Gal4-binding domain (Gal4-BD) fusions of a CuraGen Corportion proprietary
library of human sequences was used to screen multiple Gal4-AD fusion cDNA
libraries
resulting in the selection of yeast hybrid diploids in each of which the Gal4-
AD fusion
contains an individual cDNA. Each sample was amplified using the polymerase
chain
reaction (PCR) using non-specific primers at the cDNA insert boundaries. Such
PCR
product was sequenced; sequence traces were evaluated manually and edited for
corrections if appropriate. cDNA sequences from all samples were assembled
together,
sometimes including public human sequences, using bioinformatic programs to
produce a
consensus sequence for each assembly. Each assembly is included in CuraGen
Corporation's database. Sequences were included as components for assembly
when the
extent of identity with another component was at least 95% over 50 bp. Each
assembly
represents a gene or portion thereof and includes information on variants,
such as splice
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forms single nucleotide polymorphisms (SNPs), insertions, deletions and other
sequence
variations.
Physical clone: the cDNA fragment derived by the screening procedure, covering
the entire open reading frame is, as a recombinant DNA, cloned into pACT2
plasmid
(Clontech) used to make the cDNA library. The recombinant plasmid is inserted
into the
host and selected by the yeast~hybrid diploid generated during the screening
procedure by
the mating of both CuraGen Corporation proprietary yeast strains N106'
and.YULH (U. S.
Patents 6,057,101 and 6,03,693).
4. RACE: Techniques based on the polymerase chain reaction such as rapid
amplification of cDNA ends (RACE), were used to isolate or complete the
predicted
sequence of the cDNA of the invention. Usually multiple clones were sequenced
from one
or more human samples to derive the sequences for fragments. Various human
tissue
samples from different donors were used for the RACE reaction. The sequences
derived
from these procedures were included in the SeqCalling Assembly process
described in
preceding paragraphs.
5. ~ Exon Linking: 'The NOVX target sequences identified in the present
invention were subjected to the exon linking process to confirm the sequence.
PCR
primers were designed by starting at the most upstream sequence available, for
the forward
primer, and at the most downstream sequence available for the reverse primer.
In each
case, the sequence was examined, walking inward from the respective termini
toward the
coding sequence, until a suitable sequence that is either unique or highly
selective was
encountered, or, in the case of the reverse primer, until the stop codon was
reached. Such
primers were designed based on in silico predictions for the full length cDNA,
part (one or
more exons) of the DNA or.protein sequence of the target sequence, or by
translated
homology of the predicted exons to closely related human sequences from other
species.
These primers were then employed in PCR amplification based on the following
pool of
human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum,
brain -
hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal
brain, fetal
kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary
gland, pancreas,
pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal
cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting
amplicons were
gel purified, cloned and sequenced to high redundancy. The PCR product derived
from
exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting
bacterial
clone has an insert covering the entire open reading frame cloned into the
pCR2.1 vector.
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The resulting. sequences from all clones were assembled with themselves, with
other
fragments in CuraGen Corporation's database and with public ESTs. Fragments
and ESTs
were included as components for an assembly when the extent of their identity
with another
component of the assembly was at least 95% over 50 bp. In addition, sequence
traces were
evaluated manually and edited for corrections if appropriate. These procedures
provide the
sequence reported herein.
6. Physical Clone: Exons were predicted by homology and the intron/exon
boundaries were determined using standard genetic rules. Exons were further
selected and
refined by means of similarity determination using multiple BLAST (for
example, tBlastN,
BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail.
Expressed ..
sequences from both public and proprietary databases were also added when
available to
further define and complete the gene sequence. The DNA sequence was then
manually
corrected for apparent inconsistencies thereby obtaining the sequences
encoding the
full-length protein.
The PCR product derived by exon linking,-covering the entire open reading
frame,
was cloned into the pCR2.l vector from Invitrogen to provide clones used for
expression
and screening purposes.
Example C. Quantitative expression analysis of clones in various cells and
tissues
The quantitative expression of various NOV genes was assessed using microtiter
plates containing RNA samples from a variety of normal and pathology-derived
cells, cell
lines and tissues using real time quantitative PCR (RTQ-PCR) performed on an
Applied
Biosystems (Foster City, CA) ABI PRISM~ 7700 or an ABI PRISM~ 7900 HT Sequence
Detection System.
RNA integrity of all samples was determined by visual assessment of agarose
gel
electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as
a guide
(2:1 to 2.5:128s:18s) and the absence of low molecular weight RNAs
(degradation
products). Control samples to detect genomic DNA contannination included RTQ-
PCR
reactions run in the absence of reverse transcriptase using probe and primer
sets designed to
amplify across the span of a single exon.
RNA samples were normalized in reference to nucleic acids encoding
constitutively
expressed genes (i.e., (3-actin and GAPDIi). Alternatively, non-normalized RNA
samples
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were converted to single strand cDNA (sscDNA) using Superscript II (Invitrogen
Corporation, Carlsbad, CA, Catalog No. 18064-147) and random hexamers
according to
the manufacturer's instructions. Reactions containing up to 10 ~,g of total
RNA in a volume
of 20 ,ul or were scaled up to contain 50 ~,g of total RNA in a volume of 100
~,1 and were
incubated for 60 minutes at 42°C. sscDNA samples were then normalized
in reference to
nucleic acids as described above.
Probes and primers were designed according to Applied Biosystems Primer
Express
Software package (version I for Apple Computer's Macintosh Power PC) or a
similar
algorithm using the target sequence as input. Default reaction condition
settings and the
following parameters were set before selecting primers: 250 nM primer
concentration; 58°-
60° C primer melting temperature (Tm) range; 59° C primer
optimal Tm; 2° C maximum
primer difference (if probe does not have 5' G, probe Tm must be 10° C
greater than primer
Tm; and 75 by to 100 by amplicon size. The selected probes and primers were
synthesized
by Synthegen (Houston, TX). Probes were double purified by HPLC to remove
uncoupled
dye and evaluated by mass spectroscopy to verify coupling of reporter and
quencher dyes
to the 5' and 3' ends of the probe, respectively. Their final concentrations
were: 900 nM
forward and reverse primers, and 200nM probe.
Normalized RNA was spotted in individual wells of a 96 or 384-well PCR plate
(Applied Biosystems, Foster City, CA). PCR cocktails included a single gene-
specific
probe and primers set or two multiplexed probe and primers sets. PCR reactions
were done
using TaqlVlan~ One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No.
4313803) following manufacturer's instructions. Reverse transcription was
performed at
48° C for 30 minutes followed by amplification/PCR cycles: 95° C
10 min, then 40 cycles -
at 95° C for 15 seconds, followed by 60° C for 1 minute. Results
were recorded as CT
values (cycle at which a given sample crosses a threshold level of
fluorescence) and plotted
using a log scale, with the difference in RNA concentration between a given
sample and the
sample with the lowest CT value being represented as 2 to the power of delta
CT. The
percent relative expression was the reciprocal of the RNA difference
multiplied by 100. CT
values below 28 indicate high expression, between 28 and 32 indicate moderate
expression,
between 32 and 35 indicate low expression and above 35 reflect levels of
expression that
were too low to be measured reliably.
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Normalized sscDNA was analyzed by RTQ-PCR using IX TaqMan~ Universal
Master mix (Applied Biosystems; catalog No. 4324020), following the
manufacturer's
instructions. PCR amplification and analysis were done as described above.
Panels 1,1.1,1.2, and 1.3D
Panels 1, l.l, 1.2 and 1_3D included 2 control wells (genomic DNA control and
chemistry control) and 94 wells of cDNA samples from cultured cell lines and
primary
normal tissues. Cell lines were derived from carcinomas (ca) including: lung,
small cell (s
cell vary, non small cell (non-s or non-sm); breast; melanoma; colon;
prostate; glioma
(glio), astrocytoma (astro) and neuroblastoma (neuro); squamous cell (squam);
ovarian;
liver; renal; gastric and pancreatic from the American Type Culture Collection
(ATCC,
Bethesda, MD). Normal tissues were obtained from individual adults or fetuses
and
included: adult and fetal skeletal muscle, adult and fetal heart, adult and
fetal kidney, adult
and fetal liver, adult and fetal lung, brain, spleen, bone marrow, lymph node,
pancreas,
salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach,
small intestine,
colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and
adipose. The
following abbreviations are used in reporting the results: metastasis (met);
pleural effusion
(pl. eff or pl effusion) and * indicates established from metastasis.
General screening_panel_v1.4, v1.5, v1.6 and v1.7
Panels 1.4, 1.5, 1.6 and 1.7 were as described for Panels 1; 1.1, 1.2 and
1.3D, above
except that normal tissue samples were pooled from 2 to 5 different adults or
fetuses.
Panels 2D, 2.2, 2.3 and 2.4
Panels 2D, 2.2, 2.3 and 2.4 included 2 control wells and 94 wells containing
RNA
or cDNA from human surgical specimens procured through the National Cancer
Institute's
Cooperative Human Tissue Network (CHTN) or the National Disease Research
Initiative
(NDRI), Ardais (Lexington, MA) or Clinomics BioSciences (Frederick, MD).
Tissues
included human malignancies and in some cases matched adjacent normal tissue
(NAT).
Information regarding histopathological assessment of tumor differentiation
grade as well
as the clinical stage of the patient from which samples were obtained was
generally
available. Normal tissue RNA and cDNA samples were purchased from various
commercial sources such as Clontech (Palo Alto, CA), Research Genetics and
Invitrogen
(Carlsbad, CA).
HASS Panel v 1.0
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The HASS Panel v1.0 included 93 cDNA samples and two controls including: 81
samples of cultured human cancer cell lines subjected to serum starvation,
acidosis and
anoxia according to established procedures for various lengths of time; 3
human primary
cells; 9 malignant brain cancers (4 medulloblastomas and 5 glioblastomas); and
2 controls.
Cancer cell lines (ATCC) were cultured using recommended conditions and
included:
breast, prostate, bladder, pancreatic and CNS. Primary human cells were
obtained from
Clonetics (Walkersville, MD). Malignant brain samples were gifts from the
Henry Ford
Cancer Center.
ARDAIS Panel v1:0 and v1.1
The ARDAIS Panel v1.0 and v1.1 included 2 controls and 22 test samples
including: human lung adenocarcinomas, lung squamous cell carcinomas, and in
some
cases matched adjacent normal tissues (NAT) obtained from Ardais (Lexington,
MA).
Unmatched malignant and non-malignant RNA samples from lungs with gross
histopathological assessment of tumor differentiation grade and stage and
clinical state of
the patient were obtained from Ardais.
ARDAIS Prostate v1.0
ARDAIS Prostate v1.0 panel included 2 controls and 68 test samples of human
prostate malignancies and in some cases matched adjacent normal tissues (NAT)
obtained
from Ardais (Lexington, MA). RNA from unmatched malignant and non-malignant
prostate samples with gross histopathological assessment of tumor
differentiation grade and
stage and clinical state of the patient were also obtained from Ardais.
ARDAIS Kidney v1.0
ARDAIS Kidney v1.0 panel included 2 control wells and 44 test samples of human
renal cell carcinoma and in some cases matched adjacent normal tissue (NAT)
obtained
from Ardais (Lexington, MA). RNA from unmatched renal cell carcinoma and
normal
tissue with gross histopathological assessment of tumor differentiation grade
and stage and
clinical state of the patient were also obtained from Ardais.
ARDAIS Breast v1.0
ARDAIS Breast v1.0 panel included 2 control wells and 71 test samples of human
breast malignancies and in some cases matched adjacent normal tissue (NAT)
obtained
from Ardais (Lexington, MA). RNA from unmatched malignant and non-malignant
breast
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samples with gross histopathological. assessment of tumor differentiation
grade and stage
and clinical state of the patient were also obtained from Ardais.
Panel 3D, 3.1 and 3.2
Panels 3D, 3.1, and 3.2 includec~two controls, 92 cDNA samples of cultured
human
cancer cell lines and 2 samples of human primary cerebellum. Cell lines (ATCC,
National
Cancer Institute (NCI), German tumor cell bank) were cultured as recommended
and were
derived from: squamous cell carcinoma of the tongue, melanoma, sarcoma,
leukemia,
lymphoma,.and epidermoid, bladder, pancreas, kidney, breast, prostate, ovary,
uterus,
cervix, stomach, colon, lung and CNS carcinomas.
Panels 4D, 4R, and 4.1D
Panels 4D, 4R, and 4.1D included 2 control wells and 94 test samples of RNA
(Panel 4R) or cDNA (Panels 4D and 4.1D) from human cell lines or tissues
related to
inflammatory conditions. Controls included total RNA from normal tissues such
as colon,
lung (Stratagene, La Jolla, CA), thymus and kidney (Clontech, Palo Alto, CA).
Total RNA
from cirrhotic and lupus kidney was obtained from BioChain Institute, Inc.,
(Hayward,
CA). Crohn's intestinal and ulcerative colitis samples were obtained from the
National
Disease Research Interchange (NDRI, Philadelphia, PA). Cells purchased from
Clonetics
(Walkersville, MD) included: astrocytes, lung fibroblasts, dermal fibroblasts,
coronary
artery smooth muscle cells, small airway epithelium, bronchial epithelium,
microvascular
dermal endothelial cells, microvascular lung endothelial cells, human
pulmonary aortic
endothelial cells, and human umbilical vein endothelial. These primary cell
types were
activated by incubating with various cytokines (IL-1 beta ~1-5 ng/ml, TNF
alpha ~5-10
ng/ml, IFN gamma ~20-50 ng/ml,1L-4 ~5-10 ng/ml, IL-9 ~5-10 ng/ml, IL.-13 5-10
ng/ml)
or combinations of cytokines as indicated. Starved endothelial cells were
cultured in the
basal media (Clonetics, Walkersville, MD) with 0.1°lo serum.
Mononuclear cells were prepared from blood donations using Ficoll. LAK cells
were cultured in culture media [DMEM, 5% FCS (Hyclone, Logan, UT), 100 mM non
essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium
pyruvate
(Gibco), mercaptoethanol 5.5 x 10-5 M (Gibco), and 10 mM Hepes (Gibco)] and
interleukin
2 for 4-6 days. Cells were activated with 10-20 ng/ml PMA and 1-2 ~,g/ml
ionomycin, 5-10
ng/ml IL-12, 20-50 ng/ml IFN gamma or 5-10 ng/ml IL-18 for 6 hours. In some
cases,
mononuclear cells were cultured for 4-5 days in culture media with ~5 mg/ml
PHA
(phytohemagglutinin) or PWM (pokeweed mitogen; Sigma-Aldrich Corp., St. Louis,
MO).
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Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed
lymphocyte
reaction) samples were obtained by taking blood from.two donors, isolating the
mononuclear cells using Ficoll and mixing them 1:1 at a final concentration of
2x106
cells/ml in culture media. The MLR samples were taken at various time points
from 1-7
days for RNA preparation. .
Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve
VS selection columns and a Vario Magnet (Miltenyi Biotec, Auburn, CA)
according to the
manufacturer's instructions..Monocytes were differentiated into dendritic
cells by culturing
in culture media with 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days.
Macrophages were
prepared by culturing monocytes for 5-7 days in culture media with ~50 ng/ml
10% type
AB Human Serum (Life technologies, Rockville, MD) or MCSF (Macrophage colony
stimulating factor; R&D, Minneapolis, MN). Monocytes, macrophages and
dendritic cells
were stimulated for 6 or 12-14 hours with 100 ng/ml lipopolysaccharide (LPS).
Dendritic
cells were also stimulated with 10 ~tg/ml anti-CD40 monoclonal antibody
(Pharmingen,
San Diego, CA) for 6 or 12-14 hours.
CD4+ lymphocytes, CD8+ lymphocytes and NK cells were also isolated from
mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS
selection
columns and a Vario Magnet (Miltenyi Biotec, Auburn, CA) according to the
manufacturer's instructions. CD45+RA and CD45+RO CD4+ lymphocytes were
isolated
by depleting mononuclear cells of CD8+, CD56+, CD14+ and CD19+ cells using
CDB,
CD56, CD14 and CD19 Miltenyi beads and positive selection. CD45R0 Miltenyi
beads
were then used to separate the CD45+RO CD4+ lymphocytes from CD45+RA CD4+
lymphocytes. CD45+RA CD4+, CD45+RO CD4 +and: CD8+ lymphocytes were cultured in
culture media at 106 cells/ml in culture plates precoated overnight with 0.5
mg/ml anti-
CD28 (Pharmingen, San Diego, CA) and 3 ~,g/ml anti-CD3 (OKT3, ATCC) in PBS.
After
6 and 24 hours, the cells were harvested for RNA preparation. To prepare
chronically
activated CD8+ lymphocytes, isolated CD8+ lymphocytes were activated for 4
days on
anti-CD28, anti-CD3 coated plates and then harvested~and expanded in culture
media with
IL-2 (1 ng/ml). These CD8+ cells were activated again with plate bound anti-
CD3 and anti-
CD28 for 4 days and expanded as described above. RNA was isolated 6 and 24
hours after
the second activation and after 4 days of the second expansion culture.
Isolated NK cells
were cultured in culture media with 1 ng/ml IL-2 for 4-6 days before RNA was
prepared.
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B cells were prepared from minced and sieved tonsil tissue (NDRI). Tonsil
cells
were pelleted and resupended at 106 cells/ml in culture media. Cells were
activated using 5
~,glml PWM (Sigma-Aldrich Corp., St. Louis, MO) or ~10 ~g/ml anti-CD40
(Pharmingen,
San Diego, CA) and 5-10 ng/ml IL-4. Cells were harvested for RNA preparation
after 24,
48 and 72 hours.
To prepare primary and secondary Thl/Th2 and Trl cells, umbilical cord blood
CD4+ lymphocytes (Poietic Systems, German Town, MD) were cultured at 105-
106cells/ml .
in culture media with IL-2 (4 ng/ml) in 6-well Falcon plates (precoated
overnight with 10
p,g/ml anti-CD28 (Pharmingen) and 2 p.glml anti-CD3 (OKT3; ATCC) then washed
twice
with PBS).
To stimulate Thl phenotype differentiation, IL-12 (5 ng/ml) and anti-III (1
~,g/ml)
were used; for Th2 phenotype differentiation, IL-4 (5 ng/ml) and anti-IFN
gamma (1
p.g/ml) were used; and for Trl phenotype differentiation, IL-10 (5 ng/ml) was
used. After
4-5 days, the activated Thl, Th2 and Tr1 lymphocytes were washed once with
DMEM and
expanded for 4-7 days in culture media with IL-2 (1 ng/ml). Activated Thl, Th2
and Trl ..
lymphocytes were re-stimulated for 5 days with anti-CD28/CD3 and cytokines as
described
above with the addition of anti-CD95L (1 ~,g/ml) to prevent apoptosis.~After 4-
5 days, the
Thl, Th2 and Trl lymphocytes were washed and expanded in culture media with IL-
2 for
4-7 days. Activated~Th1 and Th2 lymphocytes were maintained for a maximum of
three
cycles. RNA was prepared from primary and secondary Thl, Th2 and Trl after 6
and 24
hours following the second and third activations with plate-bound anti-CD3 and
anti-CD28
mAbs and 4 days into the second and third expansion cultures.
Leukocyte cells lines Ramos, EOL-1, KU-812 were obtained from the ATCC. EOL-
1 cells were further differentiated by culturing in culture media at 5 x105
cells/ml with 0.1
mM dbcAMP for 8 days, changing the media every 3 'days and adjusting the cell
concentration to 5 x105 cells/ml. RNA was prepared from resting cells or cells
activated
with PMA (10 ng/ml) and ionomycin (1 ~Cg/ml) for 6 and 14 hours. RNA was
prepared
from resting CCD 1106 keratinocyte cell line (ATCC) or from cells activated
with ~5
ng/ml TNF alpha and 1 ng/ml IL-1 beta. RNA was prepared from resting NCI-H292,
airway epithelial tumor cell line (ATCC) or from cells activated for 6 and 14
hours in
culture media with 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13, and 25 ng/ml 1FN
gamma.
RNA was prepared by lysing approximately 10' cells/ml using Trizol (Gibco BRL)
then adding 1110 volume of bromochloropropane (Molecular Research Corporation,
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Cincinnati, OH), vortexing, incubating for 10 minutes at room temperature and
then
spinning at '14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was placed
in a 15 ml
Falcon Tube and an equal volume of isopropanol was added and left at -
20° C overnight.
The precipitated RNA was spun down at 9,000 rpm for 15 min and washed in 70%
ethanol.
The pellet was redissolved in 300 ,ul of RNAse-free water with 35 ml buffer
(Prornega,
Madison, W>) 5 ,ul DTT, 7 p.l RNAsin and 8 ~,1 DNAse and incubated at
37° C for 30.
minutes to remove contaminating genomic DNA, extracted once with phenol
chloroform
and re-precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of
100%.
ethanol. The RNA was spun down, placed in RNAse free water and stored at -
80° C.
~ AI_comprehensive panel-v1.0
Autoimmunity (AI) comprehensive panel v1.0 included two controls and 89 cDNA
test samples isolated from male (M) and female (F) surgical 'and postmortem
human tissues
that were obtained from the Backus Hospital and Clinomics (Frederick, MD).
Tissue
samples included : normal, adjacent (Adj); matched normal adjacent (match
control);~ joint
tissues (synovial (Syn) fluid, synovium, bone and cartilage, osteoarthritis
(OA), rheumatoid
arthritis (RA)); psoriatic; ulcerative colitis colon; Crohns disease colon;
and emphysrriatic,
asthmatic, allergic and chronic obstructive pulmonary disease (COPD) lung.
Pulmonary and General inflammation (PGI) panel v1.0
Pulmonary and General inflammation (PGI) panel v1.0 included two controls and
39 test samples isolated as surgical or postmortem samples. Tissue samples
include:. five
normal lung samples obtained from Maryland Brain and Tissue Bank, University
of
Maryland (Baltimore, MD), International Bioresource systems, IBS (Tuscon, AZ),
and
Asterand (Detroit, MI), five normal adjacent intestine tissues (NAT) from
Ardais
(Lexington, MA), ulcerative colitis samples (UC) from Ardais (Lexington, MA);
Crohns
disease colon from NDRI, National Disease Research Interchange (Philadelphia,
PA);
emphysematous tissue samples from Ardais (Lexington, MA) and Genomic
Collaborative
Inc. (Cambridge, MA), asthmatic tissue from Maryland Brain and Tissue Bank,
University
of Maryland (Baltimore, MD) and Genomic Collaborative Inc (Cambridge, MA) and
fibrotic tissue from Ardais (Lexinton, MA) and Genomic Collaborative
(Cambridge, MA).
Cellular OA/RA Panel
Cellular OA.RA panel includes 2 control wells and 35 test samples comprised of
cDNA generated from total RNA isolated from human cell lines or primary cells
representative of the human joint and its inflammatory condition. Cell types
included
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normal human osteoblasts (Nhost) from Clonetics (Cambrex, East Rutherford,
NJ), human
chondrosarcoma SW 1353 cells from ATCC (Manossas, VA)), human fibroblast-like
synoviocytes from Cell Applications, Inc. (San Diego, CA) and MH7A cell line
(a
rheumatoid fibroblast-like synoviocytes transformed with SV40 T antigen) .from
Riken Cell
bank ( Tsukuba Science City, Japan). These cell types were activated by
incubating with
various cytokines (IL-1 beta ~1-10 ng/ml, TNF alpha ~5-50 ng/ml, or
prostaglandin E2 for
Nhost cells) for 1, 6, 18 or 24 h. All these cells were starved for at least 5
hand cultured in
their corresponding basal medium with ~ 0.1 to 1 °~o FBS.
Minitissue OA/RA Panel
The OA/RA mini panel includes two control wells and 31 test samples comprised
of
cDNA generated from total RNA isolated from surgical and postmortem human
tissues
obtained from the University of Calgary (Alberta, Canada), NDRI (Philadelphia,
PA), and
Ardais Corporation (Lexington, MA). Joint tissue samples include synovium,
bone and
cartilage from osteoarthritic and rheumatoid arthritis patients undergoing
reconstructive
knee surgery, as well as, normal synovium samples (RNA and tissue). Visceral
normal
tissues were pooled from 2-5 different adults and included adrenal gland,
heart, kidney,
brain, colon, lung, stomach, small intestine, skeletal muscle, and ovary.
ALOS chondrosarcoma
AL05 chondrosarcoma plates included SW1353 cells (ATCC) subjected to serum
starvation and treated for 6 and 18 h with cytokines that are known to induce
MMP (1, 3
and 13) synthesis (e.g. ILlbeta). These treatments included: IL-lbeta (10
ng/ml), IL-lbeta
TNF-alpha (50 ng/ml), IL-lbeta + Oncostatin (50 ng/ml) and PMA (100 ng/ml).
Supernatants were collected and analyzed for MMP l, 3 and 13 production. RNA
was
prepared from these samples using standard procedures.
Panels 5D and SI
Panel 5D and 5I included two controls and cDNAs isolated from human tissues,
human pancreatic islets cells, cell lines, metabolic tissues obtained from
patients enrolled in
the Gestational Diabetes study (described below), and cells from different
stages of
adipocyte differentiation, including differentiated (AD), midway
differentiated (AM), and
undifferentiated (U; human mesenchymal stem cells).
Gestational Diabetes study subjects were young (18 - 40 years), otherwise
healthy
women with and without gestational. .diabetes undergoing routine (elective)
Caesarean
section. Uterine wall smooth muscle (UT), visceral (Vis) adipose, skeletal
muscle (SK),
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