U.S. patent application number 10/403676 was filed with the patent office on 2004-02-12 for therapeutic polypeptides, nucleic acids encoding same, and methods of use.
Invention is credited to Alsobrook, John II, Anderson, David, Boldog, Ferenc, Burgess, Catherine, Casman, Stacie, Edinger, Shlomit R., Gerlach, Valerie, Grosse, William, Guo, Xiaojia, Gusev, Vladimir, Ji, Weizhen, LaRochelle, William, Lepley, Denise, Li, Li, Liu, Xiaohong, MacDougall, John R., Malyankar, Uriel M., Millet, Isabelle, Padigaru, Muralidhara, Patturajan, Meera, Peyman, John A., Rastelli, Luca, Rieger, Daniel, Rothenberg, Mark E., Shimkets, Richard, Stone, David J., Taupier, Raymond JR., Vernet, Corine, Zerhusen, Bryan.
Application Number | 20040029150 10/403676 |
Document ID | / |
Family ID | 26821771 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029150 |
Kind Code |
A1 |
Alsobrook, John II ; et
al. |
February 12, 2004 |
Therapeutic polypeptides, nucleic acids encoding same, and methods
of use
Abstract
Disclosed herein are nucleic acid sequences that encode novel
polypeptides. Also disclosed are polypeptides encoded by these
nucleic acid sequences, and antibodies that immunospecifically bind
to the polypeptide, as well as derivatives, variants, mutants, or
fragments of the novel polypeptide, polynucleotide, or antibody
specific to the polypeptide. The invention further discloses
therapeutic, diagnostic and research methods for diagnosis,
treatment, and prevention of disorders involving any one of these
novel human nucleic acids and proteins.
Inventors: |
Alsobrook, John II;
(Madison, CT) ; Anderson, David; (Plantsville,
CT) ; Boldog, Ferenc; (North Haven, CT) ;
Burgess, Catherine; (Wethersfield, CT) ; Casman,
Stacie; (North Haven, CT) ; Edinger, Shlomit R.;
(New Haven, CT) ; Gerlach, Valerie; (Branford,
CT) ; Grosse, William; (Branford, CT) ; Guo,
Xiaojia; (Branford, CT) ; Gusev, Vladimir;
(Madison, CT) ; Ji, Weizhen; (Branford, CT)
; LaRochelle, William; (Madison, CT) ; Lepley,
Denise; (Branford, CT) ; Li, Li; (Branford,
CT) ; Liu, Xiaohong; (Branford, CT) ;
MacDougall, John R.; (Hamden, CT) ; Malyankar, Uriel
M.; (Branford, CT) ; Millet, Isabelle;
(Milford, CT) ; Padigaru, Muralidhara; (Branford,
CT) ; Patturajan, Meera; (Branford, CT) ;
Peyman, John A.; (New Haven, CT) ; Rastelli,
Luca; (Guilford, CT) ; Rieger, Daniel;
(Branford, CT) ; Rothenberg, Mark E.; (Clinton,
CT) ; Shimkets, Richard; (Guilford, CT) ;
Stone, David J.; (Guilford, CT) ; Taupier, Raymond
JR.; (East Haven, CT) ; Vernet, Corine; (North
Branford, CT) ; Zerhusen, Bryan; (Branford,
CT) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
26821771 |
Appl. No.: |
10/403676 |
Filed: |
March 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10403676 |
Mar 31, 2003 |
|
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|
09520781 |
Mar 8, 2000 |
|
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60123667 |
Mar 9, 1999 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 514/1.7; 514/1.9; 514/15.7; 514/16.4;
514/17.9; 514/19.5; 514/3.8; 514/4.8; 514/6.9; 514/9.8; 530/350;
536/23.5 |
Current CPC
Class: |
A61P 37/08 20180101;
A61P 31/00 20180101; A61P 9/10 20180101; A61P 25/00 20180101; A61P
17/00 20180101; A61K 2039/505 20130101; A61P 25/28 20180101; A61K
38/00 20130101; A61P 29/00 20180101; A61P 19/02 20180101; C07K
14/47 20130101; A61K 48/00 20130101; A61P 17/02 20180101; A61P
37/06 20180101; A61P 35/00 20180101; A61P 7/00 20180101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/320.1; 435/325; 514/12; 530/350; 536/23.5 |
International
Class: |
C12Q 001/68; A61K
038/17; C12P 021/02; C12N 005/06; C07K 014/47; C07H 021/04 |
Claims
What is claimed is:
1. An isolated polypeptide comprising the mature form of an amino
acid sequenced selected from the group consisting of SEQ ID NO:2n,
wherein n is an integer between 1 and 64.
2. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NO:2n, wherein n is an
integer between 1 and 64.
3. An isolated polypeptide comprising an amino acid sequence which
is at least 95% identical to an amino acid sequence selected from
the group consisting of SEQ ID NO:2n, wherein n is an integer
between 1 and 64.
4. An isolated polypeptide, wherein the polypeptide comprises an
amino acid sequence comprising one or more conservative
substitutions in the amino acid sequence selected from the group
consisting of SEQ ID NO:2n, wherein n is an integer between 1 and
64.
5. The polypeptide of claim 1 wherein said polypeptide is naturally
occurring.
6. A composition comprising the polypeptide of claim 1 and a
carrier.
7. A kit comprising, in one or more containers, the composition of
claim 6.
8. The use of a therapeutic in the manufacture of a medicament for
treating a syndrome associated with a human disease, the disease
selected from a pathology associated with the polypeptide of claim
1, wherein the therapeutic comprises the polypeptide of claim
1.
9. A method for determining the presence or amount of the
polypeptide of claim 1 in a sample, the method comprising: (a)
providing said sample; (b) introducing said sample to an antibody
that binds immunospecifically to the polypeptide; and (c)
determining the presence or amount of antibody bound to said
polypeptide, thereby determining the presence or amount of
polypeptide in said sample.
10. A method for determining the presence of or predisposition to a
disease associated with altered levels of expression of the
polypeptide of claim 1 in a first mammalian subject, the method
comprising: a) measuring the level of expression of the polypeptide
in a sample from the first mammalian subject; and b) comparing the
expression of said polypeptide in the sample of step (a) to the
expression of the polypeptide present in a control sample from a
second mammalian subject known not to have, or not to be
predisposed to, said disease, wherein an alteration in the level of
expression of the polypeptide in the first subject as compared to
the control sample indicates the presence of or predisposition to
said disease.
11. A method of identifying an agent that binds to the polypeptide
of claim 1, the method comprising: (a) introducing said polypeptide
to said agent; and (b) determining whether said agent binds to said
polypeptide.
12. The method of claim 11 wherein the agent is a cellular receptor
or a downstream effector.
13. 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 the
polypeptide of claim 1, the method comprising: (a) providing a cell
expressing the polypeptide of claim 1 and having a property or
function ascribable to the polypeptide; (b) contacting the cell
with a composition comprising a candidate substance; and (c)
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 in the absence of the substance, the
substance is identified as a potential therapeutic agent.
14. A method for screening for a modulator of activity of or of
latency or predisposition to a pathology associated with the
polypeptide of claim 1, said method comprising: (a) administering a
test compound to a test animal at increased risk for a pathology
associated with the polypeptide of claim 1, wherein said test
animal recombinantly expresses the polypeptide of claim 1; (b)
measuring the activity of said polypeptide in said test animal
after administering the compound of step (a); and (c) comparing the
activity of said polypeptide in said test animal with the activity
of said polypeptide in a control animal not administered said
polypeptide, wherein a change in the activity of said polypeptide
in said test animal relative to said control animal indicates the
test compound is a modulator activity of or latency or
predisposition to, a pathology associated with the polypeptide of
claim 1.
15. The method of claim 14, wherein said test animal is a
recombinant test animal that expresses a test protein transgene or
expresses said transgene under the control of a promoter at an
increased level relative to a wild-type test animal, and wherein
said promoter is not the native gene promoter of said
transgene.
16. A method for modulating the activity of the polypeptide of
claim 1, the method comprising contacting a cell sample expressing
the polypeptide of claim 1 with a compound that binds to said
polypeptide in an amount sufficient to modulate the activity of the
polypeptide.
17. A method of treating or preventing a pathology associated with
the polypeptide of claim 1, the method comprising administering the
polypeptide of claim 1 to a subject in which such treatment or
prevention is desired in an amount sufficient to treat or prevent
the pathology in the subject.
18. The method of claim 17, wherein the subject is a human.
19. A method of treating a pathological state in a mammal, the
method comprising 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 comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:2n, wherein n is an integer between 1 and 64, or a biologically
active fragment thereof.
20. An isolated nucleic acid molecule comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO:2n-1,
wherein n is an integer between 1 and 64.
21. The nucleic acid molecule of claim 20, wherein the nucleic acid
molecule is naturally occurring.
22. A nucleic acid molecule, wherein the nucleic acid molecule
differs by a single nucleotide from a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 2n-1, wherein n is
an integer between 1 and 64.
23. An isolated nucleic acid molecule encoding the mature form of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:2n, wherein n is an integer between 1 and
64.
24. An isolated nucleic acid molecule comprising a nucleic acid
selected from the group consisting of 2n-1, wherein n is an integer
between 1 and 64.
25. The nucleic acid molecule of claim 20, wherein said nucleic
acid molecule hybridizes under stringent conditions to the
nucleotide sequence selected from the group consisting of SEQ ID
NO: 2n-1, wherein n is an integer between 1 and 64, or a complement
of said nucleotide sequence.
26. A vector comprising the nucleic acid molecule of claim 20.
27. The vector of claim 26, further comprising a promoter operably
linked to said nucleic acid molecule.
28. A cell comprising the vector of claim 26.
29. An antibody that immunospecifically binds to the polypeptide of
claim 1.
30. The antibody of claim 29, wherein the antibody is a monoclonal
antibody.
31. The antibody of claim 29, wherein the antibody is a humanized
antibody.
32. A method for determining the presence or amount of the nucleic
acid molecule of claim 20 in a sample, the method comprising: (a)
providing said sample; (b) introducing said sample to a probe that
binds to said nucleic acid molecule; and (c) determining the
presence or amount of said probe bound to said nucleic acid
molecule, thereby determining the presence or amount of the nucleic
acid molecule in said sample.
33. The method of claim 32 wherein presence or amount of the
nucleic acid molecule is used as a marker for cell or tissue
type.
34. The method of claim 33 wherein the cell or tissue type is
cancerous.
35. A method for determining the presence of or predisposition to a
disease associated with altered levels of expression of the nucleic
acid molecule of claim 20 in a first mammalian subject, the method
comprising: a) measuring the level of expression of the nucleic
acid in a sample from the first mammalian subject; and b) comparing
the level of expression of said nucleic acid in the sample of step
(a) to the level of expression 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 expression of the nucleic acid in the first subject as
compared to the control sample indicates the presence of or
predisposition to the disease.
36. A method of producing the polypeptide of claim 1, the method
comprising culturing a cell under conditions that lead to
expression of the polypeptide, wherein said cell comprises a vector
comprising an isolated nucleic acid molecule comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO:2n-1,
wherein n is an integer between 1 and 64.
37. The method of claim 36 wherein the cell is a bacterial
cell.
38. The method of claim 36 wherein the cell is an insect cell.
39. The method of claim 36 wherein the cell is a yeast cell.
40. The method of claim 36 wherein the cell is a mammalian
cell.
41. A method of producing the polypeptide of claim 2, the method
comprising culturing a cell under conditions that lead to
expression of the polypeptide, wherein said cell comprises a vector
comprising an isolated nucleic acid molecule comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO:2n-1,
wherein n is an integer between 1 and 64.
42. The method of claim 41 wherein the cell is a bacterial
cell.
43. The method of claim 41 wherein the cell is an insect cell.
44. The method of claim 41 wherein the cell is a yeast cell.
45. The method of claim 41 wherein the cell is a mammalian
cell.
46. An isolated polypeptide comprising an amino acid sequence at
least 95% similar to SEQ ID NO: 2, wherein said amino acid sequence
comprises at least one amino acid substitution, wherein said
substitution is at amino acid position 43 when numbered in
accordance with SEQ ID NO: 2.
47. An isolated nucleic acid molecule comprising an nucleic acid
sequence at least 95% similar to SEQ ID NO: 1, wherein said nucleic
acid sequence comprises at least one nucleic acid substitution,
wherein said substitution is at nucleic acid position 135 when
numbered in accordance with SEQ ID NO: 1.
48. An isolated polypeptide comprising an amino acid sequence at
least 95% similar to SEQ ID NO: 14, wherein said amino acid
sequence comprises at least one amino acid substitution, wherein
said substitution is at the amino acid position selected from the
group consisting of 8, 54, 56, 92, 207, 240, 706, 891 and 923 when
numbered in accordance with SEQ ID NO: 14.
49. An isolated nucleic acid molecule comprising an nucleic acid
sequence at least 95% similar to SEQ ID NO: 13, wherein said
nucleic acid sequence comprises at least one nucleic acid
substitution, wherein said substitution is at the nucleic acid
position selected from the group consisting of 272, 410, 416, 523,
869, 967, 2366, 2921 and 3018 when numbered in accordance with SEQ
ID NO: 13.
50. An isolated polypeptide comprising an amino acid sequence at
least 95% similar to SEQ ID NO: 58, wherein said amino acid
sequence comprises at least one amino acid substitution, wherein
said substitution is at the amino acid position selected from the
group consisting of 23, 56, 105, 125, 160, 183 and 215 when
numbered in accordance with SEQ ID NO: 58.
51. An isolated nucleic acid molecule comprising an nucleic acid
sequence at least 95% similar to SEQ ID NO: 57, wherein said
nucleic acid sequence comprises at least one nucleic acid
substitution, wherein said substitution is at the nucleic acid
position selected from the group consisting of 181, 278, 426, 485,
591, 661 and 756 when numbered in accordance with SEQ ID NO:
57.
52. An isolated polypeptide comprising an amino acid sequence at
least 95% similar to SEQ ID NO: 80, wherein said amino acid
sequence comprises at least one amino acid substitution, wherein
said substitution is at amino acid position 219 when numbered in
accordance with SEQ ID NO: 80.
53. An isolated nucleic acid molecule comprising an nucleic acid
sequence at least 95% similar to SEQ ID NO: 79, wherein said
nucleic acid sequence comprises at least one nucleic acid
substitution, wherein said substitution is at nucleic acid position
685 when numbered in accordance with SEQ ID NO: 79.
54. An isolated polypeptide comprising an amino acid sequence at
least 95% similar to SEQ ID NO: 92, wherein said amino acid
sequence comprises at least one amino acid substitution, wherein
said substitution is at amino acid position 470 when numbered in
accordance with SEQ ID NO: 92.
55. An isolated nucleic acid molecule comprising an nucleic acid
sequence at least 95% similar to SEQ ID NO: 91, wherein said
nucleic acid sequence comprises at least one nucleic acid
substitution, wherein said substitution is at nucleic acid position
1874 when numbered in accordance with SEQ ID NO: 91.
56. An isolated polypeptide comprising an amino acid sequence at
least 95% similar to SEQ ID NO: 100, wherein said amino acid
sequence comprises at least one amino acid substitution, wherein
said substitution is at the amino acid position selected from the
group consisting of 11, 112 and 145 when numbered in accordance
with SEQ ID NO: 100.
57. An isolated nucleic acid molecule comprising an nucleic acid
sequence at least 95% similar to SEQ ID NO: 99, wherein said
nucleic acid sequence comprises at least one nucleic acid
substitution, wherein said substitution is at the nucleic acid
position selected from the group consisting of 80, 383 and 482 when
numbered in accordance with SEQ ID NO: 99.
58. An isolated polypeptide comprising an amino acid sequence at
least 95% similar to SEQ ID NO: 122, wherein said amino acid
sequence comprises at least one amino acid substitution, wherein
said substitution is at the amino acid position selected from the
group consisting of 12, 38, 54, 65, 66, 69, 80, 90, 91, 96, 100,
101, 102, 114, 122, 125, 126, 134, 135, 144, 148, 154, 155 and 156
when numbered in accordance with SEQ ID NO: 122.
59. An isolated nucleic acid molecule comprising an nucleic acid
sequence at least 95% similar to SEQ ID NO: 121, wherein said
nucleic acid sequence comprises at least one nucleic acid
substitution, wherein said substitution is at the nucleic acid
position selected from the group consisting of 35, 112, 160, 194,
197, 206, 240, 269, 273, 287, 298, 301, 305, 340, 365, 374, 376,
400, 404, 431, 442, 461, 463 and 468 when numbered in accordance
with SEQ ID NO: 121.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/520,781, filed Mar. 8, 2000, which claims priority to U.S. S No.
60/123,667, filed Mar. 9, 1999; U.S. Ser. No. 09/957,187, filed
Sep. 19, 2001; U.S. Ser. No. 09/538,092, filed Mar. 29, 2000, which
claims priority to U.S. S No. 60/127,352, filed Apr. 1, 1999; U.S.
Ser. No. 09/604,286, filed Jun. 22, 2000, which claims priority to
U.S. S No. 60/140,584, filed Jun. 23, 1999; U.S. Ser. No.
09/898,994, filed Jul. 3, 2001, which claims priority to U.S. S No.
60/216,722, filed Jul. 7, 2000; U.S. Ser. No. 09/954,342, filed
Sep. 17, 2001, which claims priority to U.S. S No. 60/233,798,
filed Sep. 19, 2000; U.S. Ser. No. 09/995,514, filed Nov. 28, 2001,
which claims priority to U.S. S No. 60/250,926, filed Nov. 30,
2000; U.S. Ser. No. 10/044,564, filed Jan. 11, 2002, which claims
priority to U.S. S No. 60/261,018, filed Jan. 11, 2001; and U.S.
Ser. No. 10/072,013, filed Feb. 8, 2002, which claims priority to
U.S. S No. 60/265,395, filed Jan. 31, 2001 and U.S. S No.
60/266,767, filed Feb. 5, 2001; and this application claims
priority to the following provisional patent applications: U.S. S
No. 60/368,996, filed Apr. 1, 2002; U.S. S No. 60/369,980, filed
Apr. 4, 2002; U.S. S No. 60/370,381, filed Apr. 5, 2002; U.S. S No.
60/370,969, filed Apr., 8, 2002; U.S. S No. 60/371,002, filed Apr.
9, 2002; U.S. S No. 60/372,002, filed Apr. 12, 2002; U.S. S No.
60/384,297, filed May 30, 2002; U.S. S No. 60/386,816, filed Jun.
7, 2002; U.S. S No. 60/389,123, filed Jun. 13, 2002; U.S. S No.
60/402,207, filed Aug. 9, 2002; and U.S. S No. 60/420,860, filed
Oct. 24, 2002; each of which is incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
[0002] 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 treating diverse pathological conditions.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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
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.
[0007] 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.
[0008] Therefore there is a need to assay for the level of a
protein effector of interest in a biological sample from such a
subject, and to compare this level with that characteristic of a
nonpathological condition. In particular, there is a need for such
an assay based on the use of an antibody that binds
immunospecifically to the antigen. There further is a need to
inhibit the activity of the protein effector in cases where a
pathological condition arises from elevated or excessive levels of
the effector based on the use of an antibody that binds
immunospecifically to the effector. Thus, there is a need for the
antibody as a product of manufacture. There further is a need for a
method of treatment of a pathological condition brought on by an
elevated or excessive level of the protein effector of interest
based on administering the antibody to the subject.
SUMMARY OF THE INVENTION
[0009] The invention is based in part upon the discovery of
isolated polypeptides including amino acid sequences selected from
mature forms of the amino acid sequences selected from the group
consisting of SEQ ID NO:2n, wherein n is an integer between 1 and
64. The novel nucleic acids and polypeptides are referred to herein
as NOVX, or NOV1, NOV2, NOV3, etc., nucleic acids and polypeptides.
These nucleic acids and polypeptides, as well as derivatives,
homologs, analogs and fragments thereof, will hereinafter be
collectively designated as "NOVX" nucleic acid or polypeptide
sequences.
[0010] The invention also is based in part upon variants of a
mature form of the amino acid sequence selected from the group
consisting of SEQ ID NO:2n, wherein n is an integer between 1 and
64, 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 ID NO:2n, wherein n is an
integer between 1 and 64. In another embodiment, the invention also
comprises variants of the amino acid sequence selected from the
group consisting of SEQ ID NO:2n, wherein n is an integer between 1
and 64, wherein any amino acid specified in the chosen sequence is
changed to a different amino acid, provided that no more than 15%
of the amino acid residues in the sequence are so changed. The
invention also involves fragments of any of the mature forms of the
amino acid sequences selected from the group consisting of SEQ ID
NO:2n, wherein n is an integer between 1 and 64, or any other amino
acid sequence selected from this group. The invention also
comprises fragments from these groups in which up to 15% of the
residues are changed.
[0011] In another embodiment, the invention encompasses
polypeptides that are naturally occurring allelic variants of the
sequence selected from the group consisting of SEQ ID NO:2n,
wherein n is an integer between 1 and 64. These allelic variants
include amino acid sequences that are the translations of nucleic
acid sequences differing by a single nucleotide from nucleic acid
sequences selected from the group consisting of SEQ ID NOS: 2n-1,
wherein n is an integer between 1 and 64. The variant polypeptide
where any amino acid changed in the chosen sequence is changed to
provide a conservative substitution.
[0012] In another embodiment, the invention comprises a
pharmaceutical composition involving a polypeptide with an amino
acid sequence selected from the group consisting of SEQ ID NO:2n,
wherein n is an integer between 1 and 64, and a pharmaceutically
acceptable carrier. In another embodiment, the invention involves a
kit, including, in one or more containers, this pharmaceutical
composition.
[0013] 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
NO:2n, wherein n is an integer between 1 and 64, wherein said
therapeutic is the polypeptide selected from this group.
[0014] 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 NO:2n,
wherein n is an integer between 1 and 64, 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.
[0015] 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 64, 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.
[0016] 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 NO:2n,
wherein n is an integer between 1 and 64, 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.
[0017] 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
NO:2n, wherein n is an integer between 1 and 64, 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.
[0018] 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
NO:2n, wherein n is an integer between 1 and 64, 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.
[0019] In another embodiment, the invention involves a method for
modulating the activity of a polypeptide with an amino acid
sequence selected from the group consisting of SEQ ID NO:2n,
wherein n is an integer between 1 and 64, 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.
[0020] 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 ID NO:2n, wherein n is an integer between 1 and 64, 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.
[0021] In another embodiment, the invention involves a method of
treating a pathological state in a mammal, the method including
administering to the mammal a polypeptide in an amount that is
sufficient to alleviate the pathological state, wherein the
polypeptide is a polypeptide having an amino acid sequence at least
95% identical to a polypeptide having the amino acid sequence
selected from the group consisting of SEQ ID NO:2n, wherein n is an
integer between 1 and 64, or a biologically active fragment
thereof.
[0022] In another embodiment, the invention involves an isolated
nucleic acid molecule comprising a nucleic acid sequence encoding a
polypeptide having an amino acid sequence selected from the group
consisting of a mature form of the amino acid sequence given SEQ ID
NO:2n, wherein n is an integer between 1 and 64, a variant of a
mature form of the amino acid sequence selected from the group
consisting of SEQ ID NO:2n, wherein n is an integer between 1 and
64, 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 NO:2n, wherein n is an integer
between 1 and 64, 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 64, 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 NO:2n, wherein n is an integer between 1
and 64, or any variant of the polypeptide wherein any amino acid of
the chosen sequence is changed to a different amino acid, provided
that no more than 10% of the amino acid residues in the sequence
are so changed; and the complement of any of the nucleic acid
molecules.
[0023] In another embodiment, the invention comprises an isolated
nucleic acid molecule having a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence selected from the
group consisting of a mature form of the amino acid sequence given
SEQ ID NO:2n, wherein n is an integer between 1 and 64, wherein the
nucleic acid molecule comprises the nucleotide sequence of a
naturally occurring allelic nucleic acid variant.
[0024] In another embodiment, the invention involves an isolated
nucleic acid molecule including a nucleic acid sequence encoding a
polypeptide having an amino acid sequence selected from the group
consisting of a mature form of the amino acid sequence given SEQ ID
NO:2n, wherein n is an integer between 1 and 64, that encodes a
variant polypeptide, wherein the variant polypeptide has the
polypeptide sequence of a naturally occurring polypeptide
variant.
[0025] In another embodiment, the invention comprises an isolated
nucleic acid molecule having a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence selected from the
group consisting of a mature form of the amino acid sequence given
SEQ ID NO:2n, wherein n is an integer between 1 and 64, wherein the
nucleic acid molecule differs by a single nucleotide from a nucleic
acid sequence selected from the group consisting of SEQ ID NOS:
2n-1, wherein n is an integer between 1 and 64.
[0026] In another embodiment, the invention includes an isolated
nucleic acid molecule having a nucleic acid sequence encoding a
polypeptide including an amino acid sequence selected from the
group consisting of a mature form of the amino acid sequence given
SEQ ID NO:2n, wherein n is an integer between 1 and 64, wherein the
nucleic acid molecule comprises a nucleotide sequence selected from
the group consisting of the nucleotide sequence selected from the
group consisting of SEQ ID NO:2n-1, wherein n is an integer between
1 and 64, a nucleotide sequence wherein one or more nucleotides in
the nucleotide sequence selected from the group consisting of SEQ
ID NO:2n-1, wherein n is an integer between 1 and 64, is changed
from that selected from the group consisting of the chosen sequence
to a different nucleotide provided that no more than 15% of the
nucleotides are so changed; a nucleic acid fragment of the sequence
selected from the group consisting of SEQ ID NO:2n-1, wherein n is
an integer between 1 and 64, and a nucleic acid fragment wherein
one or more nucleotides in the nucleotide sequence selected from
the group consisting of SEQ ID NO:2n-1, wherein n is an integer
between 1 and 64, 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.
[0027] In another embodiment, the invention includes an isolated
nucleic acid molecule having a nucleic acid sequence encoding a
polypeptide including an amino acid sequence selected from the
group consisting of a mature form of the amino acid sequence given
SEQ ID NO:2n, wherein n is an integer between 1 and 64, wherein the
nucleic acid molecule hybridizes under stringent conditions to the
nucleotide sequence selected from the group consisting of SEQ ID
NO:2n-1, wherein n is an integer between 1 and 64, or a complement
of the nucleotide sequence.
[0028] In another embodiment, the invention includes an isolated
nucleic acid molecule having a nucleic acid sequence encoding a
polypeptide including an amino acid sequence selected from the
group consisting of a mature form of the amino acid sequence given
SEQ ID NO:2n, wherein n is an integer between 1 and 64, 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.
[0029] In another embodiment, the invention includes a vector
involving the nucleic acid molecule having a nucleic acid sequence
encoding a polypeptide including an amino acid sequence selected
from the group consisting of a mature form of the amino acid
sequence given SEQ ID NO:2n, wherein n is an integer between 1 and
64. This vector can have a promoter operably linked to the nucleic
acid molecule. This vector can be located within a cell.
[0030] 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 NO:2n, wherein n is an
integer between 1 and 64, 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.
[0031] 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 ID NO:2n, wherein n is an
integer between 1 and 64, 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.
[0032] 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.
[0033] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention provides novel nucleotides and
polypeptides encoded thereby. Included in the invention are the
novel nucleic acid sequences, their encoded polypeptides,
antibodies, and other related compounds. The sequences are
collectively referred to herein as "NOVX nucleic acids" or "NOVX
polynucleotides" and the corresponding encoded polypeptides are
referred to as "NOVX polypeptides" or "NOVX proteins." Unless
indicated otherwise, "NOVX" is meant to refer to any of the novel
sequences disclosed herein. Table A provides a summary of the NOVX
nucleic acids and their encoded polypeptides.
1TABLE A Sequences and Corresponding SEQ ID Numbers SEQ ID SEQ ID
NO NO NOVX Internal (nucleic (amino Assignment Identification acid)
acid) Homology NOV1a CG50907-03 1 2 Human extracellular matrix and
cell adhesion molecule-4 (XMAD-4) - Homo sapiens NOV1b CG50907-04 3
4 Human extracellular matrix and cell adhesion molecule-4 (XMAD-4)
- Homo sapiens NOV1c CG50907-02 5 6 Human extracellular matrix and
cell adhesion molecule-4 (XMAD-4) - Homo sapiens NOV1d 170645595 7
8 Human extracellular matrix and cell adhesion molecule-4 (XMAD-4)
- Homo sapiens NOV1e 170645599 9 10 Human extracellular matrix and
cell adhesion molecule-4 (XMAD-4) - Homo sapiens NOV1f CG50907-01
11 12 Human extracellular matrix and cell adhesion molecule-4
(XMAD-4) - Homo sapiens NOV2a CG51896-04 13 14 Human semaphorin
6A-1 - Homo sapiens NOV2b 271674560 15 16 Human semaphorin 6A-1 -
Homo sapiens NOV2c 267441133 17 18 Human semaphorin 6A-1 - Homo
sapiens NOV2d 267441137 19 20 Human semaphorin 6A-1 - Homo sapiens
NOV2e 262254987 21 22 Human semaphorin 6A-1 - Homo sapiens NOV2f
260565761 23 24 Human semaphorin 6A-1 - Homo sapiens NOV2g
252324008 25 26 Human semaphorin 6A-1 - Homo sapiens NOV2h
252323542 27 28 Human semaphorin 6A-1 - Homo sapiens NOV2i
252323483 29 30 Human semaphorin 6A-1 - Homo sapiens NOV2j
CG51896-01 31 32 Human semaphorin 6A-1 - Homo sapiens NOV2k
CG51896-02 33 34 Human semaphorin 6A-1 - Homo sapiens NOV2l
CG51896-03 35 36 Human semaphorin 6A-1 - Homo sapiens NOV2m
CG51896-05 37 38 Human semaphorin 6A-1 - Homo sapiens NOV2n
CG51896-06 39 40 Human semaphorin 6A-1 - Homo sapiens NOV2o
CG51896-07 41 42 Human semaphorin 6A-1 - Homo sapiens NOV2p
CG51896-08 43 44 Human semaphorin 6A-1 - Homo sapiens NOV2q
CG51896-09 45 46 Human semaphorin 6A-1 - Homo sapiens NOV2r
CG51896-10 47 48 Human semaphorin 6A-1 - Homo sapiens NOV2s
CG51896-11 49 50 Human semaphorin 6A-1 - Homo sapiens NOV2t
CG51896-12 51 52 Human semaphorin 6A-1 - Homo sapiens NOV2u
CG51896-13 53 54 Human semaphorin 6A-1 - Homo sapiens NOV2v
CG51896-14 55 56 Human semaphorin 6A-1 - Homo sapiens NOV3a
CG52324-01 57 58 Phosphatidylethanolamine binding protein - Homo
sapiens NOV3b 249357821 59 60 Phosphatidylethanolamine binding
protein - Homo sapiens NOV3c 249357798 61 62
Phosphatidylethanolamine binding protein - Homo sapiens NOV3d
248644954 63 64 Phosphatidylethanolamine binding protein - Homo
sapiens NOV3e 248644962 65 66 Phosphatidylethanolamine binding
protein - Homo sapiens NOV3f 248645004 67 68
Phosphatidylethanolamine binding protein - Homo sapiens NOV3g
249420987 69 70 Phosphatidylethanolamine binding protein - Homo
sapiens NOV3h 248486005 71 72 Phosphatidylethanolamine binding
protein - Homo sapiens NOV3i 249421046 73 74
Phosphatidylethanolamine binding protein - Homo sapiens NOV3j
CG52324-02 75 76 Phosphatidylethanolamine binding protein - Homo
sapiens NOV3k CG52324-03 77 78 Phosphatidylethanolamine binding
protein - Homo sapiens NOV4a CG53054-02 79 80 WNT-14 protein
precursor - Homo sapiens NOV4b 170251039 81 82 WNT-14 protein
precursor - Homo sapiens NOV4c 170251076 83 84 WNT-14 protein
precursor - Homo sapiens NOV4d CG53054-01 85 86 WNT-14 protein
precursor - Homo sapiens NOV4e CG53054-03 87 88 WNT-14 protein
precursor - Homo sapiens NOV4f CG53054-04 89 90 WNT-14 protein
precursor - Homo sapiens NOV5a CG54818-01 91 92 Semaphorin 3E
precursor - Homo sapiens NOV5b CG54818-02 93 94 Semaphorin 3E
precursor - Homo sapiens NOV5c CG54818-03 95 96 Semaphorin 3E
precursor - Homo sapiens NOV5d CG54818-04 97 98 Semaphorin 3E
precursor - Homo sapiens NOV6a CG55023-01 99 100 Epigen protein
precursor - Mus musculus NOV6b 248209521 101 102 Epigen protein
precursor - Mus musculus NOV6c 317459649 103 104 Epigen protein
precursor - Mus musculus NOV6d 317459665 105 106 Epigen protein
precursor - Mus musculus NOV6e 317459901 107 108 Epigen protein
precursor - Mus musculus NOV6f 317459910 109 110 Epigen protein
precursor - Mus musculus NOV6g CG55023-02 111 112 Epigen protein
precursor - Mus musculus NOV6h CG55023-03 113 114 Epigen protein
precursor - Mus musculus NOV6i CG55023-04 115 116 Epigen protein
precursor - Mus musculus NOV6j CG55023-05 117 118 Epigen protein
precursor - Mus musculus NOV6k CG55023-06 119 120 Epigen protein
precursor - Mus musculus NOV7a CG56136-01 121 122 Interleukin 1
family member 6 (IL-1F6) (Interleukin-1 epsilon) (IL-1 epsilon)
(FIL1 epsilon) - Homo sapiens NOV7b CG56136-02 123 124 Interleukin
1 family member 6 (IL-1F6) (Interleukin-1 epsilon) (IL-1 epsilon)
(FIL1 epsilon) - Homo sapiens NOV7c CG56136-03 125 126 Interleukin
1 family member 6 (IL-1F6) (Interleukin-1 epsilon) (IL-1 epsilon)
(FIL1 epsilon) - Homo sapiens NOV7d CG56136-04 127 128 Interleukin
1 family member 6 (IL-1F6) (Interleukin-1 epsilon) (IL-1 epsilon)
(FIL1 epsilon) - Homo sapiens
[0035] 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.
[0036] Pathologies, diseases, disorders, conditions and the like
that are associated with NOVX sequences include, but are not
limited to, e.g., cardiomyopathy, atherosclerosis, hypertension,
congenital heart defects, aortic stenosis, atrial septal defect
(ASD), atrioventricular (A-V) canal defect, ductus arteriosus,
pulmonary stenosis, subaortic stenosis, ventricular septal defect
(VSD), valve diseases, tuberous sclerosis, scleroderma, obesity,
metabolic disturbances associated with obesity, transplantation,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate
cancer, diabetes, metabolic disorders, neoplasm; adenocarcinoma,
lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation,
idiopathic thrombocytopenic purpura, immunodeficiencies, graft
versus host disease, AIDS, bronchial asthma, Crohn's disease;
multiple sclerosis, treatment of Albright Hereditary
Ostoeodystrophy, infectious disease, anorexia, cancer-associated
cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's Disorder, immune disorders, hematopoietic disorders,
and the various dyslipidemias, the metabolic syndrome X and wasting
disorders associated with chronic diseases and various cancers, as
well as conditions such as transplantation and fertility.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] Additional utilities for NOVX nucleic acids and polypeptides
according to the invention are disclosed herein.
[0042] NOVX Clones
[0043] 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.
[0044] 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.
[0045] 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.
[0046] In one specific embodiment, the invention includes an
isolated polypeptide comprising an amino acid sequence selected
from the group consisting of: (a) a mature form of the amino acid
sequence selected from the group consisting of SEQ ID NO:2n,
wherein n is an integer between 1 and 64, (b) a variant of a mature
form of the amino acid sequence selected from the group consisting
of SEQ ID NO:2n, wherein n is an integer between 1 and 64, 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 1 and 64, (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 64, 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).
[0047] In another specific embodiment, the invention includes an
isolated nucleic acid molecule comprising a nucleic acid sequence
encoding a polypeptide comprising an amino acid sequence selected
from the group consisting of: (a) a mature form of the amino acid
sequence given SEQ ID NO:2n, wherein n is an integer between 1 and
64; (b) a variant of a mature form of the amino acid sequence
selected from the group consisting of SEQ ID NO:2n, wherein n is an
integer between 1 and 64, wherein any amino acid in the mature form
of the chosen sequence is changed to a different amino acid,
provided that no more than 15% of the amino acid residues in the
sequence of the mature form are so changed; (c) the amino acid
sequence selected from the group consisting of SEQ ID NO:2n,
wherein n is an integer between 1 and 64; (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 64, in which any amino
acid specified in the chosen sequence is changed to a different
amino acid, provided that no more than 15% of the amino acid
residues in the sequence are so changed; (e) a nucleic acid
fragment encoding at least a portion of a polypeptide comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:2n, wherein n is an integer between 1 and 64, 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.
[0048] 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
64; (b) a nucleotide sequence wherein one or more nucleotides in
the nucleotide sequence selected from the group consisting of SEQ
ID NO:2n-1, wherein n is an integer between 1 and 64, is changed
from that selected from the group consisting of the chosen sequence
to a different nucleotide provided that no more than 15% of the
nucleotides are so changed; (c) a nucleic acid fragment of the
sequence selected from the group consisting of SEQ ID NO:2n-1,
wherein n is an integer between 1 and 64; and (d) a nucleic acid
fragment wherein one or more nucleotides in the nucleotide sequence
selected from the group consisting of SEQ ID NO:2n-1, wherein n is
an integer between 1 and 64, 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.
[0049] NOVX Nucleic Acids and Polypeptides
[0050] One aspect of the invention pertains to isolated nucleic
acid molecules that encode NOVX polypeptides or biologically active
portions thereof. Also included in the invention are nucleic acid
fragments sufficient for use as hybridization probes to identify
NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for
use as PCR primers for the amplification and/or mutation of NOVX
nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA
generated using nucleotide analogs, and derivatives, fragments and
homologs thereof. The nucleic acid molecule may be single-stranded
or double-stranded, but preferably is comprised double-stranded
DNA.
[0051] A NOVX nucleic acid can encode a mature NOVX polypeptide. As
used herein, a "mature" form of a polypeptide or protein disclosed
in the present invention is the product of a naturally occurring
polypeptide, 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 occurring processing
steps that may take place within the cell (e.g., host cell) in
which the gene product arises. Examples of such processing steps
leading to a "mature" form of a polypeptide or protein include the
cleavage of the N-terminal methionine residue encoded by the
initiation codon of an ORF or the proteolytic cleavage of a signal
peptide or leader sequence. Thus a mature form arising from a
precursor polypeptide or protein that has residues 1 to N, where
residue 1 is the N-terminal methionine, would have residues 2
through N remaining after removal of the N-terminal methionine.
Alternatively, a mature form arising from a precursor polypeptide
or protein having residues 1 to N, in which an N-terminal signal
sequence from residue 1 to residue M is cleaved, would have the
residues from residue M+1 to residue N remaining. Further as used
herein, a "mature" form of a polypeptide or protein may arise from
a post-translational modification step other than a proteolytic
cleavage event. Such additional processes include, by way of
non-limiting example, glycosylation, myristylation or
phosphorylation. In general, a mature polypeptide or protein may
result from the operation of only one of these processes, or a
combination of any of them.
[0052] The term "probe", as utilized herein, refers to nucleic acid
sequences of variable length, preferably between at least about 10
nucleotides (nt), about 100 nt, or as many as approximately, e.g.,
6,000 nt, depending upon the specific use. Probes are used in the
detection of identical, similar, or complementary nucleic acid
sequences. Longer length probes are generally obtained from a
natural or recombinant source, are highly specific, and much slower
to hybridize than shorter-length oligomer probes. Probes may be
single- or double-stranded and designed to have specificity in PCR,
membrane-based hybridization technologies, or ELISA-like
technologies.
[0053] 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, about 4 kb, about 3 kb, about 2
kb, about 1 kb, about 0.5 kb, or about 0.1 kb, of nucleotide
sequences which naturally flank the nucleic acid molecule in
genomic DNA of the cell/tissue from which the nucleic acid is
derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium,
or of chemical precursors or other chemicals.
[0054] A nucleic acid molecule of the invention, e.g., a nucleic
acid molecule having the nucleotide sequence of SEQ ID NOS: 2n-1,
wherein n is an integer between 1 and 64, or a complement of this
nucleotide sequence, can be isolated using standard molecular
biology techniques and the sequence information provided herein.
Using all or a portion of the nucleic acid sequence of SEQ ID
NOS:2n-1, wherein n is an integer between 1 and 64, as a
hybridization probe, NOVX molecules can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL
2.sup.nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y.,
1993).
[0055] 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.
[0056] As used herein, the term "oligonucleotide" refers to a
series of linked nucleotide residues. A short oligonucleotide
sequence may be based on, or designed from, a genomic or cDNA
sequence and is used to amplify, confirm, or reveal the presence of
an identical, similar or complementary DNA or RNA in a particular
cell or tissue. Oligonucleotides comprise a nucleic acid sequence
having about 10 nt, 50 nt, or 100 nt in length, preferably about 15
nt to 30 nt in length. In one embodiment of the invention, an
oligonucleotide comprising a nucleic acid molecule less than 100 nt
in length would further comprise at least 6 contiguous nucleotides
of SEQ ID NOS:2n-1, wherein n is an integer between 1 and 64, or a
complement thereof. Oligonucleotides may be chemically synthesized
and may also be used as probes.
[0057] In another embodiment, an isolated nucleic acid molecule of
the invention comprises a nucleic acid molecule that is a
complement of the nucleotide sequence shown in SEQ ID NOS:2n-1,
wherein n is an integer between 1 and 64, or a portion of this
nucleotide sequence (e.g., a fragment that can be used as a probe
or primer or a fragment encoding a biologically-active portion of a
NOVX polypeptide). A nucleic acid molecule that is complementary to
the nucleotide sequence of SEQ ID NOS:2n-1, wherein n is an integer
between 1 and 64, is one that is sufficiently complementary to the
nucleotide sequence of SEQ ID NOS:2n-1, wherein n is an integer
between 1 and 64, that it can hydrogen bond with few or no
mismatches to a nucleotide sequence of SEQ ID NOS:2n-1, wherein n
is an integer between 1 and 64, thereby forming a stable
duplex.
[0058] 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.
[0059] 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.
[0060] A full-length NOVX clone is identified as containing an ATG
translation start codon and an in-frame stop codon. Any disclosed
NOVX nucleotide sequence lacking an ATG start codon therefore
encodes a truncated C-terminal fragment of the respective NOVX
polypeptide, and requires that the corresponding full-length cDNA
extend in the 5' direction of the disclosed sequence. Any disclosed
NOVX nucleotide sequence lacking an in-frame stop codon similarly
encodes a truncated N-terminal fragment of the respective NOVX
polypeptide, and requires that the corresponding full-length cDNA
extend in the 3' direction of the disclosed sequence.
[0061] 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.
[0062] Derivatives and analogs may be full length or other than
full length. Derivatives or analogs of the nucleic acids or
proteins of the invention include, but are not limited to,
molecules comprising regions that are substantially homologous to
the nucleic acids or proteins of the invention, in various
embodiments, by at least about 70%, 80%, or 95% identity (with a
preferred identity of 80-95%) over a nucleic acid or amino acid
sequence of identical size or when compared to an aligned sequence
in which the alignment is done by a computer homology program known
in the art, or whose encoding nucleic acid is capable of
hybridizing to the complement of a sequence encoding the proteins
under stringent, moderately stringent, or low stringent conditions.
See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley & Sons, New York, N.Y., 1993, and below.
[0063] A "homologous nucleic acid sequence" or "homologous amino
acid sequence," or variations thereof, refer to sequences
characterized by a homology at the nucleotide level or amino acid
level as discussed above. Homologous nucleotide sequences include
those sequences coding for isoforms of NOVX polypeptides. Isoforms
can be expressed in different tissues of the same organism as a
result of, for example, alternative splicing of RNA. Alternatively,
isoforms can be encoded by different genes. In the invention,
homologous nucleotide sequences include nucleotide sequences
encoding for a NOVX polypeptide of species other than humans,
including, but not limited to: vertebrates, and thus can include,
e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other
organisms. Homologous nucleotide sequences also include, but are
not limited to, naturally occurring allelic variations and
mutations of the nucleotide sequences set forth herein. A
homologous nucleotide sequence does not, however, include the exact
nucleotide sequence encoding human NOVX protein. Homologous nucleic
acid sequences include those nucleic acid sequences that encode
conservative amino acid substitutions (see below) in SEQ ID
NO:2n-1, wherein n is an integer between 1 and 64, as well as a
polypeptide possessing NOVX biological activity. Various biological
activities of the NOVX proteins are described below.
[0064] 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.
[0065] The nucleotide sequences determined from the cloning of the
human NOVX genes allows for the generation of probes and primers
designed for use in identifying and/or cloning NOVX homologues in
other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/primer typically
comprises substantially purified oligonucleotide. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 12,
25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense
strand nucleotide sequence of SEQ ID NO:2n-1, wherein n is an
integer between 1 and 64; or an anti-sense strand nucleotide
sequence of SEQ ID NO:2n-1, wherein n is an integer between 1 and
64; or of a naturally occurring mutant of SEQ ID NO:2n-1, wherein n
is an integer between 1 and 64.
[0066] 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.
[0067] "A polypeptide having a biologically-active portion of a
NOVX polypeptide" refers to polypeptides exhibiting activity
similar, but not necessarily identical to, an activity of a
polypeptide of the invention, including mature forms, as measured
in a particular biological assay, with or without dose dependency.
A nucleic acid fragment encoding a "biologically-active portion of
NOVX" can be prepared by isolating a portion of SEQ ID NO:2n-1,
wherein n is an integer between 1 and 64, 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.
[0068] NOVX Nucleic Acid and Polypeptide Variants
[0069] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequences of SEQ ID NO:2n-1,
wherein n is an integer between 1 and 64, due to degeneracy of the
genetic code and thus encode the same NOVX proteins as that encoded
by the nucleotide sequences of SEQ ID NO:2n-1, wherein n is an
integer between 1 and 64. In another embodiment, an isolated
nucleic acid molecule of the invention has a nucleotide sequence
encoding a protein having an amino acid sequence of SEQ ID NO:2n,
wherein n is an integer between 1 and 64.
[0070] In addition to the human NOVX nucleotide sequences of SEQ ID
NO:2n-1, wherein n is an integer between 1 and 64, it will be
appreciated by those skilled in the art that DNA sequence
polymorphisms that lead to changes in the amino acid sequences of
the NOVX polypeptides may exist within a population (e.g., the
human population). Such genetic polymorphism in the NOVX genes may
exist among individuals within a population due to natural allelic
variation. As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
(ORF) encoding a NOVX protein, preferably a vertebrate NOVX
protein. Such natural allelic variations can typically result in
1-5% variance in the nucleotide sequence 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.
[0071] Moreover, nucleic acid molecules encoding NOVX proteins from
other species, and thus that have a nucleotide sequence that
differs from a human SEQ ID NO:2n-1, wherein n is an integer
between 1 and 64, 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.
[0072] 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 NO:2n-1, wherein n is
an integer between 1 and 64. 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.
[0073] 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.
[0074] As used herein, the phrase "stringent hybridization
conditions" refers to conditions under which a probe, primer or
oligonucleotide will hybridize to its target sequence, but to no
other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures than shorter
sequences. Generally, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present at excess, at Tm,
50% of the probes are occupied at equilibrium. Typically, stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion (or other salts) at pH 7.0 to 8.3 and the temperature is at
least about 30.degree. C. for short probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about
60.degree. C. for longer probes, primers and oligonucleotides.
Stringent conditions may also be achieved with the addition of
destabilizing agents, such as formamide.
[0075] Stringent conditions are known to those skilled in the art
and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
Preferably, the conditions are such that sequences at least about
65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other
typically remain hybridized to each other. A non-limiting example
of stringent hybridization conditions are hybridization in a high
salt buffer comprising 6.times. SSC, 50 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured
salmon sperm DNA at 65.degree. C., followed by one or more washes
in 0.2.times. SSC, 0.01% BSA at 50.degree. C. An isolated nucleic
acid molecule of the invention that hybridizes under stringent
conditions to a sequence of SEQ ID NO:2n-1, wherein n is an integer
between 1 and 64, corresponds to a naturally-occurring nucleic acid
molecule. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0076] In a second embodiment, a nucleic acid sequence that is
hybridizable to the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:2n-1, wherein n is an integer between 1 and
64, or fragments, analogs or derivatives thereof, under conditions
of moderate stringency is provided. A non-limiting example of
moderate stringency hybridization conditions are hybridization in
6.times. SSC, 5.times. Reinhardt's solution, 0.5% SDS and 100 mg/ml
denatured salmon sperm DNA at 55.degree. C., followed by one or
more washes in 1.times. SSC, 0.1% SDS at 37.degree. C. Other
conditions of moderate stringency that may be used are well-known
within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and
Krieger, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,
Stockton Press, NY.
[0077] In a third embodiment, a nucleic acid that is hybridizable
to the nucleic acid molecule comprising the nucleotide sequences of
SEQ ID NO:2n-1, wherein n is an integer between 1 and 64, or
fragments, analogs or derivatives thereof, under conditions of low
stringency, is provided. A non-limiting example of low stringency
hybridization conditions are hybridization in 35% formamide,
5.times. SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02%
Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40.degree. C., followed by one or more
washes in 2.times. SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and
0.1% SDS at 50.degree. C. Other conditions of low stringency that
may be used are well known in the art (e.g., as employed for
cross-species hybridizations). See, e.g., Ausubel, et al. (eds.),
1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A
LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981.
Proc Natl Acad Sci USA 78: 6789-6792.
[0078] Conservative Mutations
[0079] 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 NO:2n-1, wherein n is an
integer between 1 and 64, 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 ID NO:2n, wherein n is an integer between 1 and 64. A
"non-essential" amino acid residue is a residue that can be altered
from the wild-type sequences of the NOVX proteins without altering
their biological activity, whereas an "essential" amino acid
residue is required for such biological activity. For example,
amino acid residues that are conserved among the NOVX proteins of
the invention are predicted to be particularly non-amenable to
alteration. Amino acids for which conservative substitutions can be
made are well-known within the art.
[0080] 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 NO:2n-1, wherein n is an
integer between 1 and 64, yet retain biological activity. In one
embodiment, the isolated nucleic acid molecule comprises a
nucleotide sequence encoding a protein, wherein the protein
comprises an amino acid sequence at least about 40% homologous to
the amino acid sequences of SEQ ID NO:2n, wherein n is an integer
between 1 and 64. Preferably, the protein encoded by the nucleic
acid molecule is at least about 60% homologous to SEQ ID NO:2n,
wherein n is an integer between 1 and 64; more preferably at least
about 70% homologous to SEQ ID NO:2n, wherein n is an integer
between 1 and 64; still more preferably at least about 80%
homologous to SEQ ID NO:2n, wherein n is an integer between 1 and
64; even more preferably at least about 90% homologous to SEQ ID
NO:2n, wherein n is an integer between 1 and 64; and most
preferably at least about 95% homologous to SEQ ID NO:2n, wherein n
is an integer between 1 and 64.
[0081] An isolated nucleic acid molecule encoding a NOVX protein
homologous to the protein of SEQ ID NO:2n, wherein n is an integer
between 1 and 64, can be created by introducing one or more
nucleotide substitutions, additions or deletions into the
nucleotide sequence of SEQ ID NO:2n-1, wherein n is an integer
between 1 and 64, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
[0082] Mutations can be introduced any one of SEQ ID NO:2n-1,
wherein n is an integer between 1 and 64, by standard techniques,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one
or more predicted, non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined within the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted non-essential amino acid residue in the NOVX protein is
replaced with another amino acid residue from the same side chain
family. Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of a NOVX coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for NOVX biological activity to identify mutants that
retain activity. Following mutagenesis of a nucleic acid of SEQ ID
NO:2n-1, wherein n is an integer between 1 and 64, the encoded
protein can be expressed by any recombinant technology known in the
art and the activity of the protein can be determined.
[0083] The relatedness of amino acid families may also be
determined based on side chain interactions. Substituted amino
acids may be fully conserved "strong" residues or fully conserved
"weak" residues. The "strong" group of conserved amino acid
residues may be any one of the following groups: STA, NEQK, NHQK,
NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino
acid codes are grouped by those amino acids that may be substituted
for each other. Likewise, the "weak" group of conserved residues
may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND,
SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group
represent the single letter amino acid code.
[0084] 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).
[0085] 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).
[0086] Interfering RNA
[0087] In one aspect of the invention, NOVX gene expression can be
attenuated by RNA interference. One approach well-known in the art
is short interfering RNA (siRNA) mediated gene silencing where
expression products of a NOVX gene are targeted by specific double
stranded NOVX derived siRNA nucleotide sequences that are
complementary to at least a 19-25 nt long segment of the NOVX gene
transcript, including the 5' untranslated (UT) region, the ORF, or
the 3' UT region. See, e.g., PCT applications WO00/44895,
WO99/32619, WO01/75164, WO01/92513, WO01/29058, WO01/89304,
WO02/16620, and WO02/29858, 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.
[0088] 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.
[0089] 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.
[0090] 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 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.
[0091] 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 H1. One example of
a vector system is the GeneSuppressor.TM. RNA Interference kit
(commercially available from Imgenex). The U6 and H1 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' UU overhang in the expressed siRNA,
which is similar to the 3' overhangs of synthetic siRNAs. Any
sequence less than 400 nucleotides in length can be transcribed by
these promoter, therefore they are ideally suited for the
expression of around 21-nucleotide siRNAs in, e.g., an
approximately 50-nucleotide RNA stem-loop transcript.
[0092] 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.
[0093] In general, siRNAs are chopped from longer dsRNA by an
ATP-dependent ribonuclease called DICER. DICER is a member of the
RNase III family of double-stranded RNA-specific endonucleases. The
siRNAs assemble with cellular proteins into an endonuclease
complex. In vitro studies in Drosophila suggest that the
siRNAs/protein complex (siRNP) is then transferred to a second
enzyme complex, called an RNA-induced silencing complex (RISC),
which contains an endoribonuclease that is distinct from DICER.
RISC uses the sequence encoded by the antisense siRNA strand to
find and destroy mRNAs of complementary sequence. The siRNA thus
acts as a guide, restricting the ribonuclease to cleave only mRNAs
complementary to one of the two siRNA strands.
[0094] A NOVX mRNA region to be targeted by siRNA is generally
selected from a desired NOVX sequence beginning 50 to 100 nt
downstream of the start codon. Alternatively, 5' or 3' UTRs and
regions nearby the start codon can be used but are generally
avoided, as these may be richer in regulatory protein binding
sites. UTR-binding proteins and/or translation initiation complexes
may interfere with binding of the siRNP or RISC endonuclease
complex. An initial BLAST homology search for the selected siRNA
sequence is done against an available nucleotide sequence library
to ensure that only one gene is targeted. Specificity of target
recognition by siRNA duplexes indicate that a single point mutation
located in the paired region of an siRNA duplex is sufficient to
abolish target mRNA degradation. See, Elbashir et al. 2001 EMBO J.
20(23):6877-88. Hence, consideration should be taken to accommodate
SNPs, polymorphisms, allelic variants or species-specific
variations when targeting a desired gene.
[0095] 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.
[0096] 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.
[0097] 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(N19)TT is not present
in the target sequence, an alternative target region would be
AA(N21). The sequence of the NOVX sense siRNA corresponds to
(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 the siRNA duplex is not
expected to affect targeted mRNA recognition, as the antisense
siRNA strand guides target recognition.
[0098] 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.
[0099] 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 .mu.g of the siRNA duplex is generally sufficient. Cells are
typically seeded the previous day, and are transfected at about 50%
confluence. The choice of cell culture media and conditions are
routine to those of skill in the art, and will vary with the choice
of cell type. The efficiency of transfection may depend on the cell
type, but also on the passage number and the confluency of the
cells. The time and the manner of formation of siRNA-liposome
complexes (e.g. inversion versus vortexing) are also critical. Low
transfection efficiencies are the most frequent cause of
unsuccessful NOVX silencing. The efficiency of transfection needs
to be carefully examined for each new cell line to be used.
Preferred cell are derived from a mammal, more preferably from a
rodent such as a rat or mouse, and most preferably from a human.
Where used for therapeutic treatment, the cells are preferentially
autologous, although non-autologous cell sources are also
contemplated as within the scope of the present invention.
[0100] For a control experiment, transfection of 0.84 .mu.g
single-stranded sense NOVX siRNA will have no effect on NOVX
silencing, and 0.84 .mu.g antisense siRNA has a weak silencing
effect when compared to 0.84 .mu.g 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 fraction of
lamin A/C knockdown in cells is determined the next day by such
techniques as immunofluorescence, Western blot, Northern blot or
other similar assays for protein expression or gene expression.
Lamin A/C monoclonal antibodies may be obtained from Santa Cruz
Biotechnology.
[0101] Depending on the abundance and the half life (or turnover)
of the targeted NOVX polynucleotide in a cell, a knock-down
phenotype may become apparent after 1 to 3 days, or even later. In
cases where no NOVX knock-down phenotype is observed, depletion of
the NOVX polynucleotide may be observed by immunofluorescence or
Western blotting. If the NOVX polynucleotide is still abundant
after 3 days, cells need to be split and transferred to a fresh
24-well plate for re-transfection. If no knock-down of the targeted
protein is observed, it may be desirable to analyze whether the
target mRNA (NOVX or a NOVX upstream or downstream gene) was
effectively destroyed by the transfected siRNA duplex. Two days
after transfection, total RNA is prepared, reverse transcribed
using a target-specific primer, and PCR-amplified with a primer
pair covering at least one exon-exon junction in order to control
for amplification of pre-mRNAs. RT/PCR of a non-targeted mRNA is
also needed as control. Effective depletion of the mRNA yet
undetectable reduction of target protein may indicate that a large
reservoir of stable NOVX protein may exist in the cell. Multiple
transfection in sufficiently long intervals may be necessary until
the target protein is finally depleted to a point where a phenotype
may become apparent. If multiple transfection steps are required,
cells are split 2 to 3 days after transfection. The cells may be
transfected immediately after splitting.
[0102] 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.
[0103] 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 protein
is not produced or is not produced to the extent it would be in the
absence of the treatment.
[0104] 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.sup.-) phenotype in the treated
subject sample. The NOVX.sup.- phenotype observed in the treated
subject sample thus serves as a marker for monitoring the course of
a disease state during treatment.
[0105] 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.
[0106] Production of RNAs
[0107] 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 .mu.M) in 10 mM Tris-HCl (pH 7.5) with 20 mM
NaCl were heated to 95.degree. 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).
[0108] Lysate Preparation
[0109] Untreated rabbit reticulocyte lysate (Ambion) are assembled
according to the manufacturer's directions. dsRNA is incubated in
the lysate at 30.degree. C. for 10 min prior to the 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.
[0110] In a parallel experiment made with the same conditions, the
double stranded RNA is internally radiolabeled with a .sup.32P-ATP.
Reactions are stopped by the addition of 2.times.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.
[0111] 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.
[0112] RNA Preparation
[0113] 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)).
[0114] These RNAs (20 .mu.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.degree. C. followed by
1 h at 37.degree. C.
[0115] Cell Culture
[0116] 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.times.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 techniques with positive and
negative control. A positive control is cells that naturally
express NOVX while a negative control is cells that do not express
NOVX. Base-paired 21 and 22 nt siRNAs with overhanging 3' ends
mediate efficient sequence-specific mRNA degradation in lysates and
in cell culture. Different concentrations of siRNAs are used. An
efficient concentration for suppression in vitro in mammalian
culture is between 25 nM to 100 nM final concentration. This
indicates that siRNAs are effective at concentrations that are
several orders of magnitude below the concentrations applied in
conventional antisense or ribozyme gene targeting experiments.
[0117] 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.
[0118] Antisense Nucleic Acids
[0119] Another aspect of the invention pertains to isolated
antisense nucleic acid molecules that are hybridizable to or
complementary to the nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:2n-1, wherein n is an integer
between 1 and 64, 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
NO:2n, wherein n is an integer between 1 and 64, or antisense
nucleic acids complementary to a NOVX nucleic acid sequence of SEQ
ID NO:2n-1, wherein n is an integer between 1 and 64, are
additionally provided.
[0120] In one embodiment, an antisense nucleic acid molecule is
antisense to a "coding region" of the coding strand of a nucleotide
sequence encoding a NOVX protein. The term "coding region" refers
to the region of the nucleotide sequence comprising codons which
are translated into amino acid residues. In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding the
NOVX protein. The term "noncoding region" refers to 5' and 3'
sequences which flank the coding region that are not translated
into amino acids (i.e., also referred to as 5' and 3' untranslated
regions).
[0121] Given the coding strand sequences encoding the NOVX protein
disclosed herein, antisense nucleic acids of the invention can be
designed according to the rules of Watson and Crick or Hoogsteen
base pairing. The antisense nucleic acid molecule can be
complementary to the entire coding region of NOVX mRNA, but more
preferably is an oligonucleotide that is antisense to only a
portion of the coding or noncoding region of NOVX mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of NOVX mRNA. An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20,
25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical
synthesis or enzymatic ligation reactions using procedures known in
the art. For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using
naturally-occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids (e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used).
[0122] Examples of modified nucleotides that can be used to
generate the antisense nucleic acid include: 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine,
5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 5-methoxyuracil,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
2-thiouracil, 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).
[0123] 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 pol III promoter are
preferred.
[0124] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other.
See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641.
The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl.
Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See,
e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330.
[0125] Ribozymes and PNA Moieties
[0126] 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.
[0127] In one embodiment, an antisense nucleic acid of the
invention is a ribozyme. Ribozymes are catalytic RNA molecules with
ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
as described in Haselhoff and Gerlach 1988. Nature 334: 585-591)
can be used to catalytically cleave NOVX mRNA transcripts to
thereby inhibit translation of NOVX mRNA. A ribozyme having
specificity for a NOVX-encoding nucleic acid can be designed based
upon the nucleotide sequence of a NOVX cDNA disclosed herein (i.e.,
SEQ ID NO:2n-1, wherein n is an integer between 1 and 64). 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. Pat. No. 4,987,071 to Cech, et
al. and U.S. Pat. No. 5,116,742 to Cech, et al. NOVX mRNA can also
be used to select a catalytic RNA having a specific ribonuclease
activity from a pool of RNA molecules. See, e.g., Bartel et al.,
(1993) Science 261:1411-1418.
[0128] 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.
[0129] In various embodiments, the NOVX nucleic acids can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve, e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids.
See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used
herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural 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.
[0130] PNAs of NOVX can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or
antigene agents for sequence-specific modulation of gene expression
by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs of NOVX can also be used, for example,
in the analysis of single base pair mutations in a gene (e.g., PNA
directed PCR clamping; as artificial restriction enzymes when used
in combination with other enzymes, e.g., S.sub.1 nucleases (See,
Hyrup, et al., 1996. supra); or as probes or primers for DNA
sequence and hybridization (See, Hyrup, et al., 1996, supra;
Perry-O'Keefe, et al., 1996. supra).
[0131] 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
performed as described in Hyrup, et al., 1996. supra and Finn, et
al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain
can be synthesized on a solid support using standard
phosphoramidite coupling chemistry, and modified nucleoside
analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can be used between the PNA and the 5' end of DNA.
See, e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment. See,
e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules
can be synthesized with a 5' DNA segment and a 3' PNA segment. See,
e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5:
1119-11124.
[0132] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc.
Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or
the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In addition, oligonucleotides can be modified with
hybridization triggered cleavage agents (see, e.g., Krol, et al.,
1988. BioTechniques 6:958-976) or intercalating agents (see, e.g.,
Zon, 1988. Pharm. Res. 5: 539-549). To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a
peptide, a hybridization triggered cross-linking agent, a transport
agent, a hybridization-triggered cleavage agent, and the like.
[0133] NOVX Polypeptides
[0134] A polypeptide according to the invention includes a
polypeptide including the amino acid sequence of NOVX polypeptides
whose sequences are provided in any one of SEQ ID NO:2n, wherein n
is an integer between 1 and 64. The invention also includes a
mutant or variant protein any of whose residues may be changed from
the corresponding residues shown in any one of SEQ ID NO:2n,
wherein n is an integer between 1 and 64, while still encoding a
protein that maintains its NOVX activities and physiological
functions, or a functional fragment thereof.
[0135] 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.
[0136] 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.
[0137] An "isolated" or "purified" polypeptide or protein or
biologically-active portion thereof is substantially free of
cellular material or other contaminating proteins from the cell or
tissue source from which the NOVX protein is derived, or
substantially free from chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of
cellular material" includes preparations of NOVX proteins in which
the protein is separated from cellular components of the cells from
which it is isolated or recombinantly-produced. In one embodiment,
the language "substantially free of cellular material" includes
preparations of NOVX proteins having less than about 30% (by dry
weight) of non-NOVX proteins (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-NOVX proteins, still more preferably less than about 10% of
non-NOVX proteins, and most preferably less than about 5% of
non-NOVX proteins. When the NOVX protein or biologically-active
portion thereof is recombinantly-produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
NOVX protein preparation.
[0138] 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.
[0139] Biologically-active portions of NOVX proteins include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequences of the NOVX proteins
(e.g., the amino acid sequence of SEQ ID NO:2n, wherein n is an
integer between 1 and 64) 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.
[0140] 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.
[0141] In an embodiment, the NOVX protein has an amino acid
sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 64.
In other embodiments, the NOVX protein is substantially homologous
to SEQ ID NO:2n, wherein n is an integer between 1 and 64, and
retains the functional activity of the protein of SEQ ID NO:2n,
wherein n is an integer between 1 and 64, yet differs in amino acid
sequence due to natural allelic variation or mutagenesis, as
described in detail, below. Accordingly, in another embodiment, the
NOVX protein is a protein that comprises an amino acid sequence at
least about 45% homologous to the amino acid sequence of SEQ ID
NO:2n, wherein n is an integer between 1 and 64, and retains the
functional activity of the NOVX proteins of SEQ ID NO:2n, wherein n
is an integer between 1 and 64.
[0142] Determining Homology Between Two or More Sequences
[0143] 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").
[0144] 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 NO:2n-1, wherein n is an integer
between 1 and 64.
[0145] The term "sequence identity" refers to the degree to which
two polynucleotide or polypeptide sequences are identical on a
residue-by-residue basis over a particular region of comparison.
The term "percentage of sequence identity" is calculated by
comparing two optimally aligned sequences over that region of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case
of nucleic acids) occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the region of comparison (i.e., the
window size), and multiplying the result by 100 to yield the
percentage of sequence identity. The term "substantial identity" as
used herein denotes a characteristic of a polynucleotide sequence,
wherein the polynucleotide comprises a sequence that has at least
80 percent sequence identity, preferably at least 85 percent
identity and often 90 to 95 percent sequence identity, more usually
at least 99 percent sequence identity as compared to a reference
sequence over a comparison region.
[0146] Chimeric and Fusion Proteins
[0147] 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 NO:2n, wherein n is an integer between 1 and 64, 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.
[0148] 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.
[0149] 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.
[0150] In yet another embodiment, the fusion protein is a
NOVX-immunoglobulin fusion protein in which the NOVX sequences are
fused to sequences derived from a member of the immunoglobulin
protein family. The NOVX-immunoglobulin fusion proteins of the
invention can be incorporated into pharmaceutical compositions and
administered to a subject to inhibit an interaction between a NOVX
ligand and a NOVX protein on the surface of a cell, to thereby
suppress NOVX-mediated signal transduction in vivo. The
NOVX-immunoglobulin fusion proteins can be used to affect the
bioavailability of a NOVX cognate ligand. Inhibition of the NOVX
ligand/NOVX interaction may be useful therapeutically for both the
treatment of proliferative and differentiative disorders, as well
as modulating (e.g. promoting or inhibiting) cell survival.
Moreover, the NOVX-immunoglobulin fusion proteins of the invention
can be used as immunogens to produce anti-NOVX antibodies in a
subject, to purify NOVX ligands, and in screening assays to
identify molecules that inhibit the interaction of NOVX with a NOVX
ligand.
[0151] A NOVX chimeric or fusion protein of the invention can be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini
for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments that
can subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST polypeptide). A NOVX-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the NOVX protein.
[0152] NOVX Agonists and Antagonists
[0153] The invention also pertains to variants of the NOVX proteins
that function as either NOVX agonists (i.e., mimetics) or as NOVX
antagonists. Variants of the NOVX protein can be generated by
mutagenesis (e.g., discrete point mutation or truncation of the
NOVX protein). An agonist of the NOVX protein can retain
substantially the same, or a subset of, the biological activities
of the naturally occurring form of the NOVX protein. An antagonist
of the NOVX protein can inhibit one or more of the activities of
the naturally occurring form of the NOVX protein by, for example,
competitively binding to a downstream or upstream member of a
cellular signaling cascade which includes the NOVX protein. Thus,
specific biological effects can be elicited by treatment with a
variant of limited function. In one embodiment, treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein has fewer side
effects in a subject relative to treatment with the naturally
occurring form of the NOVX proteins.
[0154] Variants of the NOVX proteins that function as either NOVX
agonists (i.e., mimetics) or as NOVX antagonists can be identified
by screening combinatorial libraries of mutants (e.g., truncation
mutants) of the NOVX proteins for NOVX protein agonist or
antagonist activity. In one embodiment, a variegated library of
NOVX variants is generated by combinatorial mutagenesis at the
nucleic acid level and is encoded by a variegated gene library. A
variegated library of NOVX variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential NOVX sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g. for
phage display) containing the set of NOVX sequences therein. There
are a variety of methods which can be used to produce libraries of
potential NOVX variants from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be performed
in an automatic DNA synthesizer, and the synthetic gene then
ligated into an appropriate expression vector. Use of a degenerate
set of genes allows for the provision, in one mixture, of all of
the sequences encoding the desired set of potential NOVX sequences.
Methods for synthesizing degenerate oligonucleotides are well-known
within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3;
Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et
al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res.
11: 477.
[0155] Polypeptide Libraries
[0156] In addition, libraries of fragments of the NOVX protein
coding sequences can be used to generate a variegated population of
NOVX fragments for screening and subsequent selection of variants
of a NOVX protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a NOVX coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double-stranded DNA
that can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes
by treatment with S.sub.1 nuclease, and ligating the resulting
fragment library into an expression vector. By this method,
expression libraries can be derived which encodes N-terminal and
internal fragments of various sizes of the NOVX proteins.
[0157] Various techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of NOVX proteins. The most widely used techniques,
which are amenable to high throughput analysis, for screening large
gene libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a new technique
that enhances the frequency of functional mutants in the libraries,
can be used in combination with the screening assays to identify
NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl.
Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein
Engineering 6:327-331.
[0158] NOVX Antibodies
[0159] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, single chain, F.sub.ab, F.sub.ab' and F.sub.(ab')2
fragments, and an F.sub.ab expression library. In general, antibody
molecules obtained from humans relates to any of the classes IgG,
IgM, IgA, IgE and IgD, which differ from one another by the nature
of the heavy chain present in the molecule. Certain classes have
subclasses as well, such as IgG.sub.1, IgG.sub.2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a
lambda chain. Reference herein to antibodies includes a reference
to all such classes, subclasses and types of human antibody
species.
[0160] An isolated protein of the invention intended to serve as an
antigen, or a portion or fragment thereof, can be used as an
immunogen to generate antibodies that immunospecifically bind the
antigen, using standard techniques for polyclonal and monoclonal
antibody preparation. The full-length protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of the antigen for use as immunogens. An antigenic peptide fragment
comprises at least 6 amino acid residues of the amino acid sequence
of the full length protein, such as an amino acid sequence of SEQ
ID NO:2n, wherein n is an integer between 1 and 64, 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.
[0161] In certain embodiments of the invention, at least one
epitope encompassed by the antigenic peptide is a region of NOVX
that is located on the surface of the protein, e.g., a hydrophilic
region. A hydrophobicity analysis of the human NOVX protein
sequence will indicate which regions of a NOVX polypeptide are
particularly hydrophilic and, therefore, are likely to encode
surface residues useful for targeting antibody production. As a
means for targeting antibody production, hydropathy plots showing
regions of hydrophilicity and hydrophobicity may be generated by
any method well known in the art, including, for example, the Kyte
Doolittle or the Hopp Woods methods, either with or without Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad.
Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157:
105-142, each incorporated herein by reference in their entirety.
Antibodies that are specific for one or more domains within an
antigenic protein, or derivatives, fragments, analogs or homologs
thereof, are also provided herein.
[0162] 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
(K.sub.D) is .ltoreq.1 .mu.M, preferably .ltoreq.100 nM, more
preferably .ltoreq.10 nM, and most preferably .ltoreq.100 pM to
about 1 pM, as measured by assays such as radioligand binding
assays or similar assays known to those skilled in the art.
[0163] 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.
[0164] Various procedures known within the art may be used for the
production of polyclonal or monoclonal antibodies directed against
a protein of the invention, or against derivatives, fragments,
analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
incorporated herein by reference). Some of these antibodies are
discussed below.
[0165] Polyclonal Antibodies
[0166] 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).
[0167] The polyclonal antibody molecules directed against the
immunogenic protein can be isolated from the mammal (e.g., from the
blood) and further purified by well known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
[0168] Monoclonal Antibodies
[0169] 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.
[0170] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes can be immunized in
vitro.
[0171] 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.
[0172] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63).
[0173] 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.
[0174] 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.
[0175] 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.
[0176] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also can be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody.
[0177] Humanized Antibodies
[0178] The antibodies directed against the protein antigens of the
invention can further comprise humanized antibodies or human
antibodies. These antibodies are suitable for administration to
humans without engendering an immune response by the human against
the administered immunoglobulin. Humanized forms of antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that are principally
comprised of the sequence of a human immunoglobulin, and contain
minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. (See also U.S.
Pat. No. 5,225,539.) In some instances, Fv framework residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Humanized antibodies can also comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
[0179] Human Antibodies
[0180] Fully human antibodies essentially relate to antibody
molecules in which the entire sequence of both the light chain and
the heavy chain, including the CDRs, arise from human genes. Such
antibodies are termed "human antibodies", or "fully human
antibodies" herein. Human monoclonal antibodies can be prepared by
the trioma technique; the human B-cell hybridoma technique (see
Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma
technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,
Inc., pp. 77-96). Human monoclonal antibodies may be utilized in
the practice of the present invention and may be produced by using
human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr
Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES
AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
[0181] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies
can be made by introducing human immunoglobulin loci into
transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks
et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature
368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild
et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature
Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol. 13 65-93 (1995)).
[0182] Human antibodies may additionally be produced using
transgenic nonhuman animals which are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. (See PCT
publication WO94/02602). The endogenous genes encoding the heavy
and light immunoglobulin chains in the nonhuman host have been
incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. The preferred
embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells which secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv molecules.
[0183] An example of a method of producing a nonhuman host,
exemplified as a mouse, lacking expression of an endogenous
immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598.
It can be obtained by a method including deleting the J segment
genes from at least one endogenous heavy chain locus in an
embryonic stem cell to prevent rearrangement of the locus and to
prevent formation of a transcript of a rearranged immunoglobulin
heavy chain locus, the deletion being effected by a targeting
vector containing a gene encoding a selectable marker; and
producing from the embryonic stem cell a transgenic mouse whose
somatic and germ cells contain the gene encoding the selectable
marker.
[0184] A method for producing an antibody of interest, such as a
human antibody, is disclosed in U.S. Pat. No. 5,916,771. It
includes introducing an expression vector that contains a
nucleotide sequence encoding a heavy chain into one mammalian host
cell in culture, introducing an expression vector containing a
nucleotide sequence encoding a light chain into another mammalian
host cell, and fusing the two cells to form a hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and
the light chain.
[0185] 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.
[0186] F.sub.ab Fragments and Single Chain Antibodies
[0187] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to an antigenic
protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of F.sub.ab
expression libraries (see e.g., Huse, et al., 1989 Science 246:
1275-1281) to allow rapid and effective identification of
monoclonal F.sub.ab fragments with the desired specificity for a
protein or derivatives, fragments, analogs or homologs thereof.
Antibody fragments that contain the idiotypes to a protein antigen
may be produced by techniques known in the art including, but not
limited to: (i) an F.sub.(ab')2 fragment produced by pepsin
digestion of an antibody molecule; (ii) an F.sub.ab fragment
generated by reducing the disulfide bridges of an F.sub.(ab')2
fragment; (iii) an F.sub.ab fragment generated by the treatment of
the antibody molecule with papain and a reducing agent and (iv)
F.sub.v fragments.
[0188] Bispecific Antibodies
[0189] 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.
[0190] 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).
[0191] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0192] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0193] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0194] Additionally, Fab' fragments can be directly recovered from
E. coli and chemically coupled to form bispecific antibodies.
Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized bispecific antibody F(ab').sub.2
molecule. Each Fab' fragment was separately secreted from E. coli
and subjected to directed chemical coupling in vitro to form the
bispecific antibody. The bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human
T cells, as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0195] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0196] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0197] Exemplary bispecific antibodies can bind to two different
epitopes, at least one of which originates in the protein antigen
of the invention. Alternatively, an anti-antigenic arm of an
immunoglobulin molecule can be combined with an arm which binds to
a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the cell expressing the particular antigen. Bispecific antibodies
can also be used to direct cytotoxic agents to cells which express
a particular antigen. These antibodies possess an antigen-binding
arm and an arm which binds a cytotoxic agent or a radionuclide
chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific
antibody of interest binds the protein antigen described herein and
further binds tissue factor (TF).
[0198] Heteroconjugate Antibodies
[0199] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0200] Effector Function Engineering
[0201] It can be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) can be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity can also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989).
[0202] Immunoconjugates
[0203] 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).
[0204] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0205] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP).
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0206] 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.
[0207] Immunoliposomes
[0208] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0209] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81(19): 1484 (1989).
[0210] Diagnostic Applications of Antibodies Directed Against the
Proteins of the Invention
[0211] Antibodies directed against a protein of the invention may
be used in methods known within the art relating to the
localization and/or quantitation of the protein (e.g., for use in
measuring levels of the protein within appropriate physiological
samples, for use in diagnostic methods, for use in imaging the
protein, and the like). In a given embodiment, antibodies against
the proteins, or derivatives, fragments, analogs or homologs
thereof, that contain the antigen binding domain, are utilized as
pharmacologically-active compounds (see below).
[0212] An antibody specific for a protein of the invention can be
used to isolate the protein by standard techniques, such as
immunoaffinity chromatography or immunoprecipitation. Such an
antibody can facilitate the purification of the natural protein
antigen from cells and of recombinantly produced antigen expressed
in host cells. Moreover, such an antibody can be used to detect the
antigenic protein (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
antigenic protein. Antibodies directed against the protein can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0213] Antibody Therapeutics
[0214] 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.
[0215] 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.
[0216] A therapeutically effective amount of an antibody of the
invention relates generally to the amount needed to achieve a
therapeutic objective. As noted above, this may be a binding
interaction between the antibody and its target antigen that, in
certain cases, interferes with the functioning of the target, and
in other cases, promotes a physiological response. The amount
required to be administered will furthermore depend on the binding
affinity of the antibody for its specific antigen, and will also
depend on the rate at which an administered antibody is depleted
from the free volume other subject to which it is administered.
Common ranges for therapeutically effective dosing of an antibody
or antibody fragment of the invention may be, by way of nonlimiting
example, from about 0.1 mg/kg body weight to about 50 mg/kg body
weight. Common dosing frequencies may range, for example, from
twice daily to once a week.
[0217] Pharmaceutical Compositions of Antibodies
[0218] 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.
[0219] 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.
[0220] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions.
[0221] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0222] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0223] ELISA Assay
[0224] An agent for detecting an analyte protein is an antibody
capable of binding to an analyte protein, preferably an antibody
with a detectable label. Antibodies can be polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof
(e.g., F.sub.ab or F.sub.(ab)2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently-labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. Included within the usage of the term "biological
sample", therefore, is blood and a fraction or component of blood
including blood serum, blood plasma, or lymph. That is, the
detection method of the invention can be used to detect an analyte
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
an analyte mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of an analyte
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence. In
vitro techniques for detection of an analyte genomic DNA include
Southern hybridizations. Procedures for conducting immunoassays are
described, for example in "ELISA: Theory and Practice: Methods in
Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press,
Totowa, N.J., 1995; "Immunoassay", E. Diamandis and T.
Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and
"Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier
Science Publishers, Amsterdam, 1985. Furthermore, in vivo
techniques for detection of an analyte protein include introducing
into a subject a labeled anti-an analyte protein antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0225] NOVX Recombinant Expression Vectors and Host Cells
[0226] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
NOVX protein, or derivatives, fragments, analogs or homologs
thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively-linked. Such
vectors are referred to herein as "expression vectors". In general,
useful expression vectors in recombinant DNA techniques are often
in the form of plasmids. In the present specification, "plasmid"
and "vector" can be used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors, such as viral
vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0227] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively-linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector,
"operably-linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell).
[0228] 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.).
[0229] The recombinant expression vectors of the invention can be
designed for expression of NOVX proteins in prokaryotic or
eukaryotic cells. For example, NOVX proteins can be expressed in
bacterial cells such as Escherichia coli, insect cells (using
baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0230] Expression of proteins in prokaryotes is most often carried
out in Escherichia coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion proteins. Fusion vectors add a number of amino acids to
a protein encoded therein, usually to the amino terminus of the
recombinant protein. Such fusion vectors typically serve three
purposes: (i) to increase expression of recombinant protein; (ii)
to increase the solubility of the recombinant protein; and (iii) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0231] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0232] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
119-128. Another strategy is to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector so that
the individual codons for each amino acid are those preferentially
utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids
Res. 20: 2111-2118). Such alteration of nucleic acid sequences of
the invention can be carried out by standard DNA synthesis
techniques.
[0233] In another embodiment, the NOVX expression vector is a yeast
expression vector. Examples of vectors for expression in yeast
Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987.
EMBO J. 6: 229-234), pMFa (Kudjan 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.).
[0234] 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).
[0235] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
EMBO J. 6: 187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0236] 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 (Banedji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985. Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the .alpha.-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
[0237] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively-linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to NOVX mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen that direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see, e.g., Weintraub, et al.,
"Antisense RNA as a molecular tool for genetic analysis,"
Reviews-Trends in Genetics, Vol. 1(1) 1986.
[0238] 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.
[0239] 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.
[0240] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0241] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Various selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding NOVX or can be introduced on a separate vector. Cells
stably transfected with the introduced nucleic acid can be
identified by drug selection (e.g., cells that have incorporated
the selectable marker gene will survive, while the other cells
die).
[0242] 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.
[0243] Transgenic NOVX Animals
[0244] 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.
[0245] A transgenic animal of the invention can be created by
introducing NOVX-encoding nucleic acid into the male pronuclei of a
fertilized oocyte (e.g., by microinjection, retroviral infection)
and allowing the oocyte to develop in a pseudopregnant female
foster animal. The human NOVX cDNA sequences, i.e., any one of SEQ
ID NOS:2n-1, wherein n is an integer between 1 and 64, can be
introduced as a transgene into the genome of a non-human animal.
Alternatively, a non-human homologue of the human NOVX gene, such
as a mouse NOVX gene, can be isolated based on hybridization to the
human NOVX cDNA (described further supra) and used as a transgene.
Intronic sequences and polyadenylation signals can also be included
in the transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be
operably-linked to the NOVX transgene to direct expression of NOVX
protein to particular cells. Methods for generating transgenic
animals via embryo manipulation and microinjection, particularly
animals such as mice, have become conventional in the art and are
described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and
4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar
methods are used for production of other transgenic animals. A
transgenic founder animal can be identified based upon the presence
of the NOVX transgene in its genome and/or expression of NOVX mRNA
in tissues or cells of the animals. A transgenic founder animal can
then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene-encoding NOVX
protein can further be bred to other transgenic animals carrying
other transgenes.
[0246] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a NOVX gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX
gene can be a human gene (e.g., the cDNA of any one of SEQ ID
NOS:2n-1, wherein n is an integer between 1 and 64), but more
preferably, is a non-human homologue of a human NOVX gene. For
example, a mouse homologue of human NOVX gene of SEQ ID NOS:2n-1,
wherein n is an integer between 1 and 64, 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).
[0247] Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous NOVX gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous NOVX protein). In the homologous
recombination vector, the altered portion of the NOVX gene is
flanked at its 5'- and 3'-termini by additional nucleic acid of the
NOVX gene to allow for homologous recombination to occur between
the exogenous NOVX gene carried by the vector and an endogenous
NOVX gene in an embryonic stem cell. The additional flanking NOVX
nucleic acid is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several
kilobases of flanking DNA (both at the 5'- and 3'-termini) are
included in the vector. See, e.g., Thomas, et al., 1987. Cell 51:
503 for a description of homologous recombination vectors. The
vector is ten introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced NOVX gene has
homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
[0248] 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.
[0249] In another embodiment, transgenic non-humans animals can be
produced that contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992.
Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355. If
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.
[0250] 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 G.sub.0 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.
[0251] Pharmaceutical Compositions
[0252] 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.
[0253] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0254] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0255] 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
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0256] 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.
[0257] 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.
[0258] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0259] 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.
[0260] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0261] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0262] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see, e.g., U.S. Pat. No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al.,
1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system.
[0263] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0264] Screening and Detection Methods
[0265] The isolated nucleic acid molecules of the invention can be
used to express NOVX protein (e.g., via a recombinant expression
vector in a host cell in gene therapy applications), to detect NOVX
mRNA (e.g., in a biological sample) or a genetic lesion in a NOVX
gene, and to modulate NOVX activity, as described further, below.
In addition, the NOVX proteins can be used to screen drugs or
compounds that modulate the NOVX protein activity or expression as
well as to treat disorders characterized by insufficient or
excessive production of NOVX protein or production of NOVX protein
forms that have decreased or aberrant activity compared to NOVX
wild-type protein (e.g.; diabetes (regulates insulin release);
obesity (binds and transport lipids); metabolic disturbances
associated with obesity, the metabolic syndrome X as well as
anorexia and wasting disorders associated with chronic diseases and
various cancers, and infectious disease(possesses anti-microbial
activity) and the various dyslipidemias. In addition, the anti-NOVX
antibodies of the invention can be used to detect and isolate NOVX
proteins 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.
[0266] The invention further pertains to novel agents identified by
the screening assays described herein and uses thereof for
treatments as described, supra.
[0267] Screening Assays
[0268] 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.
[0269] In one embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of the membrane-bound form of a NOVX protein or
polypeptide or biologically-active portion thereof. The test
compounds of the invention can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug
Design 12: 145.
[0270] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD and
most preferably less than about 4 kD. Small molecules can be, e.g.,
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules.
Libraries of chemical and/or biological mixtures, such as fungal,
bacterial, or algal extracts, are known in the art and can be
screened with any of the assays of the invention.
[0271] 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.
[0272] Libraries of compounds may be presented in solution (e.g.
Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991.
Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S.
Pat. No. 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl.
Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990.
Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla,
et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici,
1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Pat. No.
5,233,409.).
[0273] 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 .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemission or by scintillation
counting. Alternatively, test compounds can be
enzymatically-labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product. In one embodiment, the assay comprises contacting a
cell which expresses a membrane-bound form of NOVX protein, or a
biologically-active portion thereof, on the cell surface with a
known compound which binds NOVX to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with a NOVX protein,
wherein determining the ability of the test compound to interact
with a NOVX protein comprises determining the ability of the test
compound to preferentially bind to NOVX protein or a
biologically-active portion thereof as compared to the known
compound.
[0274] 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.
[0275] 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 Ca.sup.2+, diacylglycerol, IP.sub.3, etc.), detecting
catalytic/enzymatic activity of the target an appropriate
substrate, detecting the induction of a reporter gene (comprising 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] The cell-free assays of the invention are amenable to use of
both the soluble form or the membrane-bound form of NOVX protein.
In the case of cell-free assays comprising the membrane-bound form
of NOVX protein, it may be desirable to utilize a solubilizing
agent such that the membrane-bound form of NOVX protein is
maintained in solution. Examples of such solubilizing agents
include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,
3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS),
or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane
sulfonate (CHAPSO).
[0280] 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.
[0281] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either the NOVX protein or its target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated NOVX
protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with NOVX
protein or target molecules, but which do not interfere with
binding of the NOVX protein to its target molecule, can be
derivatized to the wells of the plate, and unbound target or NOVX
protein trapped in the wells by antibody conjugation. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the NOVX protein or target molecule,
as well as enzyme-linked assays that rely on detecting an enzymatic
activity associated with the NOVX protein or target molecule.
[0282] 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.
[0283] In yet another aspect of the invention, the NOVX proteins
can be used as "bait proteins" in a two-hybrid assay or three
hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos, et al.,
1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem. 268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924;
Iwabuchi, et al., 1993. Oncogene 8: 1693-1696; and Brent WO
94/10300), to identify other proteins that bind to or interact with
NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX
activity. Such NOVX-binding proteins are also likely to be involved
in the propagation of signals by the NOVX proteins as, for example,
upstream or downstream elements of the NOVX pathway.
[0284] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for NOVX is fused
to a gene encoding the DNA binding domain of a known transcription
factor (e.g., GAL-4). In the other construct, a DNA sequence, from
a library of DNA sequences, that encodes an unidentified protein
("prey" or "sample") is fused to a gene that codes for the
activation domain of the known transcription factor. If the "bait"
and the "prey" proteins are able to interact, in vivo, forming a
NOVX-dependent complex, the DNA-binding and activation domains of
the transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ) that
is operably linked to a transcriptional regulatory site responsive
to the transcription factor. Expression of the reporter gene can be
detected and cell colonies containing the functional transcription
factor can be isolated and used to obtain the cloned gene that
encodes the protein which interacts with NOVX.
[0285] The invention further pertains to novel agents identified by
the aforementioned screening assays and uses thereof for treatments
as described herein.
[0286] Detection Assays
[0287] 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.
[0288] Chromosome Mapping
[0289] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of a NOVX sequence,
i.e., of SEQ ID NOS:2n-1, wherein n is an integer between 1 and 64,
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.
[0290] Briefly, NOVX genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the NOVX
sequences. Computer analysis of the NOVX, sequences can be used to
rapidly select primers that do not span more than one exon in the
genomic DNA, thus complicating the amplification process. These
primers can then be used for PCR screening of somatic cell hybrids
containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the NOVX sequences will
yield an amplified fragment.
[0291] 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.
[0292] 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.
[0293] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical like colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases, will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see, Verma, et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC
TECHNIQUES (Pergamon Press, New York 1988).
[0294] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0295] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, e.g.,
in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line
through Johns Hopkins University Welch Medical Library). The
relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g.,
Egeland, et al., 1987. Nature, 325: 783-787.
[0296] 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.
[0297] Tissue Typing
[0298] The NOVX sequences of the invention can also be used to
identify individuals from minute biological samples. In this
technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique
bands for identification. The sequences of the invention are useful
as additional DNA markers for RFLP ("restriction fragment length
polymorphisms," described in U.S. Pat. No. 5,272,057).
[0299] 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.
[0300] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
invention can be used to obtain such identification sequences from
individuals and from tissue. The NOVX sequences of the invention
uniquely represent portions of the human genome. Allelic variation
occurs to some degree in the coding regions of these sequences, and
to a greater degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a frequency
of about once per each 500 bases. Much of the allelic variation is
due to single nucleotide polymorphisms (SNPs), which include
restriction fragment length polymorphisms (RFLPs).
[0301] Each of the sequences described herein can, to some degree,
be used as a standard against which DNA from an individual can be
compared for identification purposes. Because greater numbers of
polymorphisms occur in the noncoding regions, fewer sequences are
necessary to differentiate individuals. The noncoding sequences can
comfortably provide positive individual identification with a panel
of perhaps 10 to 1,000 primers that each yield a noncoding
amplified sequence of 100 bases. If coding sequences, such as those
of SEQ ID NOS:2n-1, wherein n is an integer between 1 and 64, are
used, a more appropriate number of primers for positive individual
identification would be 500-2,000.
[0302] Predictive Medicine
[0303] The invention also pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trials are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the invention relates
to diagnostic assays for determining NOVX protein and/or nucleic
acid expression as well as NOVX activity, in the context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby
determine whether an individual is afflicted with a disease or
disorder, or is at risk of developing a disorder, associated with
aberrant NOVX expression or activity. The disorders include
metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-associated cachexia, cancer, neurodegenerative
disorders, Alzheimer's Disease, Parkinson's Disorder, immune
disorders, and hematopoietic disorders, and the various
dyslipidemias, metabolic disturbances associated with obesity, the
metabolic syndrome X and wasting disorders associated with chronic
diseases and various cancers. The invention also provides for
prognostic (or predictive) assays for determining whether an
individual is at risk of developing a disorder associated with NOVX
protein, nucleic acid expression or activity. For example,
mutations in a NOVX gene can be assayed in a biological sample.
Such assays can be used for prognostic or predictive purpose to
thereby prophylactically treat an individual prior to the onset of
a disorder characterized by or associated with NOVX protein,
nucleic acid expression, or biological activity.
[0304] 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.)
[0305] 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.
[0306] Diagnostic Assays
[0307] An exemplary method for detecting the presence or absence of
NOVX in a biological sample involves obtaining a biological sample
from a test subject and contacting the biological sample with a
compound or an agent capable of detecting NOVX protein or nucleic
acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that
the presence of NOVX is detected in the biological sample. An agent
for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid
probe capable of hybridizing to NOVX mRNA or genomic DNA. The
nucleic acid probe can be, for example, a full-length NOVX nucleic
acid, such as the nucleic acid of SEQ ID NOS:2n-1, wherein n is an
integer between 1 and 64, 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.
[0308] 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').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a
fluorescently-labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. That is, the detection method of the invention can be
used to detect NOVX mRNA, protein, or genomic DNA in a biological
sample in vitro as well as in vivo. For example, in vitro
techniques for detection of NOVX mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of NOVX protein include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of NOVX
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of NOVX protein include introducing into a
subject a labeled anti-NOVX antibody. For example, the antibody can
be labeled with a radioactive marker whose presence and location in
a subject can be detected by standard imaging techniques.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] Prognostic Assays
[0313] 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.
[0314] 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).
[0315] The methods of the invention can also be used to detect
genetic lesions in a NOVX gene, thereby determining if a subject
with the lesioned gene is at risk for a disorder characterized by
aberrant cell proliferation and/or differentiation. In various
embodiments, the methods include detecting, in a sample of cells
from the subject, the presence or absence of a genetic lesion
characterized by at least one of an alteration affecting the
integrity of a gene encoding a NOVX-protein, or the misexpression
of the NOVX gene. For example, such genetic lesions can be detected
by ascertaining the existence of at least one of: (i) a deletion of
one or more nucleotides from a NOVX gene; (ii) an addition of one
or more nucleotides to a NOVX gene; (iii) a substitution of one or
more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement
of a NOVX gene; (v) an alteration in the level of a messenger RNA
transcript of a NOVX gene, (vi) aberrant modification of a NOVX
gene, such as of the methylation pattern of the genomic DNA, (vii)
the presence of a non-wild-type splicing pattern of a messenger RNA
transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX
protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate
post-translational modification of a NOVX protein. As described
herein, there are a large number of assay techniques known in the
art which can be used for detecting lesions in a NOVX gene. A
preferred biological sample is a peripheral blood leukocyte sample
isolated by conventional means from a subject. However, any
biological sample containing nucleated cells may be used,
including, for example, buccal mucosal cells.
[0316] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction (PCR) (see,
e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and
Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364),
the latter of which can be particularly useful for detecting point
mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl.
Acids Res. 23: 675-682). This method can include the steps of
collecting a sample of cells from a patient, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers that
specifically hybridize to 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.
[0317] Alternative amplification methods include: self sustained
sequence replication (see, Guatelli, et al., 1990. Proc. Natl.
Acad. Sci. USA 87: 1874-1878), transcriptional amplification system
(see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177); Q.beta. Replicase (see, Lizardi, et al, 1988.
BioTechnology 6: 1197), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers.
[0318] In an alternative embodiment, mutations in a NOVX gene from
a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
e.g., U.S. Pat. No. 5,493,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0319] In other embodiments, genetic mutations in NOVX can be
identified by hybridizing sample and control nucleic acids, e.g.,
DNA or RNA to high-density arrays containing hundreds or thousands
of oligonucleotide probes. See, e.g., Cronin, et al., 1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For
example, genetic mutations in NOVX can be identified in
two-dimensional arrays containing light-generated DNA probes as
described in Cronin, et al., supra. Briefly, a first hybridization
array of probes can be used to scan through long stretches of DNA
in a sample and control to identify base changes between the
sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This is
followed by a second hybridization array that allows the
characterization of specific mutations by using smaller,
specialized probe arrays complementary to all variants or mutations
detected. Each mutation array is composed of parallel probe sets,
one complementary to the wild-type gene and the other complementary
to the mutant gene.
[0320] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
NOVX gene and detect mutations by comparing the sequence of the
sample NOVX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA
74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is
also contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
(see, e.g., Naeve, et al., 1995. Biotechniques 19: 448), including
sequencing by mass spectrometry (see, e.g., PCT International
Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36: 127-162; and Griffin, et al., 1993. Appl.
Biochem. Biotechnol. 38: 147-159).
[0321] Other methods for detecting mutations in the NOVX gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See,
e.g., Myers, et al., 1985. Science 230: 1242. In general, the art
technique of "mismatch cleavage" starts by providing heteroduplexes
of formed by hybridizing (labeled) RNA or DNA containing the
wild-type NOVX sequence with potentially mutant RNA or DNA obtained
from a tissue sample. The double-stranded duplexes are treated with
an agent that cleaves single-stranded regions of the duplex such as
which will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S.sub.1 nuclease to
enzymatically digesting the mismatched regions. In other
embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to
digest mismatched regions. After digestion of the mismatched
regions, the resulting material is then separated by size on
denaturing polyacrylamide gels to determine the site of mutation.
See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an
embodiment, the control DNA or RNA can be labeled for
detection.
[0322] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in NOVX
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g.,
Hsu, et al., 1994. Carcinogenesis 15: 1657-1662. According to an
exemplary embodiment, a probe based on 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. Pat. No. 5,459,039.
[0323] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in NOVX genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc.
Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285:
125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79.
Single-stranded DNA fragments of sample and control NOVX nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In one embodiment, the subject method utilizes
heteroduplex analysis to separate double stranded heteroduplex
molecules on the basis of changes in electrophoretic mobility. See,
e.g., Keen, et al., 1991. Trends Genet. 7: 5.
[0324] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495. When DGGE
is used as the method of analysis, DNA will be modified to insure
that it does not completely denature, for example by adding a GC
clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In
a further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987.
Biophys. Chem. 265: 12753.
[0325] 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.
[0326] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification may carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl.
Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one
primer where, under appropriate conditions, mismatch can prevent,
or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech.
11: 238). In addition it may be desirable to introduce a novel
restriction site in the region of the mutation to create
cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol.
Cell Probes 6: 1. It is anticipated that in certain embodiments
amplification may also be performed using Taq ligase for
amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA
88: 189. In such cases, ligation will occur only if there is a
perfect match at the 3'-terminus of the 5' sequence, making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0327] 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.
[0328] 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.
[0329] Pharmacogenomics
[0330] Agents, or modulators that have a stimulatory or inhibitory
effect on NOVX activity (e.g., NOVX gene expression), as identified
by a screening assay described herein can be administered to
individuals to treat (prophylactically or therapeutically)
disorders (The disorders include metabolic disorders, diabetes,
obesity, infectious disease, anorexia, cancer-associated cachexia,
cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's Disorder, immune disorders, and hematopoietic
disorders, and the various dyslipidemias, metabolic disturbances
associated with obesity, the metabolic syndrome X and wasting
disorders associated with chronic diseases and various cancers.) In
conjunction with such treatment, the pharmacogenomics (i.e., the
study of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) of the
individual may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the
individual permits the selection of effective agents (e.g., drugs)
for prophylactic or therapeutic treatments based on a consideration
of the individual's genotype. Such pharmacogenomics can further be
used to determine appropriate dosages and therapeutic regimens.
Accordingly, the activity of NOVX protein, expression of NOVX
nucleic acid, or mutation content of NOVX genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual.
[0331] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See e.g.,
Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985;
Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body (altered drug action) or genetic conditions
transmitted as single factors altering the way the body acts on
drugs (altered drug metabolism). These pharmacogenetic conditions
can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common
inherited enzymopathy in which the main clinical complication is
hemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0332] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome Pregnancy Zone Protein Precursor enzymes CYP2D6 and
CYP2C19) has provided an explanation as to why some patients do not
obtain the expected drug effects or show exaggerated drug response
and serious toxicity after taking the standard and safe dose of a
drug. These polymorphisms are expressed in two phenotypes in the
population, the extensive metabolizer (EM) and poor metabolizer
(PM). The prevalence of PM is different among different
populations. For example, the gene coding for CYP2D6 is highly
polymorphic and several mutations have been identified in PM, which
all lead to the absence of functional CYP2D6. Poor metabolizers of
CYP2D6 and CYP2C19 quite frequently experience exaggerated drug
response and side effects when they receive standard doses. If a
metabolite is the active therapeutic moiety, PM show no therapeutic
response, as demonstrated for the analgesic effect of codeine
mediated by its CYP2D6-formed metabolite morphine. At the other
extreme are the so called ultra-rapid metabolizers who do not
respond to standard doses. Recently, the molecular basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
[0333] Thus, the activity of NOVX protein, expression of NOVX
nucleic acid, or mutation content of NOVX genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual. In
addition, pharmacogenetic studies can be used to apply genotyping
of polymorphic alleles encoding drug-metabolizing enzymes to the
identification of an individual's drug responsiveness phenotype.
This knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
a NOVX modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0334] Monitoring of Effects During Clinical Trials
[0335] 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.
[0336] 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.
[0337] In one embodiment, the invention provides a method for
monitoring the effectiveness of treatment of a subject with an
agent (e.g., an agonist, antagonist, protein, peptide,
peptidomimetic, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a NOVX protein, mRNA, or genomic DNA in
the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the NOVX protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the NOVX protein, mRNA, or
genomic DNA in the pre-administration sample with the NOVX protein,
mRNA, or genomic DNA in the post administration sample or samples;
and (vi) altering the administration of the agent to the subject
accordingly. For 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.
[0338] Methods of Treatment
[0339] The invention provides for both prophylactic and therapeutic
methods of treating a subject at risk of (or susceptible to) a
disorder or having a disorder associated with aberrant NOVX
expression or activity. The disorders include cardiomyopathy,
atherosclerosis, hypertension, congenital heart defects, aortic
stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal
defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis,
ventricular septal defect (VSD), valve diseases, tuberous
sclerosis, scleroderma, obesity, transplantation,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate
cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer,
fertility, hemophilia, hypercoagulation, idiopathic
thrombocytopenic purpura, immunodeficiencies, graft versus host
disease, AIDS, bronchial asthma, Crohn's disease; multiple
sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and
other diseases, disorders and conditions of the like.
[0340] These methods of treatment will be discussed more fully,
below.
[0341] Diseases and Disorders
[0342] Diseases and disorders that are characterized by increased
(relative to a subject not suffering from the disease or disorder)
levels or biological activity may be treated with Therapeutics that
antagonize (i.e., reduce or inhibit) activity. Therapeutics that
antagonize activity may be administered in a therapeutic or
prophylactic manner. Therapeutics that may be utilized include, but
are not limited to: (i) an aforementioned peptide, or analogs,
derivatives, fragments or homologs thereof; (ii) antibodies to an
aforementioned peptide; (iii) nucleic acids encoding an
aforementioned peptide; (iv) administration of antisense nucleic
acid and nucleic acids that are "dysfunctional" (i.e., due to a
heterologous insertion within the coding sequences of coding
sequences to an aforementioned peptide) that are utilized to
"knockout" endogenous function of an aforementioned peptide by
homologous recombination (see, e.g., Capecchi, 1989. Science 244:
1288-1292); or (v) modulators (i.e., inhibitors, agonists and
antagonists, including additional peptide mimetic of the invention
or antibodies specific to a peptide of the invention) that alter
the interaction between an aforementioned peptide and its binding
partner.
[0343] 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.
[0344] Increased or decreased levels can be readily detected by
quantifying peptide and/or RNA, by obtaining a patient tissue
sample (e.g., from biopsy tissue) and assaying it in vitro for RNA
or peptide levels, structure and/or activity of the expressed
peptides (or mRNAs of an aforementioned peptide). Methods that are
well-known within the art include, but are not limited to,
immunoassays (e.g., by Western blot analysis, immunoprecipitation
followed by sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis, immunocytochemistry, etc.) and/or hybridization
assays to detect expression of mRNAs (e.g., Northern assays, dot
blots, in situ hybridization, and the like).
[0345] Prophylactic Methods
[0346] 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.
[0347] Therapeutic Methods
[0348] 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, in vivo (e.g., by administering the agent to a
subject). As such, the invention provides methods of treating an
individual afflicted with a disease or disorder characterized by
aberrant expression or activity of a NOVX protein or nucleic acid
molecule. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein), or combination of agents that modulates (e.g.,
up-regulates or down-regulates) NOVX expression or activity. In
another embodiment, the method involves administering a NOVX
protein or nucleic acid molecule as therapy to compensate for
reduced or aberrant NOVX expression or activity.
[0349] Stimulation of NOVX activity is desirable in situations in
which NOVX is abnormally downregulated and/or in which increased
NOVX activity is likely to have a beneficial effect. One example of
such a situation is where a subject has a disorder characterized by
aberrant cell proliferation and/or differentiation (e.g., cancer or
immune associated disorders). Another example of such a situation
is where the subject has a gestational disease (e.g.,
preclampsia).
[0350] Determination of the Biological Effect of the
Therapeutic
[0351] 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.
[0352] In various specific embodiments, in vitro assays may be
performed with representative cells of the type(s) involved in the
patient's disorder, to determine if a given Therapeutic exerts the
desired effect upon the cell type(s). Compounds for use in therapy
may be tested in suitable animal model systems including, but not
limited to rats, mice, chicken, cows, monkeys, rabbits, and the
like, prior to testing in human subjects. Similarly, for in vivo
testing, any of the animal model system known in the art may be
used prior to administration to human subjects.
[0353] Prophylactic and Therapeutic Uses of the Compositions of the
Invention
[0354] The NOVX nucleic acids and proteins of the invention are
useful in potential prophylactic and therapeutic applications
implicated in a variety of disorders including, but not limited to:
metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-associated cancer, neurodegenerative disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders,
hematopoietic disorders, and the various dyslipidemias, metabolic
disturbances associated with obesity, the metabolic syndrome X and
wasting disorders associated with chronic diseases and various
cancers.
[0355] As an example, a cDNA encoding the NOVX protein of the
invention may be useful in gene therapy, and the protein may be
useful when administered to a subject in need thereof. By way of
non-limiting example, the compositions of the invention will have
efficacy for treatment of patients suffering from: metabolic
disorders, diabetes, obesity, infectious disease, anorexia,
cancer-associated cachexia, cancer, neurodegenerative disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders,
hematopoietic disorders, and the various dyslipidemias.
[0356] 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.
[0357] 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
Example 1
[0358] The NOV1 clone was analyzed, and the nucleotide and encoded
polypeptide sequences are shown in Table 1A.
2TABLE 1A NOV1 Sequence Analysis NOV1a, CG50907-03 SEQ ID NO:1 1914
bp DNA Sequence ORF Start: at 7 ORF Stop: end of sequence
AGATCTGCTGAGGTGTGGTGGAACCTTGTGCCGCG-
TAAGACAGTGTCTTCTGGGGAGCTGGCCACGGTAGTAC
GGCGGTTCTCCCAGACCGGCATCCAGGACTTCCTGACACTGACGCTGACAAAGCCCACTGGGCTTCTGTACGT
GGGCGCCCGAGAGGCCCTGTTTGCCTTCAGCATGGAGGCCCTGGAGCTGCAAGGAGC-
GATCTCCTGGGAGGCC CCCGTGGAGGAGAAGACTGAGTGTATCCAGAAAGGGAAGAA-
CAACCAGACCGAGTGCTTCAACTTCATCCGCT TCCTGCAGCCCTACAATGCCTCCCA-
CCTGTACGTCTGTGGCACCTACGCCTTCCAGCCCAAGTGCACCTACGT
CAACATGCTCACCTTCACTTTGGAGCATGGAGAGTTTGAAGATGGGAAGGGCAAGTGTCCCTATGACCCAGCT
AAGGGCCATGCTGGCCTTCTTGTGGATGGTGAGCTGTACTCGGCCACACTCAACAAC-
TTCCTGGGCACGGAAC CCATTATCCTGCGTAACATGGGGCCCCACCACTCCATGAAG-
ACAGAGTACCTGGCCTTTTGGCTCAACGAACC TCACTTTGTAGGCTCTGCCTATGTA-
CCTGAGAGTGTGGGCAGCTTCACGGGGGACGACGACAAGGTCTACTTC
TTCTTCAGGGAGCGGGCAGTGGAGTCCGACTGCTATGCCGAGCAGGTGGTGGCTCGTGTGGCCCGTGTCTGCA
AGGGCGATATGGGGGGCGCACGGACCCTGCAGAGGAAGTGGACCACGTTCCTGAAGG-
CGCGGCTGGCATGCTC TGCCCCGAACTGGCAGCTCTACTTCAACCAGCTGCAGGCGA-
TGCACACCCTGCAGGACACCTCCTGGCACAAC ACCACCTTCTTTGGGGTTTTTCAAG-
CACAGTGGAATGACATGTACCTGTCGGCCATCTGTGAGTACCAGTTGG
AAGAGATCCAGCGGGTGTTTGAGGGCCCTATAAGGAGTACCATGAGGAAGCCCCAGAAGTGGGACCGCTACAC
TGACCCTGTACCCAGCCCTCGGCCTGGCTCGTGCATTAACAACTGGCATCGGCGCAA-
CGCCTACACCAGCTCC CTGGAGCTACCCGACAACATCCTCAACTTCGTCAAGAAGCA-
CCCGCTGATGGAGGAGCAGGTGGGGCCTCGGT GGAGCCGCCCCCTGCTCGTGAAGAA-
GGGCACCAACTTCACCCACCTGGTGGCCGACCGGGTTACAGGACTTGA
TGGAGCCACCTATACAGTGCTGTTCATTGGCACAGGAGACGGCTGGCTGCTCAAGGCTGTGAGCCTGGGGCCC
TGGGTTCACCTGATTGAGGAGCTCCAGCTGTTTGACCAGGAGCCCATGAGAAGCCTG-
GTGAAATCTCAGAGCA AGAAGCTGCTCTTTGCCGGCTCCCGCTCTCAGCTGGTGCAG-
CTGCCCGTAACCGACTGCATGAAGTATCGCTC CTGTGCAGACTGTGTCCTCGCCCGG-
GACCCCTATTGCGCCTGGAGCGTCAACACCAGCCGCTGTGTGGCCGTG
GGTGGCCACTCTGGATCTCTACTGATCCAGCATGTGATGACCTCGGACACTTCAAACATCTGCAACCTCCGTG
GCAGTAAGAAAGTCAGGCCCACTCCCAAAAACATCACGGTGGTAACGAACACAGACC-
TAATGCTGCCCTGCCA CCTCTCCTCCAACTTGGCCCATGCCCGCTGGACCTTTGGGG-
GCCGGGACCTGCCTGCGGAACAGCCCGGGTCC TTCCTCTACGATGCCCGGCTCCAGG-
CCCTAATTGTGATGGCTGCCCAGCCCCGCCATGCCAAGGCCTACCACT
GCTTTTCAGAGGACCAGGGGGCGCAACTGGCTGCTGAAGGCTACCTTGTGGCTGTCGTGGCAGGCCCGTCGGT
GACCTTGGAGCTCGAG NOV1a, CG50907-03 Protein Sequence SEQ ID NO:2 634
aa MW at 71053.3 kD
AEVWWNLVPRKTVSSGELATVVRRFSQTGIQDFLTLTLTEPTGLLYVGAREALFAFSMEALELQGAISWEAPV
EEKTECIQKGKNNQTECFNFIRFLQPYNASHLYVCGTYAFQPKCTYVNNLTFTLEH-
GEFEDGKGKCPYDPAKG HAGLLVDGELYSATLNNFLGTEPIILRNMGPHHSMKTEYL-
AFWLNEPHFVGSAYVPESVGSFTGDDDKVYFFF RERAVESDCYAEQVVARVARVCKG-
DMGGARTLQRKWTTFLKARLACSAPNWQLYFNQLQAMHTLQDTSWHNTT
FFGVFQAQWGDMYLSAICEYQLEEIQRVFEGPYKEYHEEAQKWDRYTDPVPSPRPGSCIAAAARRHGYTSSLE
LPDNILNFVKKHPLMEEQVGPRWSRPLLVKKGTNFTHLVADRVTGLDGATYTVLFIG-
TGDGWLLKAVSLGPWV HLIEELQLFDQEPMRSLVLSQSKKLLFAGSRSQLVQLPVAD-
CMKYRSCADCVLARDPYCAWSVNTSRCVAVGG HSGSLLIQHVMTSDTSGICNLRGSK-
KVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDLPAEQPGSFL
YDARLQALVVMAAQPPHAGAYHCFSEEQGARLAAEGYLVAVVAGPSAALE NOV1b,
CG50907-04 SEQ ID NO:3 2049 bp DNA Sequence ORF Start: ATG at 7 ORF
Stop: end of sequence TGCGCCATGGCCCCACACTGGGCTGTCTGGCTGCTGGCAG-
CAAGGCTGTGGGGCCTGGGCATTGGGGCTGAGG TGTGGTGCAACCTTGTGCCGCGT-
AAGACAGTGTCTTCTAAGGAGCTGGCCACGGTAGTACGGCAATTCTCCCA
GACCGGCATCCAGGACTTCCTGACACTGACGCTGACGGAGCCCACTGGGCTTCTGTACCTGGGCGCCCGAGAG
GCCCTGTTTGCCTTCAGCATGGAGGCCCTGGAGCTGCAAGGAGCGATCTCCTGGGAG-
GCCCCCGTGGAGGAGA AGACTGACTGTATCCAGAAAGGGAAGAACAACCAGACCGAG-
TGCTTCAACTTCATCCGCTTCCTGCAGCCCTA CAATGCCTCCCACCTGTACGTCTGT-
GGCACCTACGCCTTCCAGCCCAAGTGCACCTACGTCAACATGCTCACC
TTCACTTTGGAGCATGGAGAGTTTGAAGATGGGAAGGGCAAGTGTCCCTATGACCCAGCTAAGGGCCATGCTG
GCCTTCTTGTGGATGGTGAGCTGTACTCGGCCACACTCAACAACTTCCTGGGCACGG-
AACCCATTATCCTGCG TAACATGGGGCCCCACCACTCCATGAAGACAGAGTACCTGG-
CCTTTTGGCTCAACGAACCTCACTTTGTAGGC TCTGCCTATGTACCTGAGAGTGTGG-
GCAGCTTCACGGGGGACGACGACAAGGTCTACTTCTTCTTCAGGGAGC
GGGCAGTGGAGTCCGACTGCTATGCCGAGCAGGTGGTGGCTCGTGTGGCCCGTGTCTGCAAAAGCGATATGGG
GGGCGCACGGACCCTGCAGAGGAAGTGGACCACGTTCCTGAAGGCGCGGCTGGCATG-
CTCTGCCCCGAACTGG CAGCTCTACTTCAACCAGCTGCAGGCGATGCACACCCTGCA-
GGACACCTCCTAACACAACACCACCTTCTTTG GGGTTTTTCAAGCACAGTGGGGTGA-
CATGTACCTGTCGGCCATCTGTGAGTACCAGTTGGAAGAGATCCAGCG
GGTGTTTGAGGGCCCCTATAAGGAGTACCATGAAAAGCCCAGAAAGTAAGACCGCTACACTGACCCTGTACCC
AGCCCTCGGCCTGGCTCGTGCATTAACAACTGGCATCGGCGCCACGGCTAAACCAGC-
TCCCTGGAGCTACCCG ACAACATCCTCAACTTCGTCAAGAAGCACCCGCTGATGGAG-
GAGCAGGTGGGCCCTCGGTGGAGCCGCCCCCT GCTCGTGAAGAAGGGCACCAACTTC-
ACCCACCTGGTGGCCGACCGGGTTACAGGACTTGATGGAGCCACCTAT
ACAGTGCTGTTCATTGGCACAGGAGACGGCTGGCAGCTCAAGGCTGTGAGCCTGGGGCCCTGGGTTCACCTGA
TTGAGGAGCTGCAGCTGTTTGACCAGGAGCCCATGAGAAGCCTGGTGCTATCTCAGA-
GAAAGACCCTCCAAGC TGTGCTGTGGTTCTGGCTCTGGCCTCTTCCCTGACCATGCC-
CCTGTGACCAGACCTCCCAGGCTAAGCTGCTC TTTGCCGGCTCCCGCTCTCAGCTGG-
TGCAGCTGCCCGTGGCCGACTGCATGAAGTATCGCTCCTGTGCAGACT
GTGTCCTCGCCCGGGACCCCTATTGCGCCTGGAGCGTCAACACCAGCCGCTGTGTGGCCGTAAGTGGCCACTC
TGGATCTCTACTGATCCAGCATGTGATGACCTCGGACACTTCAAACATCTGCAACCT-
CCGTGGCAGTAAGAAA GTCAGGCCCACTCCCAAAAACATCACGGTGGTGGCGGGCAC-
AGACCTGGTGCTGCCCTGCCACCTCTCCTCCA ACTTGGCCCATGCCCGCTGGACCTT-
TGGGGGCCGGGACCTGCCTGCGGAACAGCCCGGGTCCTTCCTCTACGA
TGCCCGGCTCCAGGCCCTGGTTGTGATGGCTGCCCAGCCCCGCCATCCCAAGGCCTACCACTGCTTTTCAGAG
GAGCAGGGGGCGCGGCTGGCTGCTGAAGGCTACCTTGTGGCTGTCGTGGCAGGCCCG-
TCAATGACCTTGGAGC TCGAG NOV1b, CG50907-04 Protein Sequence SEQ ID
NO:4 1495 aa MW at 56584.0 kD
MAPHWAVWLLAARLWGLGIGAEVWWNLVPRKTVSSGELATVVRRFSQTGIQDFLTLTLTEPTGLLYVGAREAL
FAFSMEALELQGAISWEAPVEEKTECIQKGAANQTECFNFIRFLQPYNASHLYVCG-
TYAFQPKCTYVNMLTFT LEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSATLNNFLG-
TEPIILRNMGPHHSMKTEYLAFWLNEPHFVGSA YVPESVGSFTGDDDKVYFFFRERA-
VESDCYAEQVVARVARVCKGDMGGARTLQRKWTTFLKARLACSAPNWQL
YFNQLQAMHTLQDTSWHNTTFFGVFQAQWGDMYLSAICEYQLEEIQRVFEGPYKEYHEEAQKWDRYTDPVPSP
RPGSCINNWHRRHGYTSSLELPDNILNFVKKHPLMEEQVGPRWSRPLLVKKGTNFTH-
LVADRVTGLDGATYTV LFIGTGDGWQLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQ-
SKTLQAVLWFWLWPLP NOV1c, CG50907-02 SEQ ID NO:5 3112 bp DNA Sequence
ORF Start: ATG at 104 ORF Stop: end of sequence
TGCTGCGGGCCCCTCTGGTTTGCTTTCTCTGGCTGTGATTTCTGACCATGTCTTTTCCCTCAGCAGGACAGCT
GGCCTGAAGCTCAGAGCCGGGGCGTGCGCCATGGCCCCACACTGGGCTGTCTGGCT-
GCTGGCAGCAAGGCTGT GGGGCCTGGGCATTGGGGCTGAGGTGTGGTGGAACCTTGT-
GCCGCGTAAGACAGTGTCTTCTGGGGAGCTGGC CACGGTAGTACGGCGGTTCTCCCA-
GACCGCCATCCAGCACTTCCTGACACTGACGCTGACGOAGCCCACTGGG
CTTCTGTACGTGGGCGCCAGGGACCATGCCTCTGCACTGGGCGTCCCTGTGTTGCTGCTGCAGGCTGTGATCT
CCTGGGAGGCCCCCGTGGAGAAGAAGACTGAGTGTATCCAGAAAGGGAAGAACAACC-
AGACCGAGTGCTTCAA CTTCATCCGCTTCCTGCAGCCCTACAATGCCTCCCACCTGT-
ACGTCTGTGGCACCTACGCCTTCCAGCCCAAG TGCACCTACGTCAACATGCTCACCT-
TCACTTTGGAGCATGGAGAGTTTGAAGATGGGAAGGGCAAGTGTCCCT
ATGACCCAGCTAAGGGCCATGCTGGCCTTCTTGTGGATGGTGAGCTGTACTCGGCCACACTCAACAACTTCCT
GGGCACGGAACCCATTATCCTGCGTAACATGGGGCCCCACCACTCCATOAAGACAGA-
GTACCTGGCCTTTTGG CTCAACGAACCTCACTTTGTAGGCTCTGCCTATGTACCTGA-
GAGTGTGGGCAGCTTCACGGGGGACGACGACA AGGTCTACTTCTTCTTCAGGGAGCG-
GGCAGTGGAGTCCGACTGCTATGCCGAGCAGGTGGTGGCTCGTGTGGC
CCCTGTCTGCAAGGGCGATATGGGGOGCGCACGGACCCTGCAGAGGAAGTGGACCACGTTCCTGAAGGCGCGG
CTGGCATGCTCTGCCCCGAACTGGCAGCTCTACTTCAACCAGCTGCAGGCGATGCAC-
ACCCTGCAGGACACCT CCTGGCACAACACCACCTTCTTTGGOGTTTTTCAAGCACAG-
TGGGGTGACATGTACCTGTCGGCCATCTGTGA GTACCAGTTGGAAGAGATCCAGCGG-
GTGTTTGAGGGCCCCTAThAGGAGTACCATGAGGAAGCCCAGAAGTGG
GACCGCTACACTGACCCTGTACCCAGCCCTCGGCCTGGCTCGTGCATTAACAACTGGCATCGGCGCCACGGCT
ACACCAGCTCCCTGGAGCTACCCGACAACATCCTCAACTTCGTCAAGAAGCACCCGC-
TGATGGAGGACCAGGT GGGGCCTCGGTGGAGCCGCCCCCTGCTCGTGAAGAAGGGCA-
CCAACTTCACCCACCTGGTGGCCGACCGGGTT ACAGGACTTGATGGAGCCACCTATA-
CAGTGCTGTTCATTGGCACAGGAGACGGCTGGCTGCTCAAGGCTGTGA
GCCTGGGGCCCTGGGTTCACCTGATTGAGGAGCTGCAGCTGTTTGACCAGGAGCCCATGAGAAGCCTGGTGCT
ATCTCAGAGCAAGAAGCTGCTCTTTGCCGGCTCCCGCTCTCAGCTGGTGCAGCTGCC-
CGTGGCCGACTGCATG AAGTATCGCTCCTGTGCAGACTGTGTCCTCGCCCGGGACCC-
CTATTGCGCCTGGAGCGTCAACACCAGCCGCT GTGTGGCCGTGGGTGGCCACTCTGG-
ATCTCTACTGATCCAGCATGTGATGACCTCGGACACTTCAGGCATCTG
CAACCTCCGTGGCAGTAAGAAAGTCAGGCCCACTCCCAAAAACATCACGGTGGTGGCGGGCACAGACCTGGTG
CTGCCCTGCCACCTCTCCTCCAACTTGGCCCATGCCCGCTGGACCTTTGGGGGCCGG-
GACCTGCCTGCGGAAC AGCCCGGGTCCTTCCTCTACGATGCCCGGCTCCAGGCCCTG-
GTTGTGATGGCTGCCCAGCCCCGCCATGCCGG GGCCTACCACTGCTTTTCAGAGGAG-
CAGGGGGCGCGGCTGGCTGCTGAAGGCTACCTTGTGGCTGTCGTGGCA
GGCCCGTCGGTGACCTTGGAGGCCCGGGCCCCCCTGGAAAACCTGGGGCTGGTGTGGCTGGCGGTGGTGGCCC
TGGGGGCTGTGTGCCTGGTGCTGCTGCTGCTGGTGCTGTCATTGCGCCGGCGGCTGC-
GGGAAGAGCTGGAGAA AGGGGCCAAGGCTACTGAGAGGACCTTGGTGTACCCCCTGG-
AGCTGCCCAAGGAGCCCACCAGTCCCCCCTTC CGGCCCTGTCCTGAACCAGATGAGA-
AACTTTGGGATCCTGTCGGTTACTACTATTCAGATGGCTCCCTTAAGA
TAGTACCTGGGCATGCCCGGTGCCAGCCCGGTGGGGGGCCCCCTTCGCCACCTCCAGGCATCCCAGGCCAGCC
TCTGCCTTCTCCAACTCGGCTTCACCTGGGGGGTGGGCGGAACTCAAATGCCAATGG-
TTACGTGCGCTTACAA CTAGGAGGGGAGGACCGGGGAGGGCTCGGGCACCCCCTGCC-
TGAGCTCGCGGATGAACTGAGACGCAAACTGC AGCAACGCCAGCCACTGCCCGACTC-
CAACCCCGAGGAGTCATCAGTATGAGGGGAACCCCCACCGCGTCGGCG
GGAAGCGTGGGAGGTGTAGCTCCTACTTTTGCACAGGCACCAGCTACCTCAGGGACATGGCACGGGCACCTGC
TCTGTCTGGGACAGATACTGCCCAGCACCCACCCGGCCATGAGGACCTGCTCTGCTC-
AGCACGGGCACTGCCA CTTGGTGTGGCTCACCAGGGCACCACCCTCGCAGAAGGCAT-
CTTCCTCCTCTCTGTGAATCACAGACACGCGG GACCCCAGCCGCCAAAACTTTTCAA-
GGCAGAAGTTTCAAGATGTGTGTTTGTCTGTATTTGCACATGTGTTTG
TGTGTGTGTGTATGTGTGTGTGCACGCGCGTGCGCGCTTGTGOCATAGCCTTCCTGTTTCTGTCAAGTCTTCC
CTTGGCCTGGGTCCTCCTGGTGAGTCATTGGAGCTATGAAGGGGAAGGGGTCGTATC-
ACTTTGTCTCTCCTAC CCCCACTGCCCCGAGTGTCGGGCAGCGATGTACATATGGAG- GTGGG
NOV1c, CG50907-02 Protein Sequence SEQ ID NO:6 833 aa MW at 92497.9
kD MAPHWAVWLLAARLWGLGIGAEVWWNLVPRKTVSSGE-
LATVVRRFSQTGIQDFLTLTLTEPTGLLAAGARDHA
SALGVPVLLLQAVISWEAPVEKKTECIQKGKNNQTECFNFIRFLQPYNASHLYVCGTYAFQPKCTYVNMLTFT
LEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSATLNNFLGTEPIILRNMGPHHSMKT-
EYLAFWLNEPHFVGSA YVPESVGSFTGDDDKVYFFFRERAVESDCYAEQVVARVARV-
CKGDMGGARTLQRKWTTFLKARLACSAPNWQL YFNQLQAMHTLQDTSWHNTTFFGVF-
QAQWGDMYLSAICEYQLEEIQRVFEGPYKEYHEEAQKWDRYTDPVPSP
RPGSCINNWHRRHGYTSSLELPDNILNFVKKHPLMEEQVGPRWSRPLLVKKGTNFTHLVADRVTGLDGATYTV
LFIGTGDGWLLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQSKKLLFAGSRSQLVQL-
PVADCMKYRSCADCVL ARDPYCAWSVNTSRCVAVGGHSGSLLIQHVMTSDTSGICNL-
RGSKKVRPTPKINTVVAGTDLVLPCHLSSNLA HARWTFGGRDLPAEQPGSFLYDARL-
QALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVVAGPSVTLEARA
PLENLGLVWLAVVAIGAVCLVLLLLVLSLRRRLREELEKGAKATERTLVYPLELPKEPTSPPFRPCPEPDEKL
WDPVGYYYSDGSLKIVPGHARCQPGGGPPSPPPGIPGQPLPSPTRLHLGGGRNSNAN-
GYVRLQLGGEDRGGLG HPLPELADELRRKLQQRQPLPDSNPEESSV NOV1d, 170645595
SEQ ID NO:7 1914 bp DNA Sequence ORF Start: at 1 ORF Stop: end of
sequence AGATCTGCTGAGGTGTGGTGGAACCTTGTGCCGCG-
TAAGACAGTGTCTTCTGGGGAGCTGGCCACGGTAGTAC
GGCGGTTCTCCCAGACCGGCATCCAGGACTTCCTGACACTGACGCTGACGGAGCCCACTGGGCTTCTGTACGT
GGGCGCCCGAGAGGCCCTGTTTGCCTTCAGCATGGAGGCCCTGGAGCTGCAAGGAGC-
GATCTCCTAAGAGGCC CCCCGTGGAGGAGAAGACTGAGTGTATCCAGAAAGGGAGAA-
CAACCAGACCGAGTGCTTCAACTTAATCCGCT TCCTGCAGCCCTACAATGCCTCCCA-
CCTGTACGTCTGTGGCACCTACGCCTTCCAGCCCAAGTGCACCTACGT
CAACATGCTCACCTTCACTTTGGAGCATGGAGAGTTTGAAGATGGGAAGGGCAAGTGTCCCTATGACCCAGCT
AAGGGCCATGCTGGCCTTCTTGTGGATGGTGAGCTGTACTCGGCCACACTCAACAAC-
TTCCTGGGCACGGAAC CCATTATCCTGCGTAACATGGGGCCCCACCACTCAATGAAG-
ACAGAGTACCTGGCCTTTTGGCTCAACGAACC TCACTTTGTAGGCTCTGCCTATGTA-
CCTGAGAGTGTGGGCAGCTTCACGGGGGACGACGACAAGGTCTACTTC
TTCTTCAGGGAGCGGGCAGTGGAGTCCGACTGCTATGCCGAGCAGGTGGTGGCTCGTGTGGCCCGTGTCTGCA
AGGGCGATATGGGGGGCGCACGGACCCTGCAGAGGAAGTGGACCACGTTCCTGAAGG-
CGCGGCTGGCATGCTC TGCCCCGAACTGGCAGCTCTACTTCAACCAGCTGCAGGCGA-
TGCACACCCTGCAGGACACCTCCTGGCACAAC ACCACCTTCTTTGGGGTTTTTCAAG-
CACAGTGGGGTGACATGTACCTGTCGGCCATCTGTCAGTACCAGTTGG
AAGAGATCCAGCGGGTGTTTGAGGGCCCCTATAAGGAGTACCATGAGGAAGCCCAGAAGTGGGACCGCTACAC
TGACCCTGTACCCAGCCCTCGGCCTGGCTCGTGCATTAACAACTGGCATCGGCGCCA-
CAACTACACCAGCTCC CTGGAGCTACCCGACAACATCCTCAACTTCGTCAAGAAGCA-
CCCGCTGATGGAGGAGCAGGTGGGGCCTCGGT GGAGCCGCCCCCTGCTCGTGAAGAA-
GGGCACCAACTTCACCCACCTGGTGGCCGACCGGGTTACAGGACTTGA
TGGAGCCACCTATACAGTGCTGTTCATTGGCACAGGAGACGGCTGGCTGCTCAAGGCTGTGAGCCTGAAGCCC
TGGGTTCACCTGATTGAGGAGCTGCAGCTGTTTGACCACGAGCCCATGAGAAGCCTG-
GTGCTATCTAAGAGCA AGAAGCTGCTCTTTGCCGGCTCCCGCTCTCAGCTGGTGCAG-
CTGCCCGTGGCCGACTGCATGAAGTATCGCTC CTGTGCAGACTGTGTCCTCGCCCGG-
GACCCCTATTGCGCCTGGAGCGTCAACACCAGCCGCTGTGTGGCCGTG
GGTGGCCACTCTGGATCTCTACTGATCCAGCATGTGATGACCTCGGACACTTCAGGCATCTGCAACCTCCGTG
GCAGTAAGAAAGTCAGGCCCACTCCCAAAAACATCACGGTGGTGGCGAACACAGACC-
TGGTGCTGCCCTGCCA CCTCTCCTCCAACTTGGCCCATGCCCGCTGGACCTTTGGGG-
GCCGGGACCTGCCTGCGGAACAGCCCAAGTCC TTCCTCTACGATGCCCGGCTCCAGG-
CCCTGGTTGTGATGGCTGCCCAGCCCCGCCATGCCGGGGCCTACCACT
GCTTTTCAGAGGAGCAGGGGGCGCGGCTGGCTGCTGAAGGCTACCTTGTGGCTGTCGTAACAGGCCCGTCGGT
GACCTTGGAGCTCGAG NOV1d, 170645595 Protein Sequence SEQ ID NO:8 638
aa MW at 71538.8 kD
RSAEVWWNLVPRKTVSSGELATVVRRFSQTGIQDFLTLTLTEPTGLLYVGAREALFAFSMEALELQGAISWEA
PVEEKTECIQKGKNNQTECFNFIRFLQPYNASHLYVCGTYAFQPKCTYVNMLTFTL-
EHGEFEDGKGKCPYDPA KGHAGLLVDGELYSATLNNFLGTEPIILRNMGPHHSMKTE-
YLAFWLNEPHFVGSAYVPESVGSFTGDDDKVYF FFRERAVESDCYAEQVVARVARVC-
KGDMGGARTLQRKWTTFLKARLACSAPNWQLYFNQLQAMAHTLQDTSWH
TTFFGVFQAQWGDMYLSAICEYQLEEIQRVFEGPYKEYHEEAQKWDRYTDPVPSPRPGSCINNWHRRHGYTSS
LELPDNILNFVKKHPLMEEQVGPRWSRPLLVKKGTNFTHLVADRVTGLDGATYTVLF-
IGTGDGWLLAAVSLGP WVHLIEELQLFDQEPMRSLVLSQSKKLLFAGSRSQLVQLPV-
ADCMKYRSCADCVLARDPYCAWSVNTSRCVAV GGHSCSLLIQHVMTSDTSGICNLRG-
SKKVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDLPAEQPGS
FLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVVACPSVTLELE NOV1e,
170645599 SEQ ID NO:9 1914 bp DNA Sequence ORF Start: at 1 ORF
Stop: end of sequence AGATCTGCTGAGGTGTGGTGGAACCTTGTGCCGCGTAAGA-
CAGTGTCTTCTGGGGAGCTGGCAACAATAGTAC GGCGGTTCTCCCAGACCGGCATC-
CAGGACTTCCTGACACTGACGCTGACAAAGCCCACTGGGCTTCTGTACGT
GGGCGCCCCAGAGGCCCTGTTTGCCTTAAGCATGGAGGCCCTGGAGCTGCAAAAAGCGATCTCCTGGGAGGCC
CCCGTGGAGAAGAAGACTGAGTGTATCCAGAAAGGGAAGAACAGCCAGACCGAGTGC-
TTCAACTTCATCCGCT TCCTGCAGCCCTACAATGCCTCCCACCTGTACGTCTGTGGC-
ACCTACGCCTCCAGCAACAAGTGCACCTACGT CAACATGCTCACCTTCACTTTGGAG-
CATGGACAGTTTGAAGATGGGAAGGGCAAGTGTCCCTATGACCCAGCT
AAGGGCCATGCTGGCCTTCTTGTGGATGGTGAGCTGTACTCGGCCACACTCAACAACTTCCTGGGCACGGAAC
CCATTATCCTGCGTAACATGGGGCCCCACCACTCCATGAAGACAGAGTACCTGGCCT-
TTTGGCTCAACGAACC TCACTTTGTAGGCTCTGCCTATGTACCTGAGAGTGTAAGAA-
GCTTCACGGAAGACGACGACAAGGTCTACTTC TTCTTCAGGGAGCGGGCAGTGGAGT-
CCGACTGCTATGCCGAGCAGGTGGTGGCTCGTGTGGCCCGTGTCTGCA
AGGGCGATATGGGGGGCGCACGGACCCTGCAGAGGAAGTGGACCACGTTCCTGAAGGCGCGGCTGGCATGCTC
TGCCCCGAACTGGCAGCTCTACTTCAACCAGCTGCAGGCGATGCAAACCCTGCAGGA-
CACCTCCTGGCACAAC ACCACCTTCTTTGGGGTTTTTCAAGCACAGTGGGGTGACAT-
GTACCTGTCGGCCATCTGTGAGTACCAGTTGG AAGAGATCCAGCGGGTGTTTGAGGC-
CCCTATAAGGAGTACAATGAGGAAGCCCAGAAGTGGGACCGCTACACA
TGACCCTGTACCCAGCCCTCGGCCTGGCTCGTGCATTAACAACTGGCATCGGCGCCACGGCTACACCAGCTCC
CTGGAGCTACCCGACAACATCCTCAACTTCGTCAAGAAGCACCCGCTGATGGAGGAG-
CAGGTGGGGCCTCGGT GGAGCCGCCCCCTGCTCGTGAAGAAGGGCACCAACTTCACC-
CACCTGGTGGCCGACCGGGTTACAGGACTTGA TGGAGCCACCTATACAGTGCTGTTC-
ATTGGCACAGGAGACGGCTGGCTGCTCAAGGCTGTGAGCCTGGGGCCC
TGGGTTCACCTGATTGAGGAGCTGCAGCTGTTTGACCAGGAGCCCATGAGAAGCCTGGTGCTATCTCAGAGCA
AGAAGCTGCTCTTTGCCGGCTCCCGCTCTCAGCTGGTGCAGCTGCCCGTGGCCGACT-
GCATGAAGTATCGCTC CTGTGCAGACTGTGTCCTCGCCCGGGACCCCTATTGCGCCT-
GGAGCGTCAACACCAGCCGCTGTGTGGCCGTG GGTGGCCACTCTGGATCTCTACTGA-
TCCAGCATGTGATGACCTCGGACACTTCAGGCATCTGCAACCTCCGTG
GCAGTAAGAAAGTCAGGCCCACTCCCAAAAACATAACGGTGGTGGCGGGCACAGACCTGGTGCTGCCCTGCCA
CCTCTCCTCCAACTTGGCCCATGCCCGCTGGACCTTTGGGGGCCGGGACCTGCCTGC-
GGAACAGCCCGGGTCC TTCCTCTACGATGCCCGGCTCCAGGCCCTGGTTGTGATGGC-
TGCCCAGCCCCGCCATGCCGGAACCTACCACT GCTTTTCACAGGAGCAGGGGGCGCA-
ACTGGCTGCTGAAGGCTACCTTGTAACTGTCGTGCCAGGCCCGTCGGT GACCTTGGAGCTCGAG
NOV1e, 170645599 Protein Sequence SEQ ID NO:10 638 aa MW at 71510.8
kD
RSAEVWWNLVPRKTVSSGELATVVRRFSQTGIQDFLTLTLTEPTGLLAAGAAEAAFAFSMEAAELQGAISWEA
PVEKKTECIQKGKNSQTECFNFIRFLQPYNASHLYVCGTYAFQPKCTYVNMLTFTL-
EHGEFEDGKGKCPYDPA KGHAGLLVDGELYSATLNNFLGTEPIILRNMGPHHSMKTE-
YLAFWLNEPHFVGSAYVPESVGSFTGDDDKVYF FFRERAVESDCYAEQVVARVARVC-
KGDMGGARTLQRKWTTFLKARLACSAPNWQLYFNQLQAMHTLQDTSWHN
TTFFGVFQAQWGDMYLSAICEYQLEEIQRVFEGPYKEYHEEAQKWDRYTDPVPSPRPGSCINNWHRRHGYTSS
LELPDNILNFVKKHPLMEEQVGPRWSRPLLVKKGTNFTHLVADRVTGLDGATYTVLF-
IGTGDGWLLKAVSLGP WVHLIEELQLFDQEPMRSLVLSQSKKLLFAGSRSQLVQLPV-
ADCMKYRSCADCVLARDPYCAWSVNTSRCVAV GGHSGSLLIQHVMTSDTSGICNLRG-
SKKVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDLPAEQPGS
FLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVVAGPSVTLELE NOV1f,
CG50907-01 SEQ ID NO:11 2739 bp DNA Sequence ORF Start: ATG at 104
ORF Stop: end of sequence TCCTGCGGGCCCCTCTGGTTTGCTTTCTC-
TGGCTGTGATTTCTGACCATGTCTTTTCCCTCAGCAGGACAGCT
GGCCTGAAGCTCAGAGCCGGGGCGTGCGCCATGGCCCCACACTGGGCTGTCTGGCTGCTGGCAGCAAGGCTGT
GGGGCCTGGGCATTGGGGCTGAGGTGTGGTGGAACCTTGTGCCGCGTAAGACAGTGT-
CTTCTGGGGAGCTGGC CACGGTAGTACGGCGGTTCTCCCAGACCGGCATCCAGGACT-
TCCTGACACTGACGCTGACGGAGCCCACTGGG CTTCTGTACGTGGGCGCCCGAGAGG-
CGCTGTTTGCCTTCAGTGTAGAGGCTCTGGAGCTGCAAGGAGCGATCT
CCTGGGAGGCCCCCGTGGAGAAGAAGACTGAGTGTATCCAGAAAGGGAAGAACAACCAGACCGAGTGCTTCAA
CTTCATCCGCTTCCTGCAGCCCTACAATGCCTCCCACCTGTACGTCTGTGGCACCTA-
CGCCTTCCAGCCCAAG TGCACCTACGTCAACATGCTCACCTTCACTTTGGAGCATAA-
AGAGTTTGAAGATGGGAAGCGCAAGTGTCCCT ATGACCCAGCTAAGGGCCATGCTGG-
CCTTCTTGTGGATGGTGAGCTGTACTCGGCCACACTCAACAACTTCCT
GGGCACGGAACCCATTATCCTCCGTAACATGGGGCCCCACCACTCCATGAAGACAGAGTACCTGGCCTTTTGG
CTCAACGAACCTCACTTTGTAGGCTCTGCCTATGTACCTGAGAGTGTGGGCAGCTTC-
ACGGGAAACGACGACA AGGTCTACTTCCTCTTCAGGGAGCGGGCAGTGGAGTCCGCC-
TGCTATGCCGAGCAGGTAATGGCTCGTGTGGC CCGTGTCTGCAAGGGCGATATGGGG-
GGCGCACGGACCCTGCAGAGGAAGTGGACCACGTTCCTGAAGGCGCGG
CTGGCATGCTCTGCCCCGAACTGGCAGCTCTACTTCAACCAGCTGCAGGCGATGCACACCCTGCAGGACACCT
CCTGGCACAACACCACCTTCTTTGGGGTTTTTCAAGCACAGTGGGGTGACATGTACC-
TGTCGGCCATCTGTGA GTACCAGTTGGAAGAGATCCAGCGGGTGTTTGAGGGCCCCT-
ATAAGGAGTACCATGAGGAAGCCCAGAAGTGG GACCGCTACACTGACCCTGTACCCA-
GCCCTCGGCCTGGCTCGTGCATTAACAACTGGCATCGGCGCCACGGCT
ACACCAGCTCCCTGGAGCTACCCGACAACATCCTCAACTTCGTCAAGAAGCACCCGCTGATGGAGGAGCAGGT
GGGGCCTCGGTGGAGCCGCCCCCTGCTCGTGAAGAAGGGCACCAACTTCACCCACCT-
GGTGGCCGACCGGGTT ACAGGACTTGATGGAGCCACCTATACAGTGCTGTTCATTGG-
CACAGGTCAGGCATGGCTGCTCAAGGCTGTGA GCCTGGGGCCCTGGGTTCACCTGAT-
TGAGGAGCTGCAGCTGTTTGACCAGGAGCCCATGAGAAGCCTGGTGCT
ATCTCAGTCGCAGAAGCTGCTCTTTGCCGGCTCCCGCTCTCAGCTGGTGCAGCTGCCCGTGGCCGACTGCATG
AAGTATCGCTCCTGTGCAGACTGTGTCCTCGCCCGGGACCCCTATTGCGCCTGGAGC-
GTCAACACCAGCCGCT GTGTGGCCGTGGGTGGCCACTCTGGGTCCTTTCTGATCCAG-
CATGTGATGACCTCGGAAACTTCAGGCATCTG CAACCTCCGTGGCAGTAAGAAAGTC-
AGGCCCACTCCCAAAAACATCACGGTGGTGGCAAGCACAGACCTGGTG
CTGCCCTGCCACCTCTCCTCCAACTTGGCCCATGCCCGCTGGACCTTTGGGAACCGGGACCTGCCTGCGGAAC
AGCCCGGGTCCTTCCTCTACGATGCCCGGCTCCAGGCCCTGGTTGTGATGGCTGCCC-
AGCCCCGCCATGCCGG GGCCTACCACTGCTTTTCAGAGGAGCACGGGGCGCGGCTGG-
CTGCTGAAGGCTACCTTGTGGCTGTCGTGGCA GGCCCGTCGGTGACCTTGGAGGCCC-
GGGCCCCCCTGGAAAACCTGGGGCTGGTGTAACTAACGGTGGTGGCCC
TGGGGGCTGTGTGCCTGGTGCTGCTGCTGCTGGTGCTGTCATTGCGCCGGCGGCTGCGGGAAGAGCTGGAGAA
AGGGGCCAAGCTACTGAGAGGACCTTGGTGTACCCCCTGGAGCTGCCCAAAAAGCCC-
ACCAGTCCCCCCCTTC CGGCCCTGTCCTGAACCAGATGAGAAACTTTGGGATCCTGT-
CGGTTACTACTATTCAGATGGCTCCCTTAAGA TAGTACCTGGGCATGCCCGGTGCCA-
GCCCGGTGGGGGGCCCCCTTCGCCACCTCCAGGAATCCCAGGCCAGCC
TCTGCCTTCTCCAACTCGGCTTCACCTGGGGGGTGGGCGGAACTCAAATGCCAATGGTTACGTGCGCTTACAA
CTAGGAGGGGAGGACCGGGGAGGGCTCGGGCACCCCCTGCCTGAGCTCGCGGATGAA-
CTGAGACGCAAACTGC AGCAACGCCAGCCACTGCCCCACTCCAACCCCGAGGAGTCA-
TCAGTATGAGGGGAACCCCCACCGCGTCGGCG GGAAGCGTGGGAGGTGTAGCTCCTA-
CTTTTGCACAGGCACCAGCTATCTCAGGGACATGGCACGGGCACCTGC
TCTGTCTGGGACAGATACTGCCCAGCACCCACCCGGCC NOV1f, CG50907-01 Protein
Sequence SEQ ID NO:12 833 aa MW at 92573.0 kD
MAPHWAVWLLAARLWGLGIGAEVWWNLVPRKTVSSGELATVVRRFSQTGIQDFLTLTLTEPTGLLAAGAAEAL
FAFSVEALELQGAISWEAPVEKKTECIQKGKNNQTECFNFIRFLQPYNASHLYVCG-
TYAFQPKCTYVNMLTFT LEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSATLNNFLG-
TEPIILRNNGPHMSMKTEYLAFWLNEPHFVGSA YVPESVGSFTGDDDKVYFLFRERA-
VESACYAEQVVARVARVCKGDMGGARTLQRKWTTFLKARLACSAPNWQL
YFNQLQAMHTLQDTSWHNTTFFGVFQAQWGDMYLSAICEYQLEEIQRVFEGPYKEYHEEAQKWDRYTDPVPSP
RPGSCINNWHRRHGYTSSLELPDNILNFVKKHPLMEEQVGPRWSRPLLVKKGTNFTH-
LVADRVTGLDGATYTV LFIGTGQAWLLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQ-
SQKLLFAGSRSQLVQLPVADCMKYRSCADCVL ARDPYCAWSVNTSRCVAVGGHSGSF-
LIQHVMTSDTSGICNLRGSKKVRPTPKNITVVAGTDLVLPCNLSSNLA
HARWTFGGRDLPAEQPGSFLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVVAGPSVTLEARA
PLENLGLVWLAAAALGAVCLVLLLLVLSLRRRLREELEKGAKATERTLVYPLELPAA-
PTSPPFRPCPEPDEKL WDPVGYYYSDGSLKIVPGHARCQPGGGPPSPPPGIPGQPLP-
SPTRLHLGGGRNSNANGYVRLQLGGEDRGGLG HPLPELADELRRKLQQRQPLPDSNP-
EESSV
[0359] A ClustalW comparison of the above protein sequences yields
the following sequence alignment shown in Table 1B.
3TABLE 1B Comparison of the NOV1 protein sequences. NOV1a
--------------------AEVWWNLVPRKTVSSGELATVVRR- FSQTGIQDFLTLTLTE (SEQ
ID NO: 2) NOV1b MAPHWAVWLLAARLWGLGIGAEVWWNLV-
PRKTVSSGELATVVRRFSQTGIQDFLTLTLTE (SEQ ID NO: 4) NOV1c
MAPHWAVWLLAARLWGLGIGAEVWWNLVPRKTVSSGELATVVRRFSQTGIQDFLTLTLTE (SEQ
ID NO: 6) NOV1d
------------------RSAEVWWNLVPRKTVSSGELATVVRRFSQTGIQDFLTLT- LTE (SEQ
ID NO: 8) NOV1e ------------------RSAEVWWNLVPRKTVSSGELATVV-
RRFSQTGIQDFLTLTLTE (SEQ ID NO: 10) NOV1f
MAPHWAVWLLAARLWGLGIGAEVWWN- LVPRKTVSSGELATVVRRFSQTGIQDFLTLTLTE (SEQ
ID NO: 12) NOV1a
PTGLLYVGAREALFAFSMEALELQGAISWEAPVEEKTECIQKGKNNQTECFNFIRFLQPY NOV1b
PTGLLYVGAREALFAFSMEALELQGAISWEAPVEEKTECIQKGKNNQTECFNFIRFLQPY NOV1c
PTGLLYVGARDHASALGVPVLLLQAVISWEAPVEKKTECIQKGKNNQTECFNFIRFLQPY NOV1d
PTGLLYVGAREALFAFSMEALELQGAISWEAPVEEKTECIQKGKNNQTECFNFIRFLQPY NOV1e
PTGLLYVGAREALFAFSMEALELQGAISWEAPVEKKTECIQKGKNSQTECFNFIRFLQPY NOV1f
PTGLLYVGAREALFAFSVEALELQGAISWEAPVEKKTECIQKGKNNQTECFNFIRFLQPY NOV1a
NASHLYVCGTYAFQPKCTYVNMLTFTLEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSAT NOV1b
NASHLYVCGTYAFQPKCTYVNMLTFTLEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSAT NOV1c
NASHLYVCGTYAFQPKCTYVNMLTFTLEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSAT NOV1d
NASHLYVCGTYAFQPKCTYVNMLTFTLEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSAT NOV1e
NASHLYVCGTYAFQPKCTYVNMLTFTLEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSAT NOV1f
NASHLYVCGTYAFQPKCTYVNMLTFTLEHGEFEDGKGKCPYDPAKGHAGLLVDGELYSAT NOV1a
LNNFLGTEPIILRNMGPHHSMKTEYLAFWLNEPHFVGSAYVPESVGSFTGDDDKV- YFFFR
NOV1b LNNFLGTEPIILRNMGPHHSMKTEYLAFWLNEPHFVCSAYVPESVGSFTGDDDKV-
YFFFR NOV1c
LNNFLGTEPIILRNMGPHHSMKTEYLAFWLNEPHFVGSAYVPESVGSFTGDDDKV- YFFFR
NOV1d LNNFLGTEPIILRNMGPNHSMKTEYLAFWLNEPHFVGSAYVPESVGSFTGDDDKV-
YFFFR NOV1e
LNNFLGTEPITLRNMGPHHSMKTEYLAFWLNEPHFVGSAYVPESVGSFTGDDDKV- YFFFR
NOV1f LNNFLGTEPIILRNMGPHHSMKTEYLAFWLNEPHFVGSAYVPESVGSFTGDDDKV-
YFLFR NOV1a ERAVESDCYAEQVVARVARVCKGDMGGARTLQRKWTTFLKARLACS-
APNWQLYFNQLQAM NOV1b
ERAVESDCYAEQVVARVARVCKGDMGGARTLQRKWTTFLKARLACS- APNWQLYFNQLOAM
NOV1c ERAVESDCYAEQVVARVARVCKGDMGGARTLQRKWTTFLKARLACS-
APNWQLYFNQLQAM NOV1d
ERAVESDCYAEQVVARVARVCKGDMGGRTLORKWTTFLKARACSAP- NWQLYFNQLQLQAM
NOV1e ERAVESDCYAEQVVARVARVCKGDMGCARTLQRKWTTFLKARLACS-
APNWQLYFNQLQAM NOV1f
ERAVESACYAEQVVARVARVCKGDMGGARTLQRKWTTFLKARLACS- APNWQLYFNQLQAA
NOV1a NTLQDTSWHNTTFFGVFQAQWGDMYLSAICEYQLEEI-
QRVFEGPYKEYHEEAQKWDRYTD NOV1b
HTLQDTSWHNTTFFGVFQAQWODMYLSAICEYQLEEI- QRVFEGPYKEYHEEAQKWDRYTD
NOV1c HTLQDTSWHNTTFFGVFQAQWGDMYLSAICEYQLEEI-
QRVFEGPYKEYHEEAQKWDRYTD NOV1d
HTLQDTSWHNTTFFGVFQAQWGDMYLSAICEYQLEEI- QRVFEGPYKEYMEEAQKWDRYTD
NOV1e HTLQDTSWMNTTFFGVFQAQWGDMYLSAICEYQLEEI-
ORVFEGPYKEYHEEAQKWDRYTD NOV1f
HTLQDTSWHNTTFFGVFQAQWGDMYLSAICEYQLEEI- QRVFEGPYKEYHEEAQKWDRYTD
NOV1a PVPSPRPGSCINNWHRRHGYTSSLELPD-
NILNFVKKHPLMEEQVGPRWSRPLLVKKGTNF NOV1b
PVPSPRPGSCINNWHRRHGYTSSLELPD- NILNFVKKHPLMEEQVGPRWSRPLLVKKGTNF
NOV1c PVPSPRPGSCINNWHRRHGYTSSLELPD-
NILNFVKKHPLMEEQVGPRWSRPLLVKKGTNF NQV1d
PVPSPRPGSCINNWHRRHGYTSSLELPD- NILNFVKKHPLMEEQVGPRWSRPLLVKKGTNF
NOV1e PVPSPRPGSCINNWHRRHGYTSSLELPD-
NILNFVKKHPLMEEQVGPRWSRPLLVKKGTNF NOV1f
PVPSPRPGSCINNWHRRHGYTSSLELPD- NILNFVKKNPLMEEQVGPRWSRPLLVKKGTNF
NOV1a THLVADRVTGLDGATYTVLFIGTGDGWLLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQS
NOV1b THLVADRVTGLDGATYTVLFIGTGDGWQLKAVSLGPWVHLIEELQLFDQEPMRSLAASQS
NOV1c TMLVADRVTGLDGATYTVLFIGTGDGWLLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQS
NOV1d THLVADRVTGLDGATYTVLFIGTGDGWLLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQS
NOV1e THLVADRVTGLDGATYTVLFIGTGDGWLLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQS
NOV1f THLVADRVTGLDCATYTVLFIGTGQAWLLKAVSLGPWVHLIEELQLFDQEPMRSLVLSQS
NOV1a KKLLFAGSRSQLVQLPVADCMKYRSCADCVLARDPYCAWSVNTSRCVAVGGHSGSLLIQH
NOV1b -KTLQAVLWFWLWPLP--------------------------------------------
NOV1c KKLLFAGSRSQLVQLPVADCMKYRSCADCVLARDPYCAWSVNTSRCVAVGGHSGSLLIQH
NOV1d KKLLFAGSRSQLVQLPVADCMKYRSCADCVLARDPYCAWSVNTSRCVAVGGHSGSLLIQN
NOV1e KKILFAGSRSQLVQLPVADCMKYRSCADCVLARDPYCAWSVNTSRCVAVGGHSGSLLIQH
NOV1f QKLLFAGSRSQLVQLPVADCMKYRSCADCVLARDPYCAWSVNTSRCVAVGGHSGSFLIQH
NOV1a VMTSDTSGICNLRGSKKVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDL-
PAEQP NOV1b
-------------------------------------------------------- -----
NOV1c VMTSDTSGICNLRGSKKVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDL-
PAEQP NOV1d
VMTSDTSGICNLRGSKKVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDL- PAEQP
NOV1e VMTSDTSGICNLRGSKKVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDL-
PAEQP NOV1f
VMTSDTSGICNLRGSKKVRPTPKNITVVAGTDLVLPCHLSSNLAHARWTFGGRDL- PAEQP
NOV1a GSFLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVV-
AGPSVTLE------ NOV1b
----------------------------------------------- --------------
NOV1c GSFLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVV-
AGPSVTLEARAPLE NOV1d
GSFLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVV- AGPSVTLELE----
NOV1e GSFLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVV-
AGPSVTLELE---- NOV1f
GSFLYDARLQALVVMAAQPRHAGAYHCFSEEQGARLAAEGYLVAVV- AGPSVTLEARAPLE
NOV1a --------------------------------------
----------------------- NOV1b
-------------------------------------- -----------------------
NOV1c NLGLVWLAVVALGAVCLVLLLLVLSLRRRLREELEKG-
AKATERTLVYPLELPKEPTSPPF NOV1d
-------------------------------------- -----------------------
NOV1e --------------------------------------
----------------------- NOV1f
NLGLVWLAVVALGAVCLVLLLLVLSLRRRLREELEKG- AKATERTLVYPLELPKEPTSPPF
NOV1a -----------------------------
-------------------------------- NOV1b
----------------------------- --------------------------------
NOV1c RPCPEPDEKLWDPVGYYYSDGSLKIVPG-
HARCQPAAGPPSPPPGIPGQPLPSPTRLHLGG NOV1d
----------------------------- --------------------------------
NOV1e -----------------------------
-------------------------------- NOV1f
RPCPEPDEKLWDPVGYYYSDGSLKIVPG- HARCQPGGGPPSPPPGIPGQPLPSPTRLHLGG
NOV1a ----------------------------------------------------- NOV1b
----------------------------------------------------- NOV1c
GRNSNANGYVRLQLGGEDRGGLGHPLPELADELRRAAQQRQPLPDSNPEESSV NOV1d
----------------------------------------------------- NOV1e
----------------------------------------------------- NOV1f
GRNSNANGYVRLQLGGEDRGGLGHPLPELADELRRAAQQRQPLPDSNPEESSV
[0360] Further analysis of the NOV1a protein yielded the following
properties shown in Table 1C.
4TABLE 1C Protein Sequence Properties NOV1a SignalP analysis: No
Known Signal Sequence Predicted PSORT II analysis: PSG: a new
signal peptide prediction method N-region: length 11; pos.chg 2;
neg.chg 1 H-region: length 5; peak value 1.15 PSG score: -3.25 GvH:
von Heijne's method for signal seq. recognition GvH score
(threshold: -2.1): -7.49 possible cleavage site: between 55 and 56
>>> Seems to have no N-terminal signal peptide ALOM: Klein
et al's method for TM region allocation Init position for
calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) . . . fixed PERIPHERAL Likelihood = 2.12 (at 616)
ALOM score: 2.12 (number of TMSs: 0) MITDISC: discrimination of
mitochondrial targeting seq R content: 1 Hyd Moment(75): 6.63 Hyd
Moment(95): 9.39 G content: 1 D/E content: 2 S/T content: 3 Score:
-5.25 Gavel: prediction of cleavage sites for mitochondrial preseq
R-10 motif at 33 VRR FS NUCDISC: discrimination of nuclear
localization signals pat4: none pat7: none bipartite: none content
of basic residues: 9.3% NLS Score: -0.47 KDEL: ER retention motif
in the C-terminus: none ER Membrane Retention Signals: none SKL:
peroxisomal targeting signal in the C-terminus: none PTS2: 2nd
peroxisomal targeting signal: none VAC: possible vacuolar targeting
motif: none RNA-binding motif: none Actinin-type actin-binding
motif: type 1: none type 2: none NMYR: N-myristoylation pattern:
none Prenylation motif: none memYQRL: transport motif from cell
surface to Golgi: none Tyrosines in the tail: none Dileucine motif
in the tail: none checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none checking 33
PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's
method for Cytoplasmic/Nuclear discrimination Prediction:
cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect
coiled-coil regions total: 0 residues Final Results (k = 9/23):
52.2%: cytoplasmic 26.1%: nuclear 21.7%: mitochondrial >>
prediction for CG50907-03 is cyt (k = 23)
[0361] A search of the NOV1a 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.
5TABLE 1D Geneseq Results for NOV1a NOV1a Identities/ Residues/
Similarities for Geneseq Protein/Organism/Length [Patent #, Match
the Matched Expect Identifier Date] Residues Region Value AAE03640
Human extracellular matrix and cell 1 . . . 634 631/634 (99%) 0.0
adhesion molecule-4 (XMAD-4) - 21 . . . 654 634/634 (99%) Homo
sapiens, 833 aa. [WO200142285-A2, 14-JUN-2001] AAE18213 Human MOL5a
protein - Homo 1 . . . 634 627/634 (98%) 0.0 sapiens, 833 aa.
[WO200206339-A2, 21 . . . 654 630/634 (98%) 24-JAN-2002] AAE18215
Human MOL5c protein - Homo 1 . . . 634 621/634 (97%) 0.0 sapiens,
833 aa. [WO200206339-A2, 21 . . . 654 624/634 (97%) 24-JAN-2002]
AAE18214 Human MOL5b protein - Homo 1 . . . 634 621/634 (97%) 0.0
sapiens, 833 aa. [WO200206339-A2, 21 . . . 654 624/634 (97%)
24-JAN-2002] ABB97963 Human protein sequence #30 - Homo 13 . . .
634 617/622 (99%) 0.0 sapiens, 886 aa. [WO200252005-A1, 86 . . .
707 620/622 (99%) 04-JUL-2002]
[0362] In a BLAST search of public sequence databases, the NOV1a
protein was found to have homology to the proteins shown in the
BLASTP data in Table 1E.
6TABLE 1E Public BLASTP Results for NOV1a NOV1a Identities/ Protein
Residues/ Similarities for Accession Match the Matched Expect
Number Protein/Organism/Length Residues Portion Value Q9C0C4
Hypothetical protein KIAA1739 - 13 . . . 634 617/622 (99%) 0.0 Homo
sapiens (Human), 963 aa 163 . . . 784 620/622 (99%) (fragment).
CAC24876 Sequence 21 from Patent WO0078802 - 1 . . . 561 559/561
(99%) 0.0 Homo sapiens (Human), 624 aa. 21 . . . 581 561/561 (99%)
CAC24871 Sequence 11 from Patent WO0078802 - 1 . . . 561 558/561
(99%) 0.0 Homo sapiens (Human), 590 aa. 21 . . . 581 560/561 (99%)
CAC24872 Sequence 13 from Patent WO0078802 - 1 . . . 564 557/565
(98%) 0.0 Homo sapiens (Human), 596 aa. 21 . . . 585 560/565 (98%)
Q64151 Semaphorin 4C precursor (Semaphorin 1 . . . 634 538/635
(84%) 0.0 I) (Sema I) (Semaphorin C-like 1) 21 . . . 655 581/635
(90%) (M-Sema F) - Mus musculus (Mouse), 834 aa.
[0363] PFam analysis predicts that the NOV1a protein contains the
domains shown in the Table 1F.
7TABLE 1F Domain Analysis of NOV1a Identities/Similarities NOV1a
for the Pfam Domain Match Region Matched Region Expect Value Sema
33 . . . 461 206/497 (41%) 1.6e-201 373/497 (75%) PSI 479 . . . 531
13/67 (19%) 0.001 37/67 (55%)
Example 2
[0364] The NOV2 clone was analyzed, and the nucleotide and encoded
polypeptide sequences are shown in Table 2A.
8TABLE 2A NOV2 Sequence Analysis NOV2a,CG51896-04 SEQ ID NO:13 4250
bp DNA Sequence ORF Start: ATG at 250 ORF Stop: end of sequence
GAACACATCGCGTTTGCATCCCAGAAAGT-
AGTCGCCGCGACTATTTCCCCCAAAGAGACAAGCACACATGTAG
GAATGACAAAGGCTTGCGAAGGAGACACCGCACCCCGCGGCCCGGAGAGATCCCCTCCATAATCCATTACTAA
ATGGGATACACGCTGTACCAGTTCGCTCCGAGCCCCGGCCGCCTGTCCGTCGATGCA-
CCGAAAAGGGTGAAGT AGAGAAATAAAGTCTCCCCGCTGAACTACTATGAGGTCAGA-
AGCCTTGCTGCTATATTTCACACTGCTACACT TTGCTGGGGCTCGTTTCCCAGAAGA-
TTCTGAGCCAATCAGTATTTCGCATTGCAACTATACAAAACAGTATCC
GGTGTTTGTGGGCCACAAGCCAGGACGGAACACCACACAGAGGCACACGCTGGACATCCAGATGATTATGATC
ATGAACGGAACCCTCTACATTGCTGCTAGGGACCATATTTATACTGTTGATATAGAC-
ACATCACACACGGAAG AAATTTATTGTAGCAAAAAACTGACATGGAAATCTAGACAG-
GCCGATGTAGACACATGCAGAATGAAGGGAAA ACATAAGGATCAGTGCCACAACTTT-
ATTAAAGTTCTTCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGA
ACTAATGCCTTCAACCCTTCCTGCAGAAACTATAAGATGGATACATTGGAACCATTCGGGGATGAATTCAGCG
GAATGCCCAGATGCCCATATGATCCCAAACATGCCAACGTTGCACTGTTTGCAGATG-
GAAAACTATACTCAGC CACAGTGACTGACTTCCTTGCCATTGACGCAGTCATTTACC-
GGAGTCTTGGAGAAAGCCCTACCCTGCGGACC GTCAAGCACGATTCAAAATGGTTGA-
AAGAACCATACTTTGTTCAAGCCGTGGATTACGGAGATTATATCTACT
TCTTCTTCAGGGAAATAGCAGTGGAGTATAACACCATGGGAAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTG
TAAGAATGATATGGGAGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCT-
GAAGGCGCGCTTGAAC TGCTCAGTTCCTGGAGACTCTCATTTTTATTTCAACATTCT-
CCAGGCAGTTACAGATGTGATTCGTATCAACG GGCGTGATGTTGTCCTGGCAACGTT-
TTCTACACCTTATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGA
CATGCTTGACATTGCCAGTGTTTTTACTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCA
GTTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCC-
TTAGAAAGATATGCAA CCTCCAATGAGTTCCCTGATGATACCCTGAACTTCATCAAG-
ACGCACCCGCTCATGGATGAGGCAGTGCCCTC CATCTTCAACAGGCCATGGTTCCTG-
AGAACAATGGTCAGATACCGCCTTACCAAAATTGCAGTGGACACAGCT
GCTGGGCCATATCAGAATCACACTGTGGTTTTTCTGGGATCAGAGAAGGGAATCATCTTGAAGTTTTTGGCCA
GAATAGGAAATAGTGGTTTTCTAAATGACAGCCTTTTCCTGGAGGAGATGAGTGTTT-
ACAACTCTGAAAAATG CAGCTATGATGGAGTCCAAGACAAAAGGATCATGGGCATGC-
AGCTCCACAGAGCAAGCAGCTCTCTGTATGTT GCGTTCTCTACCTGTGTGATAAAGG-
TTCCCCTTGGCCGGTGTGAACGACATGGGAAGTGTAAAAAAACCTGTA
TTGCCTCCAGAGACCCATATTGTGGATGGATAAAGGAAGGTGGTGCCTGCAGCCATTTATCACCCAACAGCAG
ACTGACTTTTGAGCAGGACATAGAGCGTGGCAATACAGATGGTCTGGGGGACTGTCA-
CAATTCCTTTGTGGCA CTGAATGACATTTCAACTCCTCTACCAGATAATGAAATGTC-
TTACAACACAGTGTATGGGCATTCCAGTTCCC TCTTGCCCAGCACAACCACATCAGA-
TTCGACGGCTCAAGAGGGGTATGAGTCTAGGGGAGGAATGCTGGACTG
GAAGCATCTGCTTGACTCACCTGACAGCACAGACCCTTTGGGGGCAGTGTCTTCCCATAATCACCAAGACAAG
AAGGGAGTGATTCGGGAAAGTTACCTCAAAGGCCACGACCAGCTGGTTCCCGTCACC-
CTCTTGGCCATTGCAG TCATCCTGGCTTTCGTCATGGGGCCGTCTTCTCGGGCATCA-
CCGTCTACTGCGTCTGTGATCATCAAGCGCAA AGACGTGGCTGTGGTGCAGCGCAAG-
GAGAAGGAGCTCACCCACTCGCGCCGGGGCTCCATGAGCAGCGTCACC
AAGCTCAGCGGCCTCTTTGGGGACACTCAATCCAAAGACCCAAAGCCGGAGGCCATCCTCACGCCACTCATGC
ACAACGGCAAGCTCGCCACTCCCGGCAACACGGCCAAGATGCTCATTAAAGCAGACC-
AGCACCACCTGGACCT GACGGCCCTCCCCACCCCAGAGTCAACCCCAACGCTGCAGC-
AGAAGCGGAAGCCCAGCCGCGGCAGCCGCGAG TGGGAGAGGAACCAGAACCTCATCA-
ATGCCTGCACAAAGGACATGCCCCCCATGGGCTCCCCTGTGATTCCCA
CGGACCTGCCCCTGCGGGCCTCCCCCAGCCACATCCCCAGCGTGGTGGTCCTGCCCATCACGCAGCAGGGCTA
CCAGCATGAGTACGTGGACCAGCCCAAAATGAGCGAGGTGGCCCAGATGGCGCTGGA-
GGACCAGGCCGCCACA CTGGAGTATAAGACCATCAAGGAACATCTCAGCAGCAAGAG-
TCCCAACCATGGGGTGAACCTTGTGGAGAACC TGGACAGCCTGCCCCCCAAAGTTCC-
ACAGCGGGAGGCCTCCCTGCGTCCCCCGGGAGCCTCCCTGTCTCAGAC
CGGTCTAACCAAGCGGCTGGAAATGCACCACTCCTCTTCCTACGGGGTTGACTATAAGAGGAGCTACCCCACG
AACTCGCTCACGAGAAGCCACCAGGCCACCACTCTCAAAAGAAACAACACTAACTCC-
TCCAATTCCTCTCACC TCTCCAGAAACCAGAGCTTTGGCAGGGGAGACAACCCGCCG-
CCCGCCCCGCAGAGGGTGGACTCCATCCAGGT GCACAGCTCCCAGCCATCTGGCCAG-
GCCGTGACTGTCTCGAGGCAGCCCAGCCTCAACGCCTACAACTCACTG
ACAAGGTCGGGGCTGAAGCGTACGCCCTCGCTAAAGCCGGACGTACCCCCCAAACCATCCTTTGCTCCCCTTT
CCACATCCATGAAGCCCAATGATGCGTGTACATAATCCCAGGGGGAGGGGGTCAGGT-
GTCGAACCAGCAGGCA AGGCGAGGTGCCCGCTCAGCTCAGCAAGGTTCTCAACTGCC-
TCGAGTACCCACCAGACCAAGAAGGCCTGCGG CAGAGCCGAGGACGCTGGGTCCTCC-
TCTCTGGGACACAGGGGTACTCACGAAAACTGGGCCGCGTGGTTTGGT
CAAGGTTTGCAACGGCGGGGACTCACCTTCATTCTCTTCCTTCACTTTCCCCCACACCCTACAACAGGTCGGA
CCCACAAAAGACTTCAGTTATCATCACAAACATGAGCCAAAAGCACATACCTACCCC-
ATCCCCCACCCCCACA CACACACACACATGCACACAACACATACACACACACGCACA-
GAGGTGAACAGAAACTGAAACATTTTGTCCAC AACTTCACGGGACGTGGCCAGACTG-
GGTTTGCGTTCCAACCTGCAAACACAAATACATTTTTTTTTAATCAAG
AAAATTTAAAAAGACAAAAAAAAAAGAATTCATTGATAATTCTAACTCAGACTTTAACAATGGCAGAAGTTTA
CTATGCGCAAATACTGTGAAATGCCCGCCAGTGTTACAGCTTTCTGTTGCAGCAGAT-
AAATGCCATGTTGGGC AACTATGTCATAGATTTCTGCTCCTCCTCTCTTTTAATGAA-
ATAACGTGACCGTTAACGCAAGTAACTCTTTA TTTATTGTTCACCCTTTTTTTCCTT-
AAGGAAAGGACTCTTCCAAATATCATCCTATGAACAGCTCTTCAGAAA
GCCCATTGAAAGTTAAACTATTTAACGTGAAATCCATTAACTGGAATAATTGAGTTTCTTTATTTTTACAATA
AATTCACTGAGTAAAT NOV2a, CG5 1896-04 Protein Sequence SEQ ID NO:14
1047 aa MW at 116354.6 kD
MRSEALLLYFTLLHFAGAGFPEDSEPISISHCNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAAA
DHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLLKKND-
DALFVCGTNAFNPSCRN YKMDTLEPFGDEFSGMARCPYDAKHANVALFADGKLYSAT-
VTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKE PYFVQAVDYGDYIYFFFREIAVEY-
NTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHFY
FNILQAVTDVIRINGRDVVLATFSTPYNSIPGSAVCAYDMALDIASVFTGRFKEQKSPDSTWPVPDERVPKPR
PGCCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTK-
IAVDTAAGPYQNHTVV FLGSEKGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSY-
DGVEDKRIMGMQLDRASSSLYVAFSTCVIKVP LGRCERHGKCKKTCIASRDPYCGWI-
KEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHNSFVALNDISTPLPD
NEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGCMLDWKHLLDSPDSTDPLGAVSSHAAQDKKGVIRESYLK
GHDQLVPVTLLAIAVILAFVKMGAVFSGITVYCVCDHRRKDVAVVQRKEKELTHSRG-
SMSSVTKLSGLFGDTQ SKDPKPEAILTPLMHNGKLATPGNTAKMLIKADQHHLDLTA-
LPTPESTPTLQQKRKPSRGSREWERNQNLINA CTKDMPPMGSPVIPTDLPLRASPSH-
IPSVVVLPITQQGYQHEYVDQPKMSEVAQMAAEDQAATLEYKTIAAHL
SSKSPNHGVNLVENLDSLPPKVPQREASLGPPGASLSQTGLSAALEAAHSSSYGAAYAASYPTNSLTRSHQAT
TLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRVDSIQVHSSQPSGQAVTVSRQPSL-
NAYNSLTRSGLKRTPS LKPDVPPKPSFAPLSTSMKPNDACT NOV2b, 271674560 SEQ ID
NO:15 1921 bp DNA Sequence ORF Start: at 1 ORF Stop: end of
sequence GCCGGATCCAGTATTTCGCATTGCAACTATACAAAACA-
GTATCCGGTGTTTGTGGGCCACAAGCCAGGACGGA
ACACCACACAGAGGCACAGGCTGGACATCCAGATGATTATGATCATGAACGGAACCCTCTACATTGCTGCTAG
GGACCATATTTATACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAG-
CAAAAAACTGACATGG AAATCTAGACAGGCCGATGTAGACACATGCAGAATGAAGGG-
AAAACATAAGGATGAGTGCCACAACTTTATTA AAGTTCTTCTAAAGAAAAACGATGA-
TGCATTGTTTGTCTGTGGAACTAATGCCTTCAACCCTTCCTGCAGAAA
CTATAAGATGGATACATTGGAACCATTCGGGGATGAATTCAGCGGAATGGCCAGATGCCCATATGATGCCAAA
CATGCCAACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGAAAGAC-
TTCCTTGCCATTGACG CAGTCATTTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGG-
ACCGTCAAGCACGATTCAAAATGGTTGAAAGA ACCATACTTTGTTCAAGCCGTGGAT-
TACGGAGATTATATCTACTTCTTCTTCAGGGAAATAGCAGTGGAGTAT
AACACCATGGGAAAGGTAGTTTTTCCCAAGAGTGGCTCAGGTTTGTAAGAATGATATGGAGGATCTCAAAGAG
TCCTGGAGAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTG-
GAGACTCTAATTTTTA TTTCAACATTCTCCAGGCAGTTACAGATGTGATTCGTATCA-
ACGGGCGTGATGTTGTCCTGGCAACGTTTTCT ACACCTTATAACAGCATCCCTGGGT-
CTGCAGTCTGTGCCTATGACATGCTTGACATTGCCAGTGTTTTTACTG
GGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCAGTTCCTGATGAACGAGTTCCTAAGCCCAG
GCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTT-
CCCTGATGATACCCTG AACTTCATCAAGACGCACCCGCTCATGGATGAGGCAGTGCC-
CTCCATCTTCAACAGCCCATAATTCCTGAGAA CAATGGTCAGATACCGCCTTACCAA-
AATTGCAGTGGACACAGCTGCTGGGCCATATCAGAATAACACTGTGGT
TTTTCTGGGATCACAGAAGGGAATCATCTTGAAGTTTTTGCCCAGAATAGGAAATAGTGGTTTTCTAAATGAC
AGCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGA-
GTCGAAGACAAAAGGA TCATGGGCATGCAGCTGGACAGAGCAAGCAGCTCTCTGTAT-
GTTGCGTTCTCTACCTGTGTGATAAAGGTTCC CCTTGGCCGGTGTGAACGACATGGG-
AAGTGTAAAAAAACCTGTATTGCCTCCAGAGACCCGTATTGTAAATGG
ATAAAGGAAGGTGGTGCCTGCAGCCATTTATCACCCAACAGCAGACTGACTTTTGAGCAGGGACATAGAGCTG
GCAATACAGATGGTCTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATGACATTT-
CAACTCCTCTACCAGA TAATGAAATGTCTTACAACACAGTGTATGGGCATTCCAGTT-
CCCTCTTGCCCAGCACAACCACATCAGATTCG ACGGCTCAAGAGGGGTATGAGTCTA-
GGGGAGGAATGCTGGACTGGAAGCATCTGCTTGACTCACCTGACAGCA
CAGACCCTTTCCCCGCAGTGTCTTCCCATAATCACCAAGACAAGAAGGGAGTcATTCGGGAAAGTTACCTCAA
AGGCCACGACCACGTCGACGGTG NOV2b, 271674560 Protein Sequence SEQ ID
NO:16 640 aa MW at 71799.4 kD
AGSSISHCNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAARDHIYTVDIDTSHTEEIYCSKKLTW
KSRQADVDTCRMKGKHKDECHNFIKVLLKKNDDALFVCGTNAFNPSCRAYKMDTLE-
PFGDEFSGMARCPYDAK HANVALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTV-
KHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEY NTMGKVVFPRVAQVCKNDMGGSQR-
VLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFS
TPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCACSSSLERYATSNEFPDDTL
NFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAAGPYQNHTVVFLGSEKGI-
ILKFLARIGNSGFLND SLFLEEMSVYNSEKCSYDGVEDKRIMGMQLDRASSSLYVAF-
STCVIKVPLGRCERHGKCKKTCIASRDPYCGW IKEGGACSHLSPNSRLTFEQDIERG-
NTDGLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDS
TAQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQVDG NOV2c,
267441133 SEQ ID NO:17 3106 bp DNA Sequence ORF Start: at 2 ORF
Stop: end of sequence CACCGGATCCGGTTTCCCAGAAGATTCTCAGCCAATCA-
GTATTTCGCATGGCAACTATACAAAACAGTATCCG
GTGTTTGTGGGCCACAAGCCAGGACGGAACACCACACAGAGGCACAGGCTGGACATCCAGATGATTATGATCA
TGAACGGAACCCTCTACATTGCTGCTAGGGACCATATTTATACTGTTGATATAGACA-
CATCACACACGGAAGA AATTTATTGTAGCAAAAAACTGACATGGAAATCTAGACAGG-
CCGATGTAGACACATGCAGAATGAAGGGAAAA CATAAGGATCAGTGCCACAACTTTA-
TTAAAGTTCTTCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAA
CTAATGCCTTCAACCCTTCCTGCAGAAACTATAAGATGGATACATTGGAACCATTCGGGGATGAATTCAGCGG
AATGGCCAGATGCCCATATGATGCCAAACATGCCAACGTTGCACTGTTTGCAGATGG-
AAAACTATACTCAGCC ACAGTGACTGACTTCCTTGCCATTGACGCAGTCATTTACCG-
GAGTCTTGGAGAAAGCCCTACCCTGCGGACCG TCAAGCACGATTCAAAATGGTTGAA-
AGAACCATACTTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTT
CTTCTTCAGGGAAATAGCAGTGGAGTATAACACCATGGGAAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGT
AAGAATGATATGGGAGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCTG-
AAGGCGCGCTTGAACT GCTCAGTTCCTGGAGACTCTCATTTTTATTTCAACATTCTC-
CAGGCAGTTACAGATGTGATTCGTATCAACGG GCGTGATGTTGTCCTGGCAACGTTT-
TCTACACCTTATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGAC
ATGCTTGACATTGCCAGTGTTTTTACTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCAG
TTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCCT-
TAGAAAGATATGCAAC CTCCAATGAGTTCCCTGATGATACCCTGAACTTCATCAAGA-
CGCACCCGCTCATGGATGAGGCAGTGCCCTCC ATCTTCAACAGGCCATGGTTCCTGA-
GAACAATGGTCAGATACCGCCTTACCAAAATTGCAGTGGACACAGCTG
CTGGGCCATATCAGAATCACACTGTGGTTTTTCTGGGATCAGACAAGGGAATCATCTTGAAGTTTTTGGCCAG
AATAGGAAATAGTGGTTTTCTAAATGACAGCCTTTTCCTGGAGGAGATGAGTGTTTA-
CAACTCTGAAAAATGC AGCTATGATGGAGTCGAAGACAAAAGGATCATGGGCATGCA-
GCTGGACAGAGCAAGCAGCTCTCTGTATGTTG CGTTCTCTACCTGTGTGATAAAGGT-
TCCCCTTGGCCGGTGTGAACGACATGGGAAGTGTAAAAAAACCTGTAT
TGCCTCCAGAGACCCGTATTGTGGATGGATAAAGGAAGGTGGTGCCTGCAGCCATTTATCACCCAACAGCAGA
CTGACTTTTGAGCAGGACATAGAGCGTGGCAATACAGATGGTCTGGGGGACTGTCAC-
AATTCCTTTGTGGCAC TGAATGACATTTCAACTCCTCTACCAGATAATGAAATGTCT-
TACAACACAGTGTATGGGCATTCCAGTTCCCT CTTGCCCAGCACAACCACATCAGAT-
TCGACGGCTCAAGAGGGGTATGAGTCTAGGGGAGGAATGCTGGACTGG
AAGCATCTGCTTGACTCACCTGACAGCACAGACCCTTTGGGGGCAGTGTCTTCCCATAATCACCAAGACAAGA
AGGGAGTGATTCGGGAAAGTTACCTCAAAGGCCACGACCAGCTGGTTCCCGTCACCC-
TCTTGGCCATTGCAGT CATCCTGGCTTTCGTCATGGGGGCCGTCTTCTCGGGCATCA-
CCGTCTACTGCGTCTGTGATCATCGGCGCAAA GACGTGGCTGTGGTGCAGCGCAAGG-
AGAAGGAGCTCACCCACTCGCGCCGGGGCTCCATGAGCAGCGTCACCA
AGCTCAGCGGCCTCTTTGGGGACACTCAATCCAAAGACCCAAAGCCGGAGGCCATCCTCACGCCACTCATGCA
CAACGGCAAGCTCGCCACTCCCGGCAACACGGCCAAGATGCTCATTAAAGCAGACCA-
GCACCACCTGGACCTG ACGGCCCTCCCCACCCCAGAGTCAACCCCAACGCTGCAGCA-
GAAGCGGAAGCCCAGCCGCGGCAGCCGCGAGT GGGAGAGGAACCAGAACCTCATCAA-
TGCCTGCACAAAGGACATGCCCCCCATGGGCTCCCCTGTGATTCCCAC
GGACCTGCCCCTGCCGGCCTCCCCCAGCCACATCCCCAGCGTGGTGGTCCTGCCCATCACGCAGCAGGGCTAC
CAGCATGAGTACGTGGACCAGCCCAAAATGAGCGAGGTGGCCCAGATGGCGCTGGAG-
GACCAGGCCGCCACAC TGGAGTATAACACCATCAAGGAACATCTCAGCAGCAAGAGT-
CCCAACCATGGGGTGAACCTTGTGGAGAACCT GGACAGCCTGCCCCCCAAAGTTCCA-
CAGCGGGAGGCCTCCCTGGGTCCCCCGGGAGCCTCCCTGTCTCAGACC
GGTCTAAGCAAGCGGCTGGAAATGCACCACTCCTCTTCCTACGGGGTTGACTATAAGAGGAGCTACCCCACGA
ACTCGCTCACGAGAAGCCACCAGGCCACCACTCTCAAAAGAAACAACACTAACTCCT-
CCAATTCCTCTCACCT CTCCAGAAACCACAGCTTTGGCAGGGGAGACAACCCGCCGC-
CCGCCCCGCAGAGGGTGGACTCCATCCAGGTG CACAGCTCCCAGCCATCTGGCCAGG-
CCGTGACTGTCTCGAGGCAGCCCAGCCTCAACCCCTACAACTCACTGA
CAAGGTCGGGGCTGAAGCGTACGCCCTCGCTAAAGCCGGACGTACCCCCCAAACCATCCTTTGCTCCCCTTTC
CACATCCATGAAGCCCAATCATGCGTGTACAGTCGACGGC NOV2c, 267441133 Protein
Sequence SEQ ID NO:18 1035 aa MW at 114789.6 kD
TGSGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY-
IAARDHIYTVDIDTSHTEE IYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLL-
KKNDDALFVCGTNAFNPSCRNYKMDTLEPFGDEFSG
MARCPYDAKHANVALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKEPYFVQAVDYGDYIYF
FFREFAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSKLKARLNCSVPGDSHF-
YFNILQAVTDVIRING RDVVLATFSTPYISIPGSAVCAYDMLDIASVFTGRFKEQKS-
PDSTWTPVPDERVPKPRPGCCAGSSSLERYAT SNEFPDDTLNFIKTHPLMDEAVPSI-
FNRPWFLRTMVRYRLTKIAVDTAAGPYQNHTVVFLGSEKGIILKFLAR
IGNSGFLNDSLFLEEMSVYNSEKCSYDGVEDKRIMGMQLDRASSSLYVAFSTCVIKVPLGRCERHGKCKKTCI
ASRDPYCGWIKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHNSFVALNDISTPLP-
DNEMSYNTVYGHSSSL LPSTTTSDSTAQEGYESRGGMLDWKHLLDSPDSTDPLGAVS-
SHNHQDKKGVIRESYLKGHDQLVPVTLLAIAV ILAFVMGAVFSGITVYCVCDHRRKD-
VAVVQRKEKELTHSRRGSMSSVTKLSGLFDTQSKDPKPEAILTPLMHA
NGKLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQKRKPSRGSREWERNQNLINACTAAMPPMGSPVIPT
DLPLRASPSHIPSVVVLPITQQGYQHEYVDQPKMSEVAQMAAEDQAATLEYKTIKEH-
LSSKSPNHGAALAANL DSLPPKVPQREASLGPPGASLSQTGLSKRLEMHHSSSYGAA-
YAASYPTNSLTRSHQATTLAAAATNSSNSSHL SRNQSFGRGDNPPPAPQRVDSIQVH-
SSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLS TSMKPNDACTVDG
NOV2d, 267441137 SEQ ID NO:19 2995 bp DNA Sequence ORF Start: at 2
ORF Stop: end of sequence
CACCGGATCCCTGGACATCCAGATGATTATGATCATGAACGGAACCCTCTACATTCCTGCTAGGGACCATATT
TATACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAACT-
GACATGGAAATCTAGAC AGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAA-
GGATGAGTGCCACAACTTTATTAAAGTTCTTCT AAAGAAAAACGATGATGCATTGTT-
TGTCTGTGGAACTAATGCCTTCAACCCTTCCTGCAGAAACTATAAGATG
GATACATTGGAACCATTCGGGGATGAATTCAGCGGAATGGCCAGATGCCCATATGATGCCAAACATGCCAACG
TTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTGCCA-
TTGACGCAGTCATTTA CCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGC-
ACGATTCAAAATGGTTGAAAGAACCATACTTT GTTCAAGCCGTGGATTACGGAGATT-
ATATCTACTTCTTCTTCAGGGAAATAGCAGTGGAGTATAACACCATGG
GAAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGTAAGAATGATATGGGAGGATCTCAAAGAGTCCTGGAGAA
ACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTCTCA-
TTTTTATTTCAACATT CTCCAGGCAGTTACAGATGTGATTCGTATCAACGGGCGTGA-
TGTTGTCCTAACAACGTTTTCTACACCTTATA AAAGAATCCCTGGGTCTGCAGTCTG-
TGCCTATGACATGCTTGAAATTGCCAGTGTTTTTACTGGGAGATTCAA
GGAACAGAAGTCTCCTGATTCCACCTGGACACCAGTTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGTTGC
TGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGATGAT-
ACCCTGAACTTCATCA AGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATCTTC-
AACAGGCCATGGTTCCTGAGAACAATGGTCAG ATACCGCCTTACCAAAATTGCAGTG-
GACACAGCTGCTGGGCCATATCAGAATCACACTGTGGTTTTTCTGGGA
TCAGAGAAGGGAATCATCTTGAAGTTTTTGGCCAGAATAGGATAGTAATTTTCTAAAAATGACAGCCTTTTCC
TGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGAAGACA-
AAAGGATCATGGGCAT GCAGCTGGACAGAGCAAGCAGCTCTCTGTATGTTGCGTTCT-
CTACCTGTGTGATAAAGGTTCCCCTTGGCCGG TGTGAACGACATGGGAAGTGTAAAA-
AAAACCTGTATTGCCTCCAGAGACCCGTATTGTGGATGGATAAGGAAG
GTGGTGCCTGCAGCCATTTATCACCCAACAGCAGACTGACTTAAGAGCAGGACATAGAGCGTGGCAATACAGA
TGGTCTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATGACATTTCAACTCCTCT-
ACCAGATAATGAAATG TCTTATAACACAGTGTATGGGCATTCCAGTTCCCTCTTGCC-
CAGCACAACCACATCAGATTCGACGGCTCAAG AGGGGTATGAGTCTAGGGGAGGAAT-
GCTGGACTGGAAGCATCTGCTTGACTCACCTGACAGCACAGACCCTTT
GGGGGCCAGTGTCTTCCCACAATCACCAAGACAAGAAGGGAGTGATTCGGGAAAGTTACCTCAAGGCCACGAC
CAGCTGGTTCCCGTCACCCTCTTGGCCATTGCAGTCATCCTGGCTTTCGTCATGGAA-
GCCGTCTTCTCGGGCA TCACCGTCTACTGCGTCTGTGATCATCGGCGCAAAGACGTG-
GCTGTGGTGCAGCGCAAGGAGAAGGAGCTCAC CCACTCGCGCCGGGGCTCCATGAGC-
AGCGTCACCAAGCTCAGCGGCCTCTTTGGGGACACTCAATCCAAAGAC
CCAAAGCCGGAGGCCATCCTCACGCCACTCATGCACAACGGCAAGCTCGCCACTCCCGGCAACACGGCCAAGA
TGCTCATTAAAGCAGACCAGCACCACCTAAACCTGACGGCCCTCCCCACCCCAGAGT-
CAACCCCAACGCTGCA GCAGAAGCGGAAGCCCAGCCGCGGCAGCCGCGAGTGGGAGA-
GGAACCAGAACCTCATCAATGCCTGCACAAAG GACATGCCCCCCATGGGCTCCCCTG-
TGATTCCCACGGACCTGCCCCTGCGGGCCTCCCCCAGCCACATCCCCA
GCGTGGTGGTCCTGCCCATCACGCAGCAGGGCTACCAGCATGAGTACGTGGACCAGCCCAAAATGAGCGAGGT
GGCCCAGATGGCGCTGGAGGACCAGGCCGCCACACTGGACTATAAGACCATCAACGA-
ACATCTCAGCAGCAAG AGTCCCAACCATGGGGTGAACCTTGTGGAGAACCTGGACAG-
CCTGCCCCCCAAAGTTCCACAGCGGGAGGCCT CCCTGGGTCCCCCGGGAGCCTCCCT-
GTCTCAGACCGGTCTAAGCAAGCGGCTGGAAATGCACCACTCCTCTTC
CTACGGGGTTGACTATAAGAGGAGCTACCCCACGAACTCGCTCACGAGAAGCCACCAGGCCACCACTCTCAAA
AGAAACAACACTAACTCCTCCAATTCCTCTCACCTCTCCAGAAACCAGAGCTTTGGC-
AGGGGAGACAACCCGC CGCCCGCCCCGCAGAGGGTGGACTCCATCCAGGTGCACAGC-
TCCCAGCCATCTGGCCAGGCCGTGACTGTCTC GAGGCAGCCCAGCCTCAACGCCTAC-
AACTCACTGACAAGGTCGGGGCTGAAGCGTACGCCCTCGCTAAAGCCG
GACGTACCCCCCAAACCATCCTTTGCTCCCCTTTCCACATCAATGAAGCCCAATGATGCGTGTACAGTCGACG
GC NOV2d, 267441137 Protein Sequence SEQ ID NO:20 998 aa MW at
110569.0 kD
TGSLDIQMIMIMNGTLYIAARDHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLL
KKNDDALFVCGTNAFNPSCRNYKMDTLEPFGDEFSGMARCPYDAKHANVALFADGK-
LYSATVTDFLAIDAVIY RSLGESPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFRE-
IAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEK QWTSFLKARLNCSVPGDSHFYFNI-
LQAVTDVIRINGRDVVLATFSTPAASIPGSAVCAYDMLDIASVFTGRFK
EQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERYATSNEFPDDTLNFIKTHPAADEAVPSIFNRPWFLRTMVR
YRLTKIAVDTAAGPYQNHTVVFLGSEKGHLKFLARIGAASGFLNDSLFLEEMSVYNS-
EKCSYDGVEDKRIMGM QLDRASSSLYVAFSTCVIKVPLGRCERHGKCKKTCIASRDP-
YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD GLGDCHNSFVALNDISTPLPDNEMS-
YNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDWAALLDSPDSTDPL
GAVSSHNHQDKKGVERESYLKGHDQLVPVTLLAIAVILAFVMGAVFSGITVYCVCDHRRKDVAVVQRKEKELT
HSRRGSMSSVTKLSGLFGDTQSKDPKPEAILTPLMHNGKLATPGNTAKMLIKADQHH-
LDLTALPTPESTPTLQ QKRKPSRGSREWERNQNLINACTKDMPPMGSPVIPTDLPLR-
ASPSHIPSVVVLPITQQGYQHEYVDQPKMSEV AQMALEDQAATLEYKTIKEHLSSKS-
PNHGWLVENLDSLPPKVPQREASLGPPGASLSQTGLSKRLEMHHSSSS
YGVDYKRSYPTNSLTRSHQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRAASIQVHSSQPSGQAVTVS
RQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPNDACTAAG NOV2e, 262254987
SEQ ID NO:21 1327 bp DNA Sequence ORF Start: at 2 ORF Stop: end of
sequence CACCGGATCCCTGGACATCCAGATGATT-
ATGATCATGAACGGAACCCTCTACATTGCTGCTAGGGACCATATT
TATACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAACTGACATGGAAATCTAGAC
AGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAAGGATGAGTGCCACAACT-
TTATTAAAGTTCTTCT AAAGAAAAACGATGATGCATTGTTTGTCTGTGGAACTAATG-
CCTTCAACCCTTCCTGCAGAAACTATAAGATG GATACATTGGAACCATTCGGGGATG-
AATTCAGCGGATGGCCAGAATGCCCATATGATGCCAAACATGCCAACG
TTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTGCCATTGACGCAGTCATTTA
CCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGATTCAAAATGGTT-
GAAAGAACCATACTTT GTTCAAGCCGTGGATTACGGAGATTATATCTACTTCTTCTT-
CAGGGAAATAGCAGTGGAGTATAACACCATGG GAAAGGTAGTTTTCCCAAGAGTGGC-
TCAGGTTTGTAAGAATGATATGGGAGGATCTCAAAGAGTCCTGGAGAA
ACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTCTAATTTTTATTTCAACATT
CTCCAGGCAGTTACAGATGTGATTCGTATCAACGGGCGTGATGTTGTCCTGGCAACG-
TTTTCTACACCTTATA ACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACATGCTT-
GACATTGCCAGTGTTTTTACTGGGAGATTCAA GGAACAGAAGTCTCCTGATTCCACC-
TGGACACCAGTTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGTTGC
TGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGATGATACCCTGAACTTCATCA
AGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATCTTCAACAGGCCATGGTTCC-
TGAGAACAATGGTCAG ATACCGCCTTACCAAAATTGCAGTGGACACAGCTGCTGGGC-
CATATCAGAATCACACTGTGGTTTTTCTAAGA TCAGAGAAGGGAATCATCTTGAAGT-
TTTTGGCCAGAATAGGAAATAGTGGTTTTCTAAATGACAGCCTTAACC
TGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGAAGACAAAAGGATCATGGGCAT
GCAGGTCGACGGC NOV2e, 262254987 Protein Sequence SEQ ID NO:22 442 aa
MW at 49986.5 kD
TGSLDIQMIMIMNGTLYIAARDHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLL
KKNDDALFVCGTNAFNPSCRNYKMDTLEPFGDEFSGMARCPYDAKHANVALFADGK-
LYSATVTDFLAIDAVIY RSLGESPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFRE-
IAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEK QWTSFLKARLNCSVPGDSHFYFNI-
LQAVTDVIRINGRDVVLATFSTPAASIPGSAVCAYDMLDIASVFTGRFK
EQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVR
YRLTKIAVDTAAGPYQNHTVVFLGSEKGIILKFLARIGNSGFLNDSLFLEEMSVYNS-
EKCSYDGVEDKRIMGM QVDG NOV2f, 260565761 SEQ ID NO:23 1492 bp DNA
Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCGCATCCATGAGGTCAGAAGCCTTGCTGCTATATTTCACACTGCTACACTTTGC-
TGGGGCTGGTTTCCCA GAAGATTCTGAGCCAATCAGTATTTCGCATGGCAACTATA-
CAAAACAGTATCCGGTGTTTGTGGGCCACAAGC CAGGACGGAACACCACACAGAGGC-
ACAGGCTGGACATCCAGATGATTATGATCATGAACGGAACCCTCTACAT
TGCTGCTAGGGACCATATTTATACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAA
CTGACATGGAAATCTAGACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACAT-
AAGGATGAGTGCCACA ACTTTATTAAAGTTCTTCTAAAGAAAAACGATGATCCATTG-
TTTGTCTGTGGAACTAATGCCTTCAACCCTTC CTGCAGAAACTATAAGATGGATACA-
TTGGAACCATTCGGGGATGAATTCAGCGGAATGGCCAGATGCCCATAT
GATGCCAAACATGCCAACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTG
CCATTGACGCAGTCATTTACCGGAGTCTTGCAGAAAGCCCTACCCTGCGGACCGTCA-
AGCACGATTCAAAATG GTTGAAAGAACCATACTTTGTTCAAGCCGTGGATTACGGAG-
ATTATATCTACTTCTTCTTCAGGGAAATAGCA GTGGAGTATAACACCATGGGAAAGG-
TAGTTTTCCCAAGAGTGGCTCAGGTTTGTAAGAATGATATGGGAGGAT
CTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTC
TCATTTTTATTTCAACATTCTCCAGGCAGTTACAGATGTGATTCGTATCAACGGGCG-
TGATGTTGTCCTGGCA ACGTTTTCTACACCTTATAACAGCATCCCTGGGTCTGCAGT-
CTGTGCCTATGACATGCTTGACATTGCCAGTG TTTTTACTGGGAGATTCAAGGAACA-
GAAGTCTCCTGATTCCACCTGGACACCAGTTCCTGATGAACGAGTTCC
TAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGAT
GATACCCTGAACTTCATCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATC-
TTCAACAGGCCATGGT TCCTGAGAACAATGGTCAGATACCGCCTTACCAAAATTGCA-
GTGGACACAGCTGCTGGGCCATATCAGAATCA CACTGTGGTTTTTCTGGGATCAGAG-
AAGGGAATCATCTTGAAGTTTTTGGCCAGAATAGGAAATAGTGGTTTT
CTAAATGACAGCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGAAG
ACAAAAGGATCATGCGCATGCAGGTCGACGGC NOV2f, 260565761 Protein Sequence
SEQ ID NO:24 497 aa MW at 56242.5 kD
TGSMRSEALLLYFTLLHFAGAGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQM-
IMIMNGTLYI AARDHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDE-
CHNFIKVLLKKNDDALFVCGTNAFNPS CRNYKMDTLEPFGDEFSGMARCPYDAKHAN-
VALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTVKHDSKW
LKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDS
HFYFNILQAVTDVIRINGRDVVLATFSTPYNSIPGSAVCAYDMLDIASVWTGRFKEQ-
KSPDSTWTPVPDERVP KPRPGCCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVP-
SIFNRPWFLRTMVRYRLTKIAVDTAAGPYQNH TVVFLGSEKGIILKFLARIGNSGFL-
NDSLFLEEMSVYNSEKCSYDGVEDKRIMGMQVDG NOV2g, 252324008 SEQ ID NO:25
1438 bp DNA Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCGGATCCGGTTTCCCAGAAGATTCTGAGCCAATCAGTATTTCGCATGGCAACTA-
TACAAAACAGTATCCG GTGTTTGTGGGCCACAAGCCAGGACGGAACACCACACAGA-
GGCACAGGCTGGACATCCAGATGATTATGATCA TGAACGGAACCCTCTACATTGCTG-
CTAGGGACCATATTTATACTGTTGATATAGACACATCACACACGGAAGA
AATTTATTGTAGCAAAAAACTGACATGGAAATCTAGACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAA
CATAAGGATGAGTGCCACAACTTTATTAAAGTTCTTCTAAAGAAAAACGATGATGCA-
TTGTTTGTCTGTGGAA CTAATGCCTTCAACCCTTCCTGCAGAAACTATAAGATGGAT-
ACATTGGAACCATTCGGGGATGAATTCAGCGG AATGGCCAGATGCCCATATGATGCC-
AAACATGCCAACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCC
ACAGTGACTGACTTCCTTGCCATTCACGCAGTCATTTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCG
TCAAGCACGATTCAAAATGGTTGAAAGAACCATACTTTGTTCAAGCCGTGGATTACG-
GAGATTATATCTACTT CTTCTTCAGGGAAATAGCAGTGGAGTATAACACCATGGGAA-
AGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGT AAGAATGATATGGGAGGATCTCAAA-
GAGTCCTGGAGAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAATT
GCTCAGTTCCTGGAGACTCTCATTTTTATTTCAACATTCTCCAGGCAGTTACAGATGTGATTCGTATCAACGG
CCGTGATGTTGTCCTGGCAACGTTTTCTACACCTTATAACAGCATCCCTGGGTCTGC-
AGTCTGTGCCTATGAC ATGCTTGACATTGCCAGTGTTTTTACTGGGAGATTCAAGGA-
ACAGAAGTCTCCTGATTCCACCTGGACACCAG TTCCTGATGAACGAGTTCCTAAGCC-
CAGGCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAAC
CTCCAATGAGTTCCCTGATGATACCCTGAACTTCATCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCC
ATCTTCAACAGGCCATGGTTCCTGAGAACAATGGTCAGATACCGCCTTACCAAAATT-
GCAGTGGACACAGCTG CTGGGCCATATCAGAATCACACTGTGGTTTTTCTGGGATCA-
GAGAAGGGAATCATCTTGAAGTTTTTGGCCAG AATAGGAAATAGTGGTTTTcTAAAT-
GACAGCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATCC
AGCTATGATGGAGTCGAAGACAAAAGGATCATGGGCATGCAGGTCGACGGC NOV2g,
252324008 Protein Sequence SEQ ID NO:26 479 aa MW at 54207.1 kD
TGSGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYI-
AARDHIYTVDIDTSHTEE IYCSKKLTWKSRQADVDTCRNKGKHKDECHNFIKVLLK-
KNDDALFVCGTNAFNPSCRNYKMDTLEPFGDEFSG
NARCPYDAKHANVALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKEPYFVQAVDYGDYIYF
FFREIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHF-
YFNILQAVTDVIRING RDVVLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKS-
PDSTWTPVPDERVPKPRPGCCAGSSSLERYAT SNEFPDDTLNFIKTHPLMDEAVPSI-
FNRPWFLRTMVRYRLTKIAVDTAAGPYQNHTVVFLGSEKGIILKFLAR
IGNSGFLNDSLFLEEMSVYNSEKCSYDGVEDKRIMGMQVDG NOV2h, 252323542 SEQ ID
NO:27 3055 bp DNA Sequence ORF Start: at 2 ORF Stop: end of
sequence CACCGGATCCGGTTTCCCAGAAGATTCTGAGCCAATCAGTATTT-
CGCATGGCAACTATACAAAACAGTATCCG GTGTTTGTGGGCCACAAGCCAGGACGG-
AACACCACACAGAGGCACAGGCTGGACATCCAGATGATTATGATCA
TGAACGGAACCCTCTACATTGCTGCTAGGGACCATATTTATACTGTTGATATAGACACATCACACACGGAAGA
AATTTATTGTAGCAAAAAACTGACATGGAAATCTAGACAGGCCGATGTAGACACATG-
CAGAATGAAGGGAAAA CATAAGCATGAGTGCCACAACTTTATTAAAGTTCTTCTAAA-
GAAAAACGATGATGCATTGTTTGTCTGTGGAA CTAATGCCTTCAACCCTTCCTGCAG-
AAACTATAAGATGGATACATTGGAACCATTCGGGGATGAATTCAGCGG
AATGGCCAGATGCCCATATGATGCCAAAAACATGCCAACGTTGCACTGTTTGCAGATGGAACTATACTCAGCC
ACAGTGACTGACTTCCTTGCCATTGACGCAGTCATTTACCGGAGTCTTGGAGAAAGC-
CCTACCCTGCGGACCG TCAAGCACGATTCAAAATGGTTGAAAGAACCATACTTTGTT-
CAAGCCGTGGATTACGGAGATTATATCTACTT CTTCTTCAGGGAAATAGCAGTGGAC-
TATAACACCATGGGAAAGGTAGTTTTCCCAAGAGTGGCTCACGTTTGT
AAGAATGATATGGGAGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACT
GCTCAGTTCCTGCAGACTCTCATTTTTATTTCAACATTCTCCAGGCAGTTACAGATG-
TGATTCGTATCAACGG GCGTGATGTTGTCCTGGCAACGTTTTCTACACCTTATAACA-
GCATCCCTGGGTCTGCAGTCTGTGCCTATGAC ATGCTTGACATTGCCAGTGTTTTTA-
CTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCAG
TTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAAC
CTCCAATGAGTTCCCTGATGATACCCTGAACTTCATCAAGACGCACCCGCTCATGGA-
TGAGGCAGTGCCCTCC ATCTTCAACAGGCCATGGTTCCTGAGAACAATGGTCAGATA-
CCGCCTTACCAAAATTGCAGTGGACACAGCTG CTGGGCCATATCACAATCACACTGT-
GGTTTTTCTGGGATCAGAGAAGGGAATCATCTTGAAGTTTTTGGCCAG
AATAGGAAATAGTGGTTTTCTAAATGACAGCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGC
AGCTATCATGGAGTCGAAGACAAAAGGATCATGGGCATGCAGCTGGACAGAGCAAGC-
AGCTCTCTGTATGTTG CGTTCTCTACCTGTGTGATAAAGGTTCCCCTTGGCCGGTGT-
GAACGACATGGGAAGTGAAAAAAAACCTGTAT TGCCTCCAGAGACCCATATTGTGGA-
TGGATAAAGGAAGGTGGTGCCTGCAGCCATTTATCACCCAACAGCAGA
CTGACTTTTGAGCAGGACATAGAGCGTGGCAATACAGATGGTCTGGGGGACTGTCACAATTCCTTTGTGGCAC
TGAATGGGCATTCCAGTTCCCTCTTGCCCAGCACAACCACATCAGATTCGACCGCTC-
AAGAGGGGTATGAGTC TAGGGGAGGAATGCTGGACTGGAAGCATCTGCTTGACTCAC-
CTGACAGCACAGACCCTTTGGGGGCAGTGTCT TCCCATAATCACCAAGACAAGAAGG-
GAGTGATTCGGGAAAGTTACCTCAAAGGCCACGACCAGCTGGTTCCCG
TCACCCTCTTGGCCATTGCAGTCATCCTGGCTTTCGTCATGGGGGCCGTCTTCTCCGGCATCACCGTCTACTG
CGTCTGTGATCATCGGCGCAAAGACGTGGCTGTGGTGCAGCGCAAGGAGAAGGAGCT-
CACCCACTCGCGCCGG GGCTCCATGAGCAGCGTCACCAAGCTCAGCGGCCTCTTTGG-
GGACACTCAATCCAAAGACCCAAAGCCGGAGG CCATCCTCACGCCACTCATGCACAA-
CGGCAACCTCGCCACTCCCGGCAACACGGCCAAGATGCTCATTAAAGC
AGACCAGCACCACCTGGACCTGACGGCCCTCCCCACCCCAGAGTCAACCCCAACGCTGCAGCAGAAGCGGAAG
CCCAGCCGCGGCAGCCGCGAGTGGGAGAGGAACCAGAACCTCATCAATGCCTGCACA-
AAGGACATGCCCCCCA TGGGCTCCCCTGTGATTCCCACGGACCTGCCCCTGCGGGCC-
TCCCCCAGCCACATCCCCAGCGTGGTGGTCCT GCCCATCACGCAGCAGGGCTACCAG-
CATGAGTACGTGGACCAGCCCAAAATGAGCGAGGTGGCCCAGATGCCG
CTGGAGGACCAGGCCGCCACACTGGAGTATAAGACCATCAAGGAACATCTCAGCAGCAAGAGTCCCAACCATG
GGGTGAACCTTGTGGAGAACCTGGACAGCCTGCCCCCCAAAGTTCCACAGCGGGAGG-
CCTCCCTGGGTCCCCC GGGACCCTCCCTGTCTCAGACCGGTCTAAGCAAGCGGCTGG-
AAATGCACCACTCCTCTTCCTACGGGGTTGAC TATAAGAGGAGCTACCCCACGAACT-
CGCTCACGAGAACCCACCAGGCCACCACTCTCAAAAGAAACAACACTA
ACTCCTCCAATTCCTCTCACCTCTCCAGAAACCAGAGCTTTGGCAGGGGAGACAACCCGCCGCCCGCCCCGCA
GAGGGTGGACTCCATCCAGGTGCACAGCTCCCAGCCATCTGCCCAGGCCGTGACTGT-
CTCGAGCCAGCCCAGC CTCAACGCCTACAACTCACTGACAAGGTCGGGGCTGAAGCG-
TACGCCCTCGCTAAAGCCGGACGTACCCCCCA AACCATCCTTTGCTCCCCTTTCCAC-
ATCCATGAAGCCCAATGATGCGTGTACAGTCGACGGC NOV2h, 252323542 Protein
Sequence SEQ ID NO:28 1018 aa MW at 112848.6 kD
TGSGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAARDHIYTVDIDTSHTEE
IYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLLKKNDDALFVCGTNAFNPSC-
RNYKMDTLEPFGDEFSG MARCPYDAKHANVALFADGKLYSATVTDFLAIDAVIYRSL-
GESPTLRTVKHDSKWLKEPYFVQAVDYGDYIYF FFREIAVEYNTMGKVVFPRVAQVC-
KNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRING
RDVVLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERYAT
SNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAAGPYQNHTV-
VFLGSEKGIILKFLAR IGNSGFLNDSLFLEEMSVYNSEKCSYDGVEDKRIMGMQLDR-
ASSSLYVAFSTCVIKVPLGRCERHGKCKKTCI ASRDPYCGWIKEGGACSHLSPNSRL-
TFEQDIERGNTDGLGDCHNSFVALNGHSSSLLPSTTTSDSTAQEGYES
RGGMLDWKHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAFVMGAVFSGITVYC
VCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQSKDPKPEAILTPLMHN-
GKLATPGNTAKMLIKA DQHHLDLTALPTPESTPTLQQKRKPSRGSREWERNQNLINA-
CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVL PITQQGYQHEYVDQPKMSEVAQMAL-
EDQAATLEYKTIKEHLSSKSPNHGVNLVENLDSLPPKVPQREASLGPP
GASLSQTGLSKRLEMHHSSSYGVDYKRSYPTNSLTRSHSQATTLKRNTNSSNSSHLSRNQSFGRGDNPPPAPQ
RVDSIQVHSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLST-
SMKPAAACAADG NOV2i, 252323483 SEQ ID NO:29 2944 bp DNA Sequence ORF
Start: at 2 ORF Stop: end of sequence
CACCGGATCCATGAGGTCAGAAGCCTTGCTGCTATATTTCACACTGCTACACTTTGCTGGGGCTGGTTTCCCA
GAAGATTCTGAGCCAATCAGTATTTCGCATGGCAACTATACAAAACAGTATCCAAT-
GTTTGTGGGCCACAAGC CAGGACGGAACACCACACAGAGGCACAGGCTGGACATCCA-
GATGATTATGATCATGAACGGAACCCTCTACAT TGCTGCTAGGGACCATATTTATAC-
TGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAA
CTGACATGGAAATCTAGACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAAGGATGAGTGCCACA
ACTTTATTAAAGTTCTTCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAACTA-
ATGCCTTCAACCCTTC CTGCAGAAACTATAAGATGGATACATTGGAACCATTCGGGG-
ATGAATTCAGCGGAATGGCCAGATGCCCATAT GATGCCAAACATGCCAACGTTGCAC-
TGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTG
CCATTGACGCAGTCATTTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGATTCAAAATG
GTTGAAAGAACCATACTTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTTCTT-
CTTCAGGGAAATAGCA GTGGAGTATAACACCATGGGAAAGGTAGTTTTCCCAAGAGT-
GGCTCAGGTTTGTAAGAATGATATGGGAGGAT CTCAAAGAGTCCTGGAGAAACAGTG-
GACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTC
TCATTTTTATTTCAACATTCTCCAGGCAGTTACAGATGTGATTCGTATCAAGGGGCGTGATGTTGTCCTGGCA
ACGTTTTCTACACCTTATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACATG-
CTTGACATTGCCAGTG TTTTTACTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCC-
ACCTGGACACCAGTTCCTGATGAACGAGTTCC TAAGCCCAGGCCAGGTTGCTGTGCT-
GGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGAC
GATACCCTGAACTTCATCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATCTTCAACAGGCCATGGT
TCCTGAGAACAATGGTCAGATACCGCCTTACCAAAATTGCAGTGGACACAGCTGCTG-
AACCATATAAGAATCA CACTGTGGTTTTTCTGGGATCAGAGAAGGGAATCATCTTGA-
AAGTTTTTGGCCAGAATAGGAATAGTGGTTAA CTAAATGACAGCCTTTTCCTGGAGG-
AGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGAAG
ACAAAAGGATCATCGGCATGCAGCTGGACAGAGCAAGCAGCTCTCTGTATGTTGCGTTCTCTACCTGTGTGAT
AAAGGTTCCCCTTGGCCGGTGTGAACGACATGGGAAGTGTAAAAAAACCTGTATTGC-
CTCCAGAGACCCATAT TGTGGATGGATAAAGGAAGGTGGTGCCTGCAGCCATTTATC-
ACCCAACAGCAGACTGACTTTTGAGCAGGACA TAGAGCGTGGCAATACAGATGGTCT-
GGGGGACTGTCACAATTCCTTTGTGGCACTGAATGGAGTGATTCGGGA
AAGTTACCTCAAAGGCCACGACCAGCTGGTTCCCGTCACCCTCTTGGCCATTGCAGTCATCCTGGCTTTCGTC
ATGGGGGCCGTCTTCTCGGGCATCACCGTCTACTGCGTCTGTGATCATCGGCGCAAA-
GACGTGGCTGTGGTGC AGCGCAAGGAGAAGGAGCTCACCCACTCGCGCCGGGGCTCC-
ATGAGCAGCGTCACCAAGCTCAGCGGCCTCTT TGGGGACACTCAATCCAAAGACCCA-
AAGCCGGAGGCCATCCTCACGCCACTCATGCACAACGGCAAGCTCGCC
ACTCCCGGCAACACGGCCAAGATGCTCATTAAAGCAGACCAGCACCACCTGGACCTGACGGCCCTCCCCACCC
CAGAGTCAACCCCAACGCTGCAGCAGAAGCGGAAGCCCAGCCGCGGCAGCCGCGAGT-
GGGAGAGGAACCAGAA CCTCATCAATGCCTGCACAAAGGACATGCCCCCCATGGGCT-
CCCCTGTGATTCCCACGGACCTGCCCCTGCGG CCCTCCCCCAGCCACATCCCCAGCG-
TGGTGGTCCTGCCCATCACGCAGCAAAGCTACCAGCATGAGTACGTGG
ACCAGCCCAAAATGAGCGAGGTGGCCCAGATGGCGCTGGAGGACCAGGCCGCCACACTGGAGTATAAGACCAT
CAAGGACATCTCAGCAGCAAGAGTCCCAACCATGGGGTGAACCTTGTGGAGAACCTG-
GACAGCCTGCCCCCCC AAAGTTCCACAGCGGGAGGCCTCCCTGGGTCCCCCGGGAGC-
CTCCCTGTCTCAGACCGGTCTAAGCAAGCGGC TGGAAATGCACCACTCCTCTTCCTA-
CGGGGTTGACTATAAGAGGAGCTACCCCACGAACTCGCTCACGAGAAG
CCACCAGGCCACCACTCTCAAAAGAAACAACACTAACTCCTCCAATTCCTCTCACCTCTCCAGAAACCAGAGC
TTTGGCAGGGGAGACAACCCGCCGCCCGCCCCGCAGAGGGTGGACTCCATCCAGGTG-
CACAGCTCCCACCCAT CTGGCCAGGCCGTGACTGTCTCGAGGCAGCCCAGCCTCAAC-
GCCTACAACTCACTGACAAGGTCGGGGCTGAA GCGTACGCCCTCGCTAAAGCCGGAC-
GTACCCCCCAAACCATCCTTTGCTCcCCTTTCCACATCCATGAAGCCC
AATGATGCGTGTACAGTCGACGGC NOV2i, 252323483 Protein Sequence SEQ ID
NO:30 981 aa MW at 109048.9 kD
TGSMRSEALLLYFTLLHFAGAGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYI
AARDHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRNKGKHKDECHNFIKVLLK-
KNDDALFVCGTNAFNPS CRNYKMDTLEPFGDEFSGMARCPYDAKHANVALFADGKLY-
SATVTDFLAIDAVIYRSLGESPTLRTVKHDSKW LKEPYFVQAVDYGDYIYFFFREIA-
VEYNTMGKVVFPRVAQVCKNDNGGSQRVLEKQWTSFLKARLNCSVPGDS
HFYFNILQAVTDVIRIKGRDVVLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVP
KPRPGCCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYR-
LTKIAVDTAAGPYQNH TVVFLGSEKGIILKFLARIGNSGFLNDSLFLEEMSVYNSEK-
CSYDGVEDKRIMGMQLDRASSSLYVAFSTCVI KVPLGRCERHGKCKKTCIASRDPYC-
GWIKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCNNSFVALNGVIRE
SYLKGHDQLVPVTLLAIAVILAFVMGAVFSGITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLF
GDTQSKDPKPEAILTPLMHNGKLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQ-
KRKPSRGSREWERNQN LINACTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQ-
GYQHEYVDQPKMSEVAQMALEDQAATLEYKTI KEHLSSKSPNHGVNLVENLDSLPPK-
VPQREASLGPPGASLSQTGLSKRLEMHHSSSYGVDYKRSYPTNSLTRS
HQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRVDSIQVHSSQPSGQAVTVSRQPSLNAYNSLTRSGLK
RTPSLKPDVPPKPSFAPLSTSMKPNDACTVDG NOV2j, CG51896-01 SEQ ID NO:31
3498 bp DNA Sequence ORF Start: ATG at 214 ORF Stop: end of
sequence GCGACTATTTCCCCCAAAGAGACAAGCACACATGTA-
GGAATGACAAAGGCTTGCGAAGGAGAGACCGCAGCCC
GCGGCCCGGAGAGATCCCCTCGATAATGGATTACTAAATGGGATACACGCTGTACCAGTTCGCTCCGAGCCCC
GGCCGCCTGTCCGTCGATGCACCGAAAAGGGTGAAGTAGAGAAATAAAGTCTCCCCG-
CTGAACTACTATGAGG TCAGAAGCCTTGCTGCTATATTTCACACTGCTACACTTTGC-
TGGGGCTGGTTTCCCAGAAGATTCTGAGCCAA TCAGTATTTCGCATGGCAACTATAC-
AAAACAGTATCCGGTGTTTGTGGGCCACAAGCCAGGACGGAACACCAC
ACAGAGGCACAGGCTGGACATCCAGATGATTATGATCATGAACGGAACCCTCTACATTGCTGCTAGGCACCAT
ATTTATACTGTTCATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAA-
CTGACATGGAAATCTA GACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACAT-
AAGGATGAGTGCCACAACTTTATTAAAGTTCT TCTAAAGAAAAACGATGATGCATTG-
TTTGTCTGTGGAACTAATGCCTTCAACCCTTCCTGCAGAAACTATAAG
ATGGATACATTGGAACCATTCGGGGATGAATTCAGCGGAATGGCCAGATCCCCATATGATGCCAAACATGCCA
ACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTG-
CCATTGACGCAGTCAT TTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCA-
AGCACGATTCAAAATGGTTGAAAGAACCATAC TTTGTTCAAGCCGTGGATTACGGAG-
ATTATATCTACTTCTTCTTCAGGGAAATAGCAGTGGAGTATAACACCA
TGGGAAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGTAAGAATGATATGGGAGGATCTCAAAGAGTCCTGGA
GAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTC-
TCATTTTTATTTCAAC ATTCTCCAGGCAGTTACAGATGTGATTCGTATCAACGCGCG-
TGATGTTGTCCTGGCAACGTTTTCTACACCTT ATAACAGCATCCCTGGGTCTGCAGT-
CTGTGCCTATGACATGCTTGACATTGCCAGTGTTTTTACTGGGAGATT
CAAGGAACAGAAGTCTCCTGATTCCACCTCGACACCAGTTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGT
TGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGAT-
GATACCCTGAACTTCA TCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATC-
TTCAACAGGCCATGGTTCCTGAGAACAATGGT CAGATACCGCCTTACCAAAATTGCA-
GTCGACACAGCTGCTGGGCCATATCAGAATCACACTGTGGTTTTTCTG
GGATCAGAGAAGCGAATCATCTTGAAGTTTTTCGCCAGAATAGGAAATAGTGGTTTTCTAAATGACAGCCTTT
TCCTGGACGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGAAG-
ACAAAAGGATCATGGG CATGCAGCTGGACAGAGCAAGCAGCTCTCTGTATGTTGCGT-
TCTCTACCTGTGTGATAAAGGTTCCCCTTGGC CGGTGTGAACGACATGGGAAGTGTA-
AAAAAACCTGTATTGCCTCCAGAGACCCATATTGTGCATGGATAAAGG
AAGGTGGTGCCTGCAGCCATTTATCACCCAACAGCAGACTGACTTTTGACCAGGACATACAGCGTGGCAATAC
AGATGGTCTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATGGGCATTCCAGTTC-
CCTCTTGCCCAGCACA ACCACATCAGATTCGACGGCTCAAGAGGGGTATGAGTCTAG-
GCGAGGAATCCTGGACTGGAAGCATCTGCTTG ACTCACCTGACAGCACAGACCCTTT-
GGUGGCAGTGTCTTCCCATAATCACCAAGACAAGAAGGGAGTGATTCG
GGAAAGTTACCTCAAAGGCCACGACCAGCTGGTTCCCGTCACCCTCTTGGCCATTGCAGTCATCCTGGCTTTC
GTCATGGGGGCCGTCTTCTCGGGCATCACCGTCTACTGCCTCTGTGATCATCGGCGC-
AAAGACGTGGCTGTGG TGCAGCGCAAGOAGAAGGAGCTCACCCACTCGCGCCGGGGC-
TCCATGAGCAGCGTCACCAAGCTCAGCGGCCT CTTTGGGGACACTCAATCCAAAGAC-
CCAAAGCCGGAGGCCATCCTCACGCCACTCATGCACAACGGCAAGCTC
GCCACTCCCGGCAACACGGCCAAGATGCTCATTAAAGCAGACCAGCACCACCTGGACCTGACAACCCTCCCCA
CCCCAGAGTCAACCCCAACGCTGCAGCAGAAGCGGGAACCCAGCCGCGGCACCCGCG-
AGTGGGAGAGGAACCA GAACCTCATCAATGCCTGCACAAAGGACATCCCCCCCATGG-
GCTCCCCTGTGATTCCCACGGACCTGCCCCTG CGGGCCTCCCCCAGCCACATCCCCA-
GCGTGGTGGTCCTGCCCATCACGCAGCAGGGCTACCAGCATGAGTACG
TGGACCAGCCCAAAATGAGCGAGGTGGCCCAGATGGCGCTGGAGGACCAAACCGCAACACTGGAGTATAAGAC
CATCAAGGAACATCTCAGCAGCAAGAGTCCCAACCATGGGGTGAACCTTGTGGAGAA-
CCTGGACAGCCTGCCC CCCAAAGTTCCACAGCGGGAGGCCTCCCTGGGTCCCCCGGG-
AGCCTCCCTGTCTCAGACCGCTCTAAGCAAGC GGCTGGAAATGCACCACTCCTCTTC-
CTACGGGGTTGACTATAAGAGGAGCTACCCCACGAACTCGCTCACGAG
AAGCCACCTGACCACCTACTCTCATCAGAAGCAACACTAACCCCGACAATTCANCTCTGACTTCAAAGGGACC
AGAGCTTTGGCAGGGGAGACAACCCGCCGCCCGCCCCGCAGAGGGTGGACTCCATCC-
AGGTGCACAGCTCCCA GCCATCTGGCCAGGCCGTGACTGTCTCGAGGCAGCCCAGCC-
TCAACGCCTACAACTCACTGACAAAATCGGGG CTGAAGCGTACGCCCTCGCTAAAGC-
CGGACGTACCCCCCAAACCATCCTTTGCTCCCCTTTCCACATCCATGA
AGCCCAATGATGCGTGTACATAATCCCAGGGGGAGGGGGTCAGGTGTCGAACCAGCAGGCAAGGCGAGGTGTC
CGCTCAGCTCAGCAAGGTTCTCAACTGCCTCGAGTACCCACCAAACCAAAAAGGCCT-
GCGGCAGAACCGAGGG ACGCTGGGTCCTCCTCTCTGGGACACAGGGGTACTCACGCT-
GGGCCGCGTAATTTGGTGAAAG NOV2j, CG5 1896-01 Protein Sequence SEQ ID
NO:32 939 aa MW at 104828.0 kD
MRSEALLLYFTLLHFAGAGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAAR
DHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLLKKND-
DALFVCGTNAFNPSCRN YKMDTLEPFGDEFSGMARCPYDAKHANVALFADGKLYSAT-
VTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKE PYFVQAVDYGDYIYFFFREIAVEY-
NTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHFY
FNILQAVTDVIRINGRDVVLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPR
PGCCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTK-
IAVDTAAGPYQNHTVV FLGSEKGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSY-
DGVEDKRIMGMQLDRASSSLYVAFSTCVIKVP LGRCERIIGKCKKTCIASRDPYCGW-
IKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHNSFVAANGHSSSLP
STTTSDSTAQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPAALAAIAVIL
AFVMGAVFSGITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQS-
DPKPEAILTPLMHNGA KLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQAAEPS-
RGTREWEAAQNLINACTAAMPPMGSPVIPTDL PLRASPSHIPSVVVLPITQQGYQHE-
YVDQPKMSEVAQMALEDQATLEYKTIKEHLSSKSPNIIGVNLVENLDS
LPPAAPQREASLGPPGASLSQTGLSKAAEMHHSSSYGAAYAASYPTNSLTRSHLTTYSHQKQH
NOV2k, CG51896-02 SEQ ID NO:33 1878 bp DNA Sequence ORF Start: at 1
ORF Stop: end of sequence GGTTTCCCAGAAGATTCTGAGCCAATCA-
GTATTTCGCATGGCAACTATACAAAACAGTATCCGGTGTTTGTGG
GCCACAAGCCAGGACGGAACACCACACAGAGGCACAGGCTGGACATCCAGATGATTATGATCATGAACGGAAC
CCTCTACATTGCTGCTAGGGACCATATTTATACTGTTGATATAGACACATCACACAC-
GGAAGAAATTTATTGT AGCAAAAAACTGACATGGAAATCTAGACAGGCCGATGTAGA-
CACATGCAGAATGAAGGGAAAACATAAGGATG AGTGCCACAACTTTATTAAAGTTCT-
TCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAACTAATGCCTT
CAACCCTTCCTGCAGAAACTATAAGATGGATACATTGGAACCATTCGGGGATGAATTCAGCGGAATGGCCAGA
TGCCCATATGATGCCAAACATGCCAACGTTGCACTGTTTGCAGATGGAAAACTATAC-
TCAGCCACAGTGACTG ACTTCCTTGCCATTGACGCAGTCATTTACCGGAGTCTTGGA-
CAAAGCCCTACCCTGCGGACCGTCAAGCACGA TTCAAAATGGTTGAAAGAACCATAC-
TTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTTCTTCTTCAGG
GAAATAGCAGTGGAGTATAACACCATAAGAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGTAAGAAATGATA
TGGGAGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCTGAAGGCGCGCT-
TGAACTGCTCAGTTCC TGGAGACTCTCATTTTTATTTCAACATTCTCCAGGCAGTTA-
CAGATGTGATTCGTATCAACGGGCGTGATGTT GTCCTGGCAACGTTTTCTACACCTT-
ATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACATGCTTGACA
TTGCCAGTGTTTTTACTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCAGTTCCTGATGA
ACGAGTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATA-
TGCAACCTCCAATGAG TTCCCTGATGATACCCTGAACTTCATCAAGACGCACCCGCT-
CATGGATGAAACAGTGCCCTCCATCTTCAACA GGCCATGGTTCCTGAGAACAATGGT-
CAGATACCGCCTTACCAAAATTGCAGTGGACACAGCTGCTGGGCCATA
TCAGAATCACACTGTGGTTTTTCTGGGATCAGAGAAGGGAATCATCTTGAAGTTTTTGGCCAGAATAGGAAAT
AGTGGTTTTCTAAATGACACCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTCAA-
AATGCAGCTATGAATG GAGTCGAAGACAAAAGGATCATGGGCATGCAGCTGGACAGA-
CCAAGCAGCTCTCTGTATGTTGCGTTCTCTAC CTGTGTGATAAAGGTTCCCCTTGGC-
CGGTGTGAACGACATGGGAAGTGTAAAAAAACCTGTATTGCCTCCAGA
GACCCATATTGTGGATGGATAAAGGAAGGTGGTGCCTCCAGCCATTTATCACCCAACAGCAGACTGACTTTTG
AGCAGGACATAGAGCGTGGCAATACAGATAATCTGGGGGACTGTCACAATTCCTTTG-
TGGCACTGAATGGGCA TTCCAGTTCCCTCTTGCCCAGCACAACCACATCAGATTCGA-
CGGCTCAAGAGGGGTATGAGTCTAGGGGAGGA ATGCTGGACTGGAAGCATCTGCTTG-
ACTCACCTGACAGCACAGACCCTTTGGGGGCAGTGTCTTCCCATAATC
ACCAAGACAAGAAGGGAGTGATTCGGGAAAGTTACCTCAAAGGCCACGACCAG NOV2k,
CG51896-02 Protein Sequence SEQ ID NO:34 626 aa MW at 70297.8 kD
GFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAAR-
DHIYTVDIDTSHTEEIYC SKKLTWKSRQADVDTCRMKGKHKDECHNPIKVLLKKND-
DALFVCGTNAFNPSCRNYKMDTLEPFGDEFSGMAA
CPYDAKHANVALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKEPFVQAVDYGDYIYFFFER
EIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHFYFN-
ILQAVTDVIRINGRDV VLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDS-
TWTPVPDERVPKPRPGCCAGSSSLERYATSNE FPDDTLNFIKTHPLMDEAVPSIFMR-
PWFLRTMVRYRLTKIAAATAAGPYQAATAAFLGSEKGIILKFLARIGN
SGFLNDSLFLEEMSVYNSEKCSYDGVEDKRIMGMQLDAASSSLYVAFSTCVIKVPLGRCERHGKCKKTCIASR
DPYCGWIKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHNSFVALNGHSSSLLPST-
TTSDSTAQEGYESRGG MLDWKHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHD- Q
NOV2l, CG51896-03 SEQ ID NO:35 1908 bp DNA Sequence ORF Start: at 1
ORF Stop: end of sequence
GGTTTCCCAGAAGATTCTGAGCCAATCAGTATTTCGCATGGCAACTATACAAAACAGTATCCGGTGTTTGTGG
GCCACAAGCCAGGACGGAACACCACACAGAGGCACAGGCTGGACATCCAGATGATT-
ATGATCATGAACGGAAC CCTCTACATTGCTGCTAGGGACCATATTTATACTGTTGAT-
ATAGACACATCACACACGGAAGAAATTTATTGT AGCAAAAAACTGACATGGAAATCT-
AGACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAAGGATG
AGTGCCACAACTTTATTAAAGTTCTTCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAACTAATGCCTT
CAACCCTTCCTGCAGAAACTATAAGATGGATACATTGGAACCATTCGGGGATGAATT-
CAGCGGAATGGCCAGA TGCCCATATGATGCCAAACATGCCAACGTTGCACTGTTTGC-
AGATGGAAAACTATACTCAGCCACAGTGACTG ACTTCCTTGCCATTGACGCAGTCAT-
TTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGA
TTCAAAATGGTTGAAAGAACCATACTTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTTCTTCTTCAGG
GAAATAGCAGTGGAGTATAACACCATGGGAAAGGTAGTTTTCCCAAGAGTGGCTCAG-
GTTTGTAAGAATGATA TGGCAGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCG-
TTCCTGAAGGCGCGCTTGAACTGCTCAGTTCC TGGAGACTCTCATTTTTTATTTCAC-
ATTCTCCAGGCAGTTACAGATGTGATTCGTATCAACAAGCGTGATGTT
GTCCTGGCAACGTTTTCTACACCTTATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACATGCTTGACA
TTGCCAGTGTTTTTACTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGA-
CACAAGTTCCTGATGA ACGAGTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCAT-
CCTCCTTAGAAAGATATGCAACCTCCAATGAG TTCCCTGATGATACCCTGAACTTCA-
TCAAGACGCACCCCCTCATGGATGAGGCAGTGCCCTCCATCTTCAAAA
GGCCATGGTTCCTGAGAACAATGGTCAGATGCAGCTATGATGGAGTCGAAGACAAAAGGATCATGGGCATGCA
GCTGGACAGAGCAAGCAGCTCTCTGTATGTTGCGTTCTCTACCTGTGTGATAAAGGT-
CCCCCTTGGCCGGTGT GAACGACATGCGAAGTGTAAAAAAAACCTGTATTGCCTCCA-
GAGACCCATATTGTGGATGGATAAGGAAGGTG GTGCCTGCAGCCATTTATCACCCAA-
CAGCGACTGACTTTTGAGCAGGACATAGAGCGTGGCAAATACAGATGG
TCTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATAAGCATTCCAGTTCCCTCTTGCCCAGCACAACCACA
TCAGATTCGACGGCTCAAGAGGGGTATGAGTCTAGGGGAGGAATGCTGGACTGGAAG-
CATCTGCTTGACTCAC CTGACAGCACAGACCCTTTGGGGGCAGTGTCTTCCCATAAT-
CACCAAGACAAGAAGGGAGTGATTCGGGAAAG TTACCTCAAAGGCCACGACCAGCTG-
GTTCCCGTCACCCTCTTGGCCATTGCAGTCATCCTAACTTTCGTCATG
GGGGCCGTCTTCTCGGGCATCACCGTCTACTGCGTCTGTGATCATCGGCGCAAAAACGTGGCTGTGGTGCAGC
GCAAGGAGAAGCAGCTCACCCACTCGCGCCGGGGCTCCATGAGCAGCGTCACCAAGC-
TCAGCGGCCTCTTTAA GGACACTCAA NOV2l, CG51896-03 Protein Sequence SEQ
ID NO:36 636 aa MW at 71237.1 kD
GFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAARDHTYTVDI-
DTSHTEEIYC SKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLLKKNDDALFVCGT-
NAFNPSCRNYKMDTLEPFGDEFSGMAR CPYDAKHANVALFAADGKLYSATVTDFLAI-
DAVIYRSLGESPTLRTVKHDSKWKEPYFVQAVDYGDYIYFFFR
EIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDV
VLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPG-
CCAGSSSLERYATSNE FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRCSYDGVE-
DKRIMGMQLDAASSSLAAAFSTAAIAAPLGRC ERHGKCKKTCIASRDPYCGWIKEGG-
ACSHLSPNSRLTFEQDIERGNTDGLGDCHNSFVALNGHSSSLLPSTTT
SDSTAQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSIINHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAAA
GAVFSGITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQ NOV2m,
CG51896-05 SEQ ID NO:37 54 bp DNA Sequence OFR Start: at 1 ORF
Stop: at 55 GGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGATTC-
AAAATGGTTGAAAGAA NOV2m, GG51896-05 Protein Sequence SEQ ID NO:38 18
aa MW at 2111.4 kD GESPTLRTVKHDSKWLKE NOV2n, CG51896-06 SEQ ID
NO:39 54 bp DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
GGAGAGAAAGCCCTACCCTGCGGA- CCGTCAAGCACGATTCAAAATGGTTGAAAGAA NOV2n,
CG5 1896-06 Protein Sequence SEQ ID NO:40 18 aa MW at 2111.4 kD
GESPTLRTVKHDSKWLKE NOV2o, CG51896-07 SEQ ID NO:41 51 bp DNA
Sequence ORF Start: at 1 ORF Stop: end of sequence
TCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGATGATACC NOV2o,
CG51896-07 Protein Sequence SEQ ID NO:42 17 aa MW at 1918.9 kD
SSSLERYATSNEFPDDT NOV2p, CG51896-08 SEQ ID NO:43 60 bp DNA Sequence
ORF Start: at 1 ORF Stop: end of sequence
GAGGAGATGAGTGTTTACAACTCTGAAAAATGCAQCTATGATCGAGTCGAAGACAAA- AGG
NOV2p, CG51896-08 Protein Sequence SEQ ID NO:44 20 aa MW at 2368.5
kD EEMSVYNSEKCSYDGVEDKR NOV2q, CG51896-09 SEQ ID NO:45 3983 bp DNA
Sequence ORF Start: ATG at 214 ORF Stop: end of sequence
GCGACTATTTCCCCCAAAGAGACAAGCAC-
ACATGTAGGAATGACAAAGCCTTGCGAAGGAGAGAGCGCAGCCC
GCGGCCCGGAGAGATCCCCTCGATAATGGATTACTAAATGGGATACACGCTGTACCAGTTCGCTCCGAGCCCC
GCCCGCCTGTCCGTCGATCCACCGAAAGGGTGAAGTAGAOAAATAAAGTCTCCCCGC-
TGAAACTACTATGAAA TCAGAAGCCTTGCTGCTATATTTCACACTGCTACACTTTGC-
TGGGGCTGGTTTCCCAGAAGATTCTGAGCCAA TCAGTATTTCGCATGGCAACTATAC-
AAAACAGTATCCGGTGTTTGTGGGCCACAAGCCAGGACGGAACACCAC
ACAGAGGCACAGGCTGGACATCCAGATGATTATGATCATGAACGGAACCCTCTACATTGCTGCTAGGGACCAT
ATTTATACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAA-
CTGACATGGAAATCTA GACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACAT-
AAGGATGAGTGCCACAACTTTATTAAAGTTCT TCTAAAGAAAAACGATGATGCATTG-
TTTGTCTGTGGAACTAATGCCTTCAACCCTTCCTGCAGAAACTATAAG
ATGGATACATTGGAACCATTCGGGGATGAATTCAGCGGAATGGCCAGATGCCCATATGATGCCAAACATGCCA
ACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTG-
CCATTGACGCAGTCAT TTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCA-
AGCACGATTCAAAATGGTTGAAAGAACCATAC TTTGTTCAAGCCGTGGATTACGGAG-
ATTATATCTACTTCTTCTTCAGGGAAATAGCACTGGAGTATAACACCA
TGGGAAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGTAAGAATGATATGGGAGGATCTCAAAGAGTCCTGGA
GAAACGGTGCACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTC-
TCATTTTTATTTCAAC ATTCTCCAGGCAGTTACAGATGTGATTCGTATCAACGCGCG-
TGATGTTGTCCTGGCAACGTTTTCTACACCTT ATAACAGCATCCCTGGGTCTGCAGT-
CTGTGCCTATGACATGCTTGACATTGCCAGTGTTTTTACTGGGAGATT
CAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCAGTTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGT
TGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGAT-
GATACCCTGAACTTCA TCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATC-
TTCAACAGGCCATGGTTCCTGAGAACAATGGT CAGATGCAGCTATGATGGAGTCGAA-
GACAAAAGGATCATGGGCATGCAGCTGGACAGAGCAAGCAGCTCTCTG
TATGTTGCGTTCTCTACCTGTGTGATAAAGGTTCCCCTTGGCCGGTGTGAACGACATGGGAAGTGTAAAAAAA
CCTGTATTGCCTCCAGAGACCCATATTGTGGATGGATAAAGGAAGGTAATGCCTGCA-
GCCATTTATCACCCAA CAGCAGACTGACTTTTGAGCAGGACATAGAGCGTGGCAATA-
CAGATGGTCTGGGGGACTGTCACAATTCCTTT CTGGCACTGAATGGGCATTCCAGTT-
CCCTCTTGCCCACCACAACCACATCAGATTCGACGGCTCAAGAGGGGT
ATGAGTCTAGGGGAGGAATGCTGGACTGGAAGCATCTGCTTGACTCACCTGACAGCACAGACCCTTTGGGGGC
AGTGTCTTCCCATAATCACCAAGACAAGAAGGGAGTGATTCGGGAAAGTTACCTCAA-
AGGCCACGACCAGCTG GTTCCCGTCACCCTCTTGGCCATTGCAGTCATCCTGGCTTT-
CGTCATGGGGGCCGTCTTCTCGGGCATCACCG TCTACTGCGTCTGTGATCATCGGCG-
CAAAGACGTGGCTGTGGTGCAGCGCAAGGAGAAGGAGCTCACCCACTC
GCGCCGGGGCTCCATGAGCAGCGTCACCAAGCTCAGCGGCCTCTTTGGGGACACTCAATCCAAAGACCCAAAG
CCGGAGGCCATCCTCACGCCACTCATGCACAACGGCAAGCTCGCCACTCCCGGCAAC-
ACGGCCAAGATGCTCA TTAAAGCAGACCAGCACCACCTGGACCTGACGGCCCTCCCC-
ACCCCAGAGTCAACCCCAACGCTGCAGCAGAA GCGGAAGCCCAGCCGCGGCAGCCGC-
GAGTGGGAGAGGAACCAGAACCTCATCAATGCCTGCACAAAGGACATG
CCCCCCATGGGCTCCCCTGTGATTCCCACGGACCTGCCCCTGCGGGCCTCCCCCAGCCACATCCCCAGCGTGG
TGGTCCTGCCCATCACGCAGCAGGGCTACCAGCATGAGTACGTGGACCAGCCCAAAA-
TGAGCGAGGTAACCCA GATGGCGCTCGAGGACCAGGCCGCCACACTGGAGTATAAGA-
CCATCAAGGAACATCTCAGCAGCAAGAGTCCC AACCATGGGGTGAACCTTGTGGAGA-
ACCTGGACAGCCTGCCCCCCAAAGTTCCACAGCGGGAAACCTCCCTAA
GTCCCCCGGGAGCCTCCCTGTCTCAGACCGGTCTAAGCAAGCGGCTGGAAATGCACCACTCCTCTTCCTACAA
GGTTGACTATAAGAGGAGCTACCCCACGAACTCGCTCACGAGAAGCCACCAGGCCAC-
CACTCTCAAAAGAAAC AACACTAACTCCTCCAATTCCTCTCACCTCTCCAGAAACCA-
GAGCTTTGGCAGGGGAGACAACCCGCCGCCCG CCCCGCAGAGGGTGGACTCCATCCA-
GGTGCACAGCTCCCAGCCATCTGGCCAGGCCGTGACTGTCTCGAAACA
GCCCAGCCTCAACGCCTACAACTCACTGACAAGGTCGGGGCTGAAGCGTACGCCCTCGCTAAAGCCGGACGTA
CCCCCCAAACCATCCTTTGCTCCCCTTTCCACATCCATGAAGCCCAATGATGCGTGT-
ACATAATCCCAGAAGG AGGGGGTCAGGTGTCGAACCAGCAGGCAAGGCGAGGTGCCC-
GCTCAGCTCAGCAAGGTTCTCAACTGCCTCGA GTACCCACCAGACCAAGAAGGCCTG-
CGGCACAGCCGAGGACGCTGGGTCCTCCTCTCTGGGACACAGAAGTAC +E,usn
TCACGAAAACTGGCCCGCGTGGTTTGGTGAAGGTTTCCAACGGCGGGGACTCACCTTCATTCTCTTCCTTCAC
TTTCCCCCACACCCTACAACAGGTCGCACCCACAAAAGACTTCAGTTATCATCACAA-
ACATGAGCCAAAACCA CATACCTACCCCATCCCCCACCCCCACACACACACACATGC-
ACACAACACATACACACACACGCACAGAGGTG AACACAAACTGAAACATTTTGTCCA-
CAACTTCACGGGACGTGGCCAGACTGGGTTTGCGTTCCAACCTGCAAA
ACACAAATACATTTTTTAAAATCAAGAAAATTTAAAAAGACAAAAAAAAGAATTCATTCATAATTCTAACTCA
GACTTTAACAATGGCAGAAGTTTACTATGCGCAAATACTGTGAAATGCCCGCCAGTG-
TTACAGCTTTCTGTTG CAGCAGATAAATGCCATGTTGGGCAGCTATGTCATAGATTT-
CTGCTCCTCCTCTCTTGTCATAGATTTCTGCT CCTCCTCTCTTGTCATAGATTTCTG-
CTCCTCCTCTCTTGTCATAGATTTCTGCTCCTCCTCTCTTGTCATAGA
TTTCTGCTCCTCCTCTCTTGTCATAGATTTCTCCTCCTCCTCTCTTGTCATAGATTTCTOCTCCTCCTCTCTT
GTCATAGATTTCTGCTCCTCCTCTCTTGTCATAGATTTCTG NOV2q, CG51896-09 Protein
Sequence SEQ ID NO:46 971 aa MW at 107846.1 kD
MRSEALLLYFTLLHFACAGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRH-
RLDIQMIMIMNGTLYIAAR DHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRNK-
GKHKDECHNFIKVLLKKNDDALFVCGTNAFNPSCRN
YKDDTLEPFGDEFSGMARCPYDAKHANVALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKE
PYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKRWTSF-
LKARLNCSVPGDSHFY FNILQAVTDVIRINGRDVVLATFSTPYNSIPGSAVCAYDML-
DIASVFTGRFKEQKSPDSTWTPVPDERVPKPR PGCCAGSSSLERYATSNEFPDDTLN-
FTKTHPLMDEAVPSIFNRPWFLRTMVRCSYDGVEDKRIMGMQLDRASS
SLYVAFSTCVIKVPLCRCERHGKCKKTCIASRDPYCGWIKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHN
SFVALNGHSSSLLPSTTTSDSTAQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSHNI-
QDKKGVIRESYLKGHD QLVPVTLLAIAVILAFVMGAVFSGITVYCVCDHRRKDVAVV-
QRKEKELTHSRRGSMSSVTKLSGLFGDTQSKD PKPEAILTPLMHNGKLATPGNTAKM-
LIKADQHILDLTALPTPESTPTLQQKRKPSRGSREWERNQNLINACTK
DMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQGYQHEYVDQPKMSEVAQMALEDQAATLEYKTIKEHLSSK
SPNHGVNLVENLDSLPPKVPQREASLGPPGASLSQTGLSKRLEMHHSSSYGVDYKRS-
YPTNSLTRSHQATTLK RNNTNSSNSSHLSRNQSFGRGDNPPPAPQRVDSIQVHSSQP-
SGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKP DVPPKPSFAPLSTSMKPNDACT NOV2r,
CG51896-10 SEQ ID NO:47 3165 bp DNA Sequence ORF Start: ATG at 13
ORF Stop: end of sequence
CAGCGCGGATCCATGAGGTCAGAAGCCTTGCTGCTGTATTTCACACTGCTACACTTTGCTGGGGCTGGTTTCC
CAGAAGATTCTGAGCCAATCAGTATTTCGCATGGCAACTATACAAAACAGTATCCG-
GTGTTTGTGGGCCACAA GCCAGGACGGAACACCACACAGAGGCACAGGCTGGACATC-
CAGATGATTATGATCATGAACGGAACCCTCTAC ATTGCTGCTAGGGACCATATTTAT-
ACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAA
AACTGACATGGAAATCTAGACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAAGGATGAGTGCCA
CAACTTTATTAAAGTTCTTCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAAC-
TAATGCCTTCAACCCT TCCTGCAGAAACTATAAGATGGATACATTGGAACCATTCGG-
GGATGAATTCAGCGGAATGGCCAGATGCCCAT ATGATGCCAAACATGCCAACGTTGC-
ACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCT
TGCCATTGACGCAGTCATTTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGATTCAAAA
TGGTTGAAAGAACCATACTTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTTC-
TTCTTCAGGGAAATAG CAGTGGAGTATAACACCATGGGAAAGGTAGTTTTCCCAAGA-
GTGGCTCAGGTTTGTAAGAATGATATGGGAGG ATCTCAAAGAGTCCTGGAGAAACAG-
TGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGAC
TCTCATTTTTATTTCAACATTCTCCAGCCAGTTACAGATGTGATTCGTATCAACGGGCGTGATGTTGTCCTGG
CAACGTTTTCTACACCTTATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACA-
TGCTTGACATTGCCAG TGTTTTTACTGGGAGATTCAAGGAACAGAAGTCTCCTGATT-
CCACCTGGACACCAGTTCCTGATGAACGAGTT CCTAAGCCCAGGCCAGGTTGCTGTG-
CTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTG
ATGATACCCTGAACTTCATCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATCTTCAACAGGCCATG
GTTCCTGAGAACAATGGTCAGATACCGCCTTACCAAAATTGCAGTGGACACAGCTGC-
TGGGCCATATCAGAAT CACACTGTGGTTTTTCTGGGATCAGAGAAGGGAATCATCTT-
GAAGTTTTTGGCCAGAATAGGAAATAGTGGTT TTCTAAATGACAGCCTTTTCCTGGA-
GGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGA
AGACAAAACGATCATGGGCATGCAGCTGGACAGAGCAAGCAGCTCTCTGTATGTTGCGTTCTCTACCTGTGTG
ATAAAGGTTCCCCTTGGCCGGTGTGAACGACATGGGAAGTGTAAAAAAACCTGTATT-
GCCTCCAGAGACCCGT ATTGTGGATGGATAAAGGAAGGTGGTGCCTGCAGCCATTTA-
TCACCCAACAGCAGACTGACTTTTGAGCAGGA CATAGAGCGTGGCAATACAGATGGT-
CTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATGACATTTCAACT
CCTCTACCAGATAATGAAATGTCTTACAACACAGTGTATGGGCATTCCAGTTCCCTCTTGCCCAGCACAACCA
CATCAGATTCGACGGCTCAAGAGGGGTATGAGTCTAGGGGAGGAATGCTGGACTGGA-
AGCATCTGCTTGACTC ACCTGACAGCACAGACCCTTTGGGGGCAGTGTCTTCCCATA-
ATCACCAAGACAAGAACGGAGTGATTCGGGAA AGTTACCTCAAAGGCCACGACCAGC-
TGGTTCCCGTCACCCTCTTGGCCATTGCAGTCATCCTGGCTTTCGTCA
TGGGGGCCGTCTTCTCGGGCATCACCGTCTACTGCGTCTGTGATCATCGGCGCAAAGACGTGGCTGTGGTGCA
GCGCAAGGAGAAGGAGCTCACCCACTCCCGCCGGGGCTCCATGAGCAGCGTCACCAA-
GCTCAGCGGCCTCTTT GGGGACACTCAATCCAAAGACCCAAAGCCGGAGGCCATCCT-
CACGCCACTCATCCACAACGGCAAGCTCGCCA CTCCCGGCAACACGGCCAAGATGCT-
CATTAAAGCAGACCAGCACCACCTGGACCTGACGGCCCTCCCCACCCC
AGAGTCAACCCCAACGCTGCAGCAGAAGCGGAAGCCCAGCCGCGGCAGCCGCGAGTGGGAGAGGAACCAGAAC
CTCATCAATGCCTGCACAAAGGACATCCCCCCCATGGGCTCCCCTGTGATTCCCACG-
GACCTGCCCCTGCGGG CCTCCCCCAGCCACATCCCCAGCGTGGTGGTCCTGCCCATC-
ACGCAGCAGGGCTACCAGCATGAGTACGTGCA CCAGCCCAAAATGAGCGAGGTGGCC-
CAGATGGCGCTGCAGGACCAGGCCGCCACACTGGAGTATAAGACCATC
AAGGAACATCTCAGCAGCAAGAGTCCCAACCATGGGGTGAACCTTGTGGAGAACCTGGACAGCCTGCCCCCCA
AAGTTCCACACCGGGAGGCCTCCCTGGGTCCCCCGGGAGCCTCCCTGTCTCAGACCG-
GTCTAAGCAAGCGGCT GGAAATGCACCACTCCTCTTCCTACGGGGTTGACTATAAGA-
GGAGCTACCCCACGAACTCGCTCACGAGAAGC CACCAGGCCACCACTCTCAAAAGAA-
ACAACACTAACTCCTCCAATTCCTCTCACCTCTCCAGAAACCAGAGCT
TTGGCAGGGGAGACAACCCGCCGCCCGCCCCGCAGAGGGTGGACTCCATCCAGGTGCACAGCTCCCAGCCATC
TGGCCAGCCCGTGACTGTCTCGAGGCAGCCCAGCCTCAACGCCTACAACTCACTGAC-
AAGGTCGGGGCTGAAG CGTACGCCCTCGCTAAAGCCGGACGTACCCCCCAAACCATC-
CTTTGCTCCCCTTTCCACATCCATGAAGCCCA ATGATGCGTGTACAGTCGACGCGCT- G
NOV2r, CG51896-10 Protein Sequence SEQ ID NO:48 1047 aa MW at
116308.5 kD MRSEALLLYFTLLHFAGAGFPEDSEPISISHGNY-
TKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAAR
DHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRNKGKHKDECHNFIKVLLKKNDDAIFVCGTNAFNPSCRN
YKMDTLEPFGDEFSGMARCPYDAKHANVALFADGKLYSATVTDFLAIDAVIYRSLGE-
SPTLRTVKHDSKWLKE PYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKN-
DMGGSQRVLEKQWTSFLKARLNCSVPGDSHFY FNILQAVTDVIRINGRDVVLATFST-
PYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPR
PGCCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAAGPYQNHTVV
FLGSEKGIILKFLARIGNSGFLMDSLFLEEMSVYNSEKCSYDGVEDKRIMGMQLDRA-
SSSLYVAFSTCVIKVP LGRCERHGKCKKTCIASRDPYCGWIKEGGACSHLSPNSRLT-
FEQDIERGNTDGLGDCHNSFVALNDISTPLPD NEMSYNTVYGHSSSLLPSTTTSDST-
AQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLK
GHDQLVPVTLLAIAVILAFVMGAVFSGITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQ
SKDPKPEAILTPLMHNGKLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQKRKP-
SRGSREWERNQNLINA CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQGYQH-
EYVDQPKMSEVAQMALEDQAATLEYKTIKEHL SSKSPNHGVNLVENLDSLPPKVPQR-
EASLGPPGASLSQTGLSKRLEMHHSSSYGVDYKRSYPTNSLTRSHQAT
TLKRNNTNSSNSSHLSRNQSFGRGDAAPPPAPQRVDSIQVHSSQPSCQAVTVSQPSLNAYNSLTRSGLKRTPS
LKPDVPPKPSFAPLSTSMKPNDACT NOV2s, CG51896-1 SEQ ID NO:49 1948 bp DNA
Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCGGATCCGGTTTCCCAGAAGATTCTGAGCCAATCAGTATTT-
CGCATGGCAACTATACAAAACAGTATCCG GTGTTTGTGGGCCACAAGCCAGGACGG-
AACACCACACAGAGGCACAGGCTGGACATCCAGATGATTATGATCA
TGAACGGAACCCTCTACATTGCTGCTAGGGACCATATTTATACTGTTGATATAGACACATCACACACGGAAGA
AATTTATTGTAGCAAAAAACTGACATGGAAATCTAGACAGGCCGATGTAGACACATG-
CAGAATGAAGGGAAAA CATAAGGATGAGTGCCACAACTTTATTAAAGTTCTTCTAAA-
GAAAAACGATGATGCATTGTTTGTCTGTGGAA CTAATCCCTTCAACCCTTCCTGCAG-
AAACTATAAGATGGATACATTGGAACCATTCGGGGATGAATTCAGCGG
AATGGCCAGATGCCCATATGATGCCAAACATGCCAACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCC
ACAGTGACTGACTTCCTTGCCATTGACGCAGTCATTTACCGGAGTCTTGGAGAAAGC-
CCTACCCTGCGGACCG TCAAGCACGATTCAAAATGGTTGAAAGAACCATACTTTGTT-
CAAGCCGTGGATTACGGAGATTATATCTACTT CTTCTTCAGGGAAATAGCAGTGCAG-
TATAACACCATGGGAAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGT
AAGAATGATATGGGAGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACT
GCTCAGTTCCTGGAGACTCTCATTTTTATTTCAACATTCTCCAGGCAGTTACAGATG-
TGATTCGTATCAACGG GCGTGATGTTGTCCTGGCAACGTTTTCTACACCTTATAACA-
GCATCCCTGGGTCTGCAGTCTGTGCCTATGAC ATGCTTGACATTGCCAGTGTTTTTA-
CTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCAG
TTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAAC
CTCCAATGAGTTCCCTGATGATACCCTGAACTTCATCAAGACGCACCCGCTCATGGA-
TGAGGCAGTGCCCTCC ATCTTCAACAGGCCATGGTTCCTGAGAACAATGGTCAGATA-
CCGCCTTACCAAAATTGCAGTGGACACAGCTG CTGGGCCATATCAGAATCACACTGT-
GGTTTTTCTGGGATCACAGAAGGGPATCATCTTGAAGTTTTTGGCCAG
AATAGGAAATAGTGGTTTTCTAAATGACAGCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGC
AGCTATGATGGAGTCGAAGACAAAAGGATCATGGGCATGCAGCTGGACAGAGCAAGC-
AGCTCTCTGTATGTTG CGTTCTCTACCTGTGTGATAAACGTTCCCCTTGGCCGGTGT-
GAACGACATGGGAAGTGTAAAAAAACCTGTAT TGCCTCCAGAGACCCGTATTGTGGA-
TGGATAAAGGAAGGTGGTGCCTGCAGCCATTTATCACCCAACAGCAGA
CTGACTTTTGACCAGCACATAGAGCGTGGCAATACAGATGGTCTGGGGGACTGTCACAATTCCTTTGTGGCAC
TGAATGACATTTCAACTCCTCTACCAGATAATGAAATGTCTTATAACACAGTGTATG-
GGCATTCCAGTTCCCT CTTGCCCAGCACAACCACATCAGATTCGACGGCTCAAGAGG-
GGTATGAGTCTAGGGGAGGAATGCTGGACTGG AAGCATCTGCTTGACTCACCTGACA-
GCACAGACCCTTTGGGGGCAGTGTCTTCCCACAATCACCAAGACAAGA
AGGGAGTGATTCGGGAAAGTTACCTCAAAGGCCACGACCAGGTCGACGGC NOV2s,
CG51896-11 Protein Sequence SEQ ID NO:50 649 aa MW at 72755.3 kD
TGSGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLYIAARDHIYT-
VDIDTSHTEE IYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLLKKNDDALFV-
CGTNAFNPSCRNYKMDTLEPFGDEFSG MARCPYDAKHAIWALFADGKLYSATVTDFL-
AIDAVIYRSLGESPTLRTVKHDSKWLKEPYFVQAVDYGDYIYF
FFREIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRING
RDVVLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKP-
RPGCCAGSSSLERYAT SNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLT-
KIAVDTAAGPYQNHTVVFLGSEKGIILKFLAR IGNSGFLNDSLFLEEMSVYNSEKCS-
YDGVEDKRIMGMQLDRASSSLYVAFSTCVIKVPLGRCERHGKCKKTCI
ASRDPYCGWIKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSL
LPSTTTSDSTAQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYL- KGHDQVDG
NOV2t, CG51896-12 SEQ ID NO:51 2583 bp DNA Sequence ORF Start: at 1
ORF Stop: end of sequence
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG-
GTGGACGTGAGCCACGA AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG-
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAG GAGCAGTACAACAGCACGTACCGT-
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA-
CCAGGTCAGCCTGACC TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA-
GTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGA-
CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG
CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG
AGCCTCTCCCTGTCTCCGGGTAAAGGCGGCGGCGGCGGCGGCGGCGGCGGTTTCCCA-
GAAGATTCTGAGCCAA TCAGTATTTCGCATGGCAACTATACAAAACAGTATCCGGTG-
TTTGTGGGCCACAAGCCAGGACGGAACACCAC ACAGAGGCACAGGCTGGACATCCAG-
ATGATTATGATCATGAACGGAACCCTCTACATTGCTGCTAGGGACCAT
ATTTATACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAACTGACATGGAAATCTA
GACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAAGGATGAGTGCCACA-
ACTTTATTAAAGTTCT TCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAACTA-
ATGCCTTCAACCCTTCCTGCAGAAACTATAAG ATGGATACATTGGAACCATTCGGGG-
ATGAATTCAGCGGAATGGCCAGATGCCCATATGATGCCAAACATGCCA
ACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTGCCATTGACGCAGTCAT
TTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGATTCAAAATG-
GTTGAAAGAACCATAC TTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTTCTT-
CTTCAGGGAAATAGCAGTGGAGTATAACACCA TGGGAAAGGTAGTTTTCCCAAGAGT-
GGCTCAGGTTTGTAAGAATGATATGGGAGGATCTCAAAGAGTCCTGGA
GAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTCTCATTTTTATTTCAAC
ATTCTCCAGGCAGTTACAGATGTGATTCGTATCAACGGGCGTGATGTTGTCCTGGCA-
ACGTTTTCTACACCTT ATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACATG-
CTTGACATTGCCAGTGTTTTTACTGGGAGATT CAAGGAACAGAAGTCTCCTGATTCC-
ACCTGGACACCAGTTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGT
TGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGATGATACCCTGAACTTCA
TCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATCTTCAACAGGCCATGGT-
TCCTGAGAACAATGGT CAGATACCGCCTTACCAAAATTGCAGTGGACACAGCTGCTG-
GGCCATATCAGAATCACACTGTGGTTTTTCTG GGATCAGAGAAGGGAATCATCTTGA-
AGTTTTTGGCCAGAATAGGAAATAGTGGTTTTCTAAATGACAGCCTTT
TCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGAAGACAAAAGGATCATGGG
CATGCACCTGGACAGAGCAAGCAGCTCTCTGTATGTTGCGTTCTCTACCTGTGTGAT-
AAAGGTTCCCCTTGGC CGGTGTGAACGACATGGGAAGTGTAAAAAAACCTGTATTGC-
CTCCAGAGACCCATATTGTGGATGGATAAAGG AAGGTGGTGCCTGCAGCCATTTATC-
ACCCAACAGCAGACTGACTTTTGAGCAGGACATAGAGCGTGGCAATAC
AGATGGTCTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATGGGCATTCCAGTTCCCTCTTGCCCAGCACA
ACCACATCAGATTCGACGGCTCAAGAGGGGTATGAGTCTAGGGGAGGAATGCTGGAC-
TGGAAGCATCTGCTTG ACTCACCTGACAGCACAGACCCTTTGGGGGCAGTGTCTTCC-
CATAATCACCAAGACAAGAAGGGAGTGATTCG GGAAAGTTACCTCAAAGGCCACGAC- CAG
NOV2t, CG51896-12 Protein Sequence SEQ ID NO:52 861 aa MW at
96283.9 kD DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT-
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF-
SCSVMHEALHNHYTQK SLSLSPGKGGGGGGGGGFPEDSEPISISHGNYTKQYPVFVG-
HKPGRNTTQRHRLDIQMIMIMNGTLYIAARDH IYTVDIDTSHTEEIYCSKKLTWKSR-
QADVDTCRMKGKHKDECHNFIKVLLKKNDDALFVCGTNAFNPSCRNYK
MDTLEPFGDEFSGMARCPYDAKHANVALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKEPY
FVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLK-
ARLNCSVPGDSHFYFN ILQAVTDVIRINGRDVVLATFSTPYNSIPGSAVCAYDMLDI-
ASVFTGRFKEQKSPDSTWTPVPDERVPKPRPG CCAGSSSLERYATSNEFPDDTLNFI-
KTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAAGPYQNHTVVFL
GSEKGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYDGVEDKRIMGMQLDRASSSLYVAFSTCVIKVPLG
RCERHGKCKKTCIASRDPYCGWIKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHN-
SFVALNGHSSSLLPST TTSDSTAQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSHNH-
QDKKGVIRESYLKGHDQ NOV2u, CG51896-13 SEQ ID NO:53 2634 bp DNA
Sequence ORF Start: at 1 ORF Stop: end of sequence
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG-
GTGGACGTGAGCCACGA AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG-
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAG GAGCAGTACAACAGCACGTACCGT-
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA-
CCAGGTCAGCCTGACC TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA-
GTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGA-
CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG
CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG
AGCCTCTCCCTGTCTCCGGGTAAAGGCGGCGGCGGCGGCGGCGGCGGCGGTTTCCCA-
GAAGATTCTGAGCCAA TCAGTATTTCGCATGGCAACTATACAAAACAGTATCCGGTG-
TTTGTGGGCCACAAGCCAGGACGGAACACCAC ACAGAGGCACAGGCTGGACATCCAG-
ATGATTATGATCATGAACGGAACCCTCTACATTGCTGCTAGGGACCAT
ATTTATACTGTTGATATAGACACATCACACACGGAAGAAATTTATTGTAGCAAAAAACTGACATGGAAATCTA
GACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAAGGATGAGTGCCACA-
ACTTTATTAAAGTTCT TCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAACTA-
ATGCCTTCAACCCTTCCTGCAGAAACTATAAG ATGGATACATTGGAACCATTCGGGG-
ATGAATTCAGCGGAATGGCCAGATGCCCATATGATGCCAAACATGCCA
ACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTGCCATTGACGCAGTCAT
TTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGATTCAAAATG-
GTTGAAAGAACCATAC TTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTTCTT-
CTTCAGGGAAATAGCAGTGGAGTATAACACCA TGGGAAAGGTAGTTTTCCCAAGAGT-
GGCTCAGGTTTGTAAGAATGATATGGGAGGATCTCAAAGAGTCCTGGA
GAAACACTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGAGACTCTCATTTTTATTTCAAC
ATTCTCCAGGCAGTTACAGATGTGATTCGTATCAACGGGCGTGATGTTGTCCTGGCA-
ACGTTTTCTACACCTT ATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACATC-
CTTGACATTGCCAGTGTTTTTACTGGGAGATT CAAGGAACAGAAGTCTCCTGATTCC-
ACCTGGACACCAGTTCCTGATGAACGAGTTCCTAAGCCCAGGCCAGGT
TGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCCCTGATGATACCCTGAACTTCA
TCAAGACGCACCCGCTCATGGATGAGGCAGTGCCCTCCATCTTCAACAGGCCATGGT-
TCCTGAGAACAATGGT CAGATACCGCCTTACCAAAATTGCAGTGGACACAGCTGCTG-
OGCCATATCAGAATCACACTGTGGTTTTTCTG GGATCAGAGAAGGCAATCATCTTGA-
AGTTTTTGGCCAGAATAGGAAATAGTGGTTTTCTAAATGACAGCCTTT
TCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGGAGTCGAAGACAAAAGGATCATGGG
CATGCAGCTGGACAGAGCAAGCAGCTCTCTGTATGTTGCGTTCTCTACCTGTGTGAT-
AAAGGTTCCCCTTGGC CGGTGTGAACGACATGGGAAGTGTAAAAAAACCTGTATTGC-
CTCCAGAGACCCGTATTGTGGATGGATAAAGC AAGGTGGTGCCTGCAGCCATTTATC-
ACCCAACAGCAGACTGACTTTTGAGCAGGACATAGAGCGTGGCAATAC
AGATGGTCTGGGGGACTGTCACAATTCCTTTGTCGCACTGAATGACATTTCAACTCCTCTACCAGATAATGAA
ATGTCTTATAACACAGTGTATGGGCATTCCAGTTCCCTCTTGCCCAGCACAACCACA-
TCAGATTCGACGGCTC AAGAGGGGTATGAGTCTAGGGGAGGAATGCTGGACTGGAAG-
CATCTGCTTGACTCACCTGACAGCACAGACCC TTTGGCGGCAGTGTCTTCCCACAAT-
CACCAAGACAAGAAGGGAGTGATTCGGGAAAGTTACCTCAAAGGCCAC GACCAG NOV2u,
CG51896-13 Protein Sequence SEQ ID NO:54 878 aa MW at 98225.0 kD
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV-
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKE-
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK
SLSLSPGKGGGGGGGGGFPEDSEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQ-
MIMIMNGTLYIAARDH IYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDE-
CHNFIKVLLKKNDDALFVCGTNAFNPSCRNYK MDTLEPFGDEFSGMARCPYDAKHAN-
VALFADGKLYSATVTDFLAIDAVIYRSLGESPTLRTVKHDSKWLKEPY
FVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMGGSQRVLEKQWTSFLKARLNCSVPGDSHFYFN
ILQAVTDVIRINGRDVVLATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDS-
TWTPVPDERVPKPRPG CCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVPSIFNR-
PWFLRTMVRYRLTKIAVDTAAGPYQNHTVVFL GSEKGIILKFLARIGNSGFLNDSLF-
LEEMSVYNSEKCSYDGVEDKRIMGMQLDRASSSLYVAFSTCVIKVPLG
RCERHGKCKKTCIASRDPYCGWIKEGGACSHLSPNSRLTFEQDIERGNTDGLGDCHNSFVALNDISTPLPDNE
MSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDWKHLLDSPDSTDPLCAVSSHN-
HQDKKGVIRESYLKGH DQ NOV2v, CG51896-14 SEQ ID NO:55 2113 bp DNA
Sequence ORF Start: at 1 ORF Stop: end of sequence
GCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGT-
TCCACTGGTGACGGTT TCCCAGAAGATTCTGAGCCAATCAGTATTTCGCATCGCAA-
CTATACAAAACAGTATCCGGTGTTTGTGGGCCA CAAGCCAGGACGGAACACCACACA-
GAGGCACAGGCTGGACATCCAGATGATTATGATCATGAACGGAACCCTC
TACATTGCTGCTAGGGACCATATTTATACTGTTGATATAGACACATCACACACGGAGGAAATTTATTGTAGCA
AAAAACTGACATGGAAATCTAGACAGGCCGATGTAGACACATGCAGAATCAAGGGAA-
AACATAACGATGAGTG CCACAACTTTATTAAAGTTCTTCTAAAGAAAAACGATGATG-
CATTGTTTGTCTGTGGAACTAATGCCTTCAAC CCTTCCTGCAGAAACTATAAGATGG-
ATACATTGGAACCATTCGGGGATGAATTCAGCGGAATGGCCAGATGCC
CATATGATGCCAAACATGCCAACGTTGCACTGTTTGCAGATGGAAAACTATACTCAGCCACAGTGACTGACTT
CCTTGCCATTGACGCAGTCATTTACCGGAGTCTTGGAGAAAGCCCTACCCTGCGGAC-
CGTCAAGCACGATTCA AAATGGTTGAAAGAACCATACTTTGTTCAAGCCGTGGATTA-
CGGAGATTATATCTACTTCTTCTTCAGGGAAA TTGCACTGGAGTATAACACCATGGG-
AAACGTAGTTTTCCCAAGAGTGGCTCACGTTTGTAAGAATGATATGGG
AGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCTGAAGGCGCGCTTGAACTGCTCAGTTCCTGGA
GACTCTCATTTTTATTTCAACATTCTCCAGGCAGTTACAGATGTGATTCGTATCAAC-
GGGCGTGATGTTGTCC TGGCAACGTTTTCTACACCTTATAACAGCATCCCTGGGTCT-
GCAGTCTGTGCCTATGACATGCTTGACATTGC CAGTGTTTTTACTGGGAGATTCAAG-
GAACAGAAGTCTCCTGATTCCACCTGGACACCAGTTCCTGATGAACGA
GTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATATGCAACCTCCAATGAGTTCC
CTGATGATACCCTGAACTTCATCAAGACGCACCCGCTCATGGATCAGGCAGTGCCCT-
CCATCTTCAACAGGCC ATGGTTCCTGACAACAATGGTCAGATACCGCCTTACCAAAA-
TTGCAGTGGACACAGCTGCTGGGCCATATCAG AATCACACTGTGGTTTTTCTGGGAT-
CAGAGAAGGGAATCATCTTGAAGTTTTTGGCCAGAATAGGAAATAGTG
GTTTTCTAAATGACAGCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCTATGATGCAGT
CGAAGACAAAAGGATCATGGGCATGCAGCTGGACAGAGCAAGCAGCTCTCTGTATGT-
TGCGTTCTCTACCTGT GTGATAAAGGTTCCCCTTGGCCGGTGTGAACGACATGGGAA-
GTGTAAAAAAACCTGTATTGCCTCCAGAGACC CGTATTGTGGATGGATAAAGGAAGG-
TGGTGCCTGCAGCCATTTATCACCCAACAGCAGACTGACTTTTGAGCA
GGACATAGAGCGTGGCAATACAGATGGTCTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATGACATTTCA
ACTCCTCTACCAGATAATGAAATGTCTTACAACACAGTGTATGGGCATTCCAGTTCC-
CTCTTGCCCAGCACAA CCACATCAGATTCGACGGCTCAAGAGGGGTATGAGTCTAGG-
GGAGGAATGCTGGACTGGAAGCATCTGCTTGA CTCACCTCACAGCACAGACCCTTTC-
GGGGCAGTGTCTTCCCATAATCACCAAGACAAGAAGGGAGTGATTCGG
GAAAGTTACCTCAAAGGCCACGACCAGTGACTCGAGGACTACAAGGATGACGATGACAAGGATTACAAAGACG
ACGATGATAAGGACTATAAGGATCATGACGACAAATAATAGCAATTCCTCGACGCTG-
CATAGGGTTACA NOV2v, CG51896-14 Protein Sequence SEQ ID NO:56 666 aa
MW at 74752.7 kD ATMETDTLLLWVLLLWVPGSTGDGFPEDS-
EPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTL
YIAARDHIYTVDIDTSHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNFIKVLLKKNDDALFVCGTNAFN
PSCRNYKMDTLEPFGDEFSGMARCPYDAKHANVALFADGKLYSATVTDFLAIDAVIY-
RSLGESPTLRTVKHDS KWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVA-
QVCKNDMGGSQRVLEKQWTSFLKARLNCSVPG DSHFYFNILQAVTDVIRINGRDVVL-
ATFSTPYNSIPGSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDER
VPKPRPGCCAGSSSLERYATSNEFPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAAGPYQ
NHTVVFLGSEKGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYDGVEDKRIMGM-
QLDRASSSLYVAFSTC VIKVPLGRCERHGKCKKTCIASRDPYCGWIKEGGACSHLSP-
NSRLTFEQDIERGNTDGLCDCHNSFVALNDIS TPLPDNEMSYNTVYGHSSSLLPSTT-
TSDSTAQEGYESRGGMLDWKHLLDSPDSTDPLGAVSSHNHQDKKGVIR ESYLKGHDQ
[0365] A ClustalW comparison of the above protein sequences yields
the following sequence alignment shown in Table 2B.
9TABLE 2B Comparison of the NOV2 protein sequences. NOV2a
--------------------------------------------- ---------------- (SEQ
ID NO: 14) NOV2b ----------------------------
--------------------------------- (SEQ ID NO: 16) NOV2c
------------------------------------------------------------ (SEQ
ID NO: 18) NOV2d
--------------------------------------------------------- ---- (SEQ
ID NO: 20) NOV2e -----------------------------------------
-------------------- (SEQ ID NO: 22) NOV2f
------------------------- ------------------------------------ (SEQ
ID NO: 24) NOV2g
------------------------------------------------------------ (SEQ
ID NO: 26) NOV2h
--------------------------------------------------------- ---- (SEQ
ID NO: 28) NOV2i -----------------------------------------
-------------------- (SEQ ID NO: 30) NOV2j
------------------------- ------------------------------------ (SEQ
ID NO: 32) NOV2k
------------------------------------------------------------ (SEQ
ID NO: 34) NOV2l
--------------------------------------------------------- ---- (SEQ
ID NO: 36) NOV2m -----------------------------------------
-------------------- (SEQ ID NO: 38) NOV2n
------------------------- ------------------------------------ (SEQ
ID NO: 40) NOV2o
------------------------------------------------------------ (SEQ
ID NO: 42) NOV2p
--------------------------------------------------------- ---- (SEQ
ID NO: 44) NOV2q -----------------------------------------
-------------------- (SEQ ID NO: 46) NOV2r
------------------------- ------------------------------------ (SEQ
ID NO: 48) NOV2s
------------------------------------------------------------ (SEQ
ID NO: 50) NOV2t
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN- WYVD (SEQ
ID NO: 52) NOV2u DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT-
CVVVDVSHEDPEVKFNWYVD (SEQ ID NO: 54) NOV2v
------------------------- ------------------------------------ (SEQ
ID NO: 56) NOV2a
------------------------------------------------------------ NOV2b
------------------------------------------------------------ NOV2c
------------------------------------------------------------ MOV2d
------------------------------------------------------------ NOV2e
------------------------------------------------------------ NOV2f
------------------------------------------------------------ NOV2g
------------------------------------------------------------ NOV2h
------------------------------------------------------------ NOV2i
------------------------------------------------------------ NOV2j
------------------------------------------------------------ NOV2k
------------------------------------------------------------ NOV2l
------------------------------------------------------------ NOV2m
------------------------------------------------------------ NOV2n
------------------------------------------------------------ NOV2o
------------------------------------------------------------ NOV2p
------------------------------------------------------------ NOV2q
------------------------------------------------------------ NOV2r
------------------------------------------------------------ NOV2s
------------------------------------------------------------ NOV2t
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK NOV2u
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK NOV2v
------------------------------------------------------------ NOV2a
------------------------------------------------------------ NOV2b
------------------------------------------------------------ NOV2c
------------------------------------------------------------ NOV2d
------------------------------------------------------------ NOV2e
------------------------------------------------------------ NOV2f
------------------------------------------------------------ NOV2g
------------------------------------------------------------ NOV2h
------------------------------------------------------------ NOV2i
------------------------------------------------------------ NOV2j
------------------------------------------------------------ NOV2k
------------------------------------------------------------ NOV2l
------------------------------------------------------------ NOV2m
------------------------------------------------------------ NOV2n
------------------------------------------------------------ NOV2o
------------------------------------------------------------ NOV2p
------------------------------------------------------------ NOV2q
------------------------------------------------------------ NOV2r
------------------------------------------------------------ NOV2s
------------------------------------------------------------ NOV2t
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS NOV2u
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS NOV2v
------------------------------------------------------------ NOV2a
----------------------------------MRSEALLLYFTLLHFAG---A- GFPED
NOV2b --------------------------------------------------------
----- NOV2c
-------------------------------------------------TG---S- GFPED
NOV2d --------------------------------------------------------
----- NOV2e
-------------------------------------------------------- -----
NOV2f ------------------------------TGSMRSEAILLLYFTLLHFAG---A-
GFPED NOV2g
-------------------------------------------------TG---S- GFPED
NOV2h -------------------------------------------------TG---S-
GFPED NOV2i
-----------------------------TGSMRSEALLLYLAATLLHFAG---A- GFPED
NOV2j ----------------------------------MRSEALLLYFTLLHFAG---A-
GFPED NOV2k
-------------------------------------------------------- GFPED
NOV2l --------------------------------------------------------
GFPED NOV2m
-------------------------------------------------------- -----
NOV2n --------------------------------------------------------
----- NOV2o
-------------------------------------------------------- -----
NOV2p --------------------------------------------------------
----- NOV2q
----------------------------------MRSEALLLYFTLLHFAG---A- GFPED
NOV2r ----------------------------------MRSEALLLYFTLLHFAG---A-
GFPED NOV2s
-------------------------------------------------TG---S- GFPED
NOV2t DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGG-
GFPED NOV2u
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGG- GFPED
NOV2v --------------------------------ATMETDTLLLWVLLLWVPGSTGD-
GFPED NOV2a SEPISISHCNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY-
IAARDHIYTVDIDT NOV2b
-AGSSISHCNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY- IAARDHIYTVDIDT
NOV2c SEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY-
IAARDHIYTVDIDT NOV2d
-----------------------------TGSLDIQMIMIMNGTLY- IAARDHIYTVDIDT
NOV2e -----------------------------TGSLDIQMIMIMNGTLY-
IAARDHIYTVDIDT NOV2f
SEPISISHGNYTKQYPVFVGNKPGRNTTQRHRLDIQMINIMNGTLY- IAARDNIYTVDIDT
NOV2g SEPISISHONYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY-
IAARDHIYTVDIDT NOV2h
SEPISISHGNYTKQYPVFVGNKPGRNTTQRHRLDIQMINIMNGTLY- IAARDHIYTVDIDT
NOV2i SEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY-
IAARDHIYTVDIDT NOV2j
SEPISISHGNYTKQYPVFVGNKPGRNTTQRHRLDIQMIMIMNGTLY- IAARDHIYTVDIDT
NOV2k SEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY-
IAARDHIYTVDIDT NOV2l
SEPISISHGNYTKQYPVFVGNKPGRNTTQRHRLDIQMIMIMNGTLY- IAARDHIYTVDIDT
NOV2m -----------------------------------------------
-------------- NOV2n
----------------------------------------------- --------------
NOV2o -----------------------------------------------
-------------- NOV2p
----------------------------------------------- --------------
NOV2q SEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQHIMIMNGTLY-
IAARDHIYTVDIDT NOV2r
SEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY- IAARDHIYTVDIDT
NOV2s SEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY-
IAARDHIYTVDIDT NOV2t
SEPISISHGNYTKQYPVFVGHKPGRNTTQRHRLDIQMIMIMNGTLY- IAARDHIYTVDIDT
NOV2u SEPISISHGNYTKQYPVFVGHKPGRNTTQRNRLDIQMIMINNGTLY-
IAARDHIYTVDIDT NOV2v
SEPISISHGNYTKQYPVFVGHKPORNTTQRHRLDIQMIMIMNGTLY- IAARDHIYTVDIDT
NOV2a SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2b
SHTEEIYCSKKLTWKSRQADVDTCRMKGKNKDECHNF- IKVLLKKNDDAIFVCGTNAFNPS
NOV2c SHTEEIYCSKKLTWKSRQADVDTCRMKGKNKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2d
SHTEEIYCSKKLTWKSRQADVDTCRMKGKNKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2e SHTEEIYCSKKLTWKSRQADVDTCRMKGKNKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2f
SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2g SHTEEIYCSKKLTWKSRQADVDTCRMKOKHKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2h
SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2i SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2j
SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2k SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECMNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2l
SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2m --------------------------------------
----------------------- NOV2n
-------------------------------------- -----------------------
NOV2o --------------------------------------
----------------------- NOV2p
-------------------------------------- -----------------------
NOV2q SHTEEIYCSKKLTWKSRQADVDTCRMKGKMKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2r
SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2s SHTEEIYCSKKLTWKSRQADVDTCRNKGKHKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2t
SHTEEIYCSKKLTWKSRQADVDTCRMKGKHKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2u SHTEEIYCSKKLTWKSRQADVDTCRNKGKHKDECHNF-
IKVLLKKNDDALFVCGTNAFNPS NOV2v
SHTEEIYCSKKLTWKSRQADVDTCRNKGKHKDECHNF- IKVLLKKNDDALFVCGTNAFNPS
NOV2a CRNYKMDTLEPFGDEFSGMARCPYDAKN-
ANVALFADOKLYSATVTDFLAIDAVIYRSLGE NOV2b
CRNYKMDTLEPFGDEFSGMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2c CRNYKMDTLEPFGDEFSGMARCPYDAKH-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2d
CRNYKMDTLEPFGDEFSGMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2e CRNYKNDTLEPFGDEFSGMARCPYDAKH-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2f
CRNYKMDTLEPFGDEFSGMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2g CRNYKMDTLEPFGDEFSGMARCPYDAKH-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2h
CRNYKMDTLEPFGDEFSGMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2i CRNYKMDTLEPFGDEFSGMARCPYDAKH-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2j
CRNYKMDTLEPFGDEFSGMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2k CRNYKMDTLEPFGDEFSGMARCPYDAKN-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2l
CRNYKMDTLEPFGDEFSGMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2m -----------------------------
------------------------------GE NOV2n
----------------------------- ------------------------------GE
NOV2o -----------------------------
-------------------------------- NOV2p
----------------------------- --------------------------------
NOV2q CRNYKMDTLEPFGDEFSGMARCPYDAKH-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2r
CRNYKMDTLEPFGDEFSCMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2s CRNYKMDTLEPFGDEFSGMARCPYDAKH-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2t
CRNYKMDTLEPFGDEFSGMARCPYDAKH- ANVALFADGKLYSATVTDFLAIDAVIYRSLGE
NOV2u CRNYKMDTLEPFGDEFSGMARCPYDAKH-
ANVALFADGKLYSATVTDFLAIDAVIYRSLGE NOV2v
CRNYAADTLEPFGDEFSGMARCPYDAAA- ANVALFADGKLYSATAADFAAIDAVIYRSLGE
NOV2a SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMG
NOV2b SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCAADMG
NOV2c SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCAADMG
NOV2d SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMG
NOV2e SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCAADMG
NOV2f SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMG
NOV2g SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMG
NOV2h SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCAADMG
NOV2i SPTLRTVAADSKWLKEPYFVQAVDYGDYIYFFFREIAVEYAAMGKVVFPRVAQVCAADMG
NOV2j SPTLRTVKNDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMCKVVFPRVAQVCAADMG
NOV2k SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMCKVVFPRVAQVCAADMG
NOV2l SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMCKVVFPRVAQVCAADMG
NOV2m SPTLRTVKHDSKWLKE--------------------------------------------
NOV2n SPTLRTVKHDSKWLKE--------------------------------------------
NOV2o ------------------------------------------------------------
NOV2p ------------------------------------------------------------
NOV2q SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMG
NOV2r SPTLRTVKHDSKWLKEPYFVQAVDYODYIYFFFREIAVEYNTMGKVVFPRVAQVCKNDMG
NOV2s SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCAADMG
NOV2t SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCAADMG
NOV2u SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAQVCAADMG
NOV2v SPTLRTVKHDSKWLKEPYFVQAVDYGDYIYFFFREIAVEYNTMGKVVFPRVAOVCAADMG
NOV2a GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPYNSIP
NOV2b GSQRVLEKQWTSFLKARLNCSVPGDSNFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2c GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGAAAAAATFSTPAASIP
NOV2d GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2e GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2f GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2g GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2h GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINCRDVVLATFSTPAASIP
NOV2i GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINCRDVVLATFSTPAASIP
NOV2j GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2k GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2l GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVVLATFSTPAASIP
NOV2m ------------------------------------------------------------
NOV2n ------------------------------------------------------------
NOV2o ------------------------------------------------------------
NOV2p ------------------------------------------------------------
NOV2q GSQRVLEKRWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDAALATFSTPYNSIP
NOV2r GSQRVLEKRWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDAALATFSTPYNSIP
NOV2s GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVAAATFSTPYNSIP
NOV2t GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVAAATFSTPYNSIP
NOV2u GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVAAATFSTPAASIP
NOV2v GSQRVLEKQWTSFLKARLNCSVPGDSHFYFNILQAVTDVIRINGRDVAAATFSTPYNSIP
NOV2a GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY-
ATSNE NOV2b
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY- ATSNE
NOV2c GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY-
ATSNE NOV2d
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY- ATSNE
NOV2e GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPRPRPGCCAGSSSLERY-
ATSNE NOV2f
GSAVCAYDMLDTASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY- ATSNE
NOV2g GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY-
ATSNE NOV2h
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY- ATSNE
NOV2i GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY-
ATSNE NOV2j
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPOCCAGSSSLERY- ATSNE
NOV2k GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY-
ATSNE NOV2l
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY- ATSNE
NOV2m --------------------------------------------------------
----- NOV2n
-------------------------------------------------------- -----
NOV2o ------------------------------------------------SSSLERY-
ATSNE NOV2p
-------------------------------------------------------- -----
NOV2q GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPCCCACSSSLERY-
ATSNE NOV2r
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY- ATSNE
NOV2s GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY-
ATSNE NOV2t
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY- ATSNE
NOV2u GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCAGSSSLERY-
ATSNE NOV2v
GSAVCAYDMLDIASVFTGRFKEQKSPDSTWTPVPDERVPKPRPGCCACSSSLERY- ATSNE
NOV2a FPDDTLNFIKTNPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA-
GPYQNHTVVFLGSE NOV2b
FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2c FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA-
GPYQNHTVVFLGSE NOV2d
FPDDTLNFIKTHTPLMDAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2e FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA-
GPYQNHTVVFLGSE NOV2f
FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2g FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA-
GPYQNHTVVFLCSE NOV2h
FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2i FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTNVRYRLTKIAVDTAA-
GPYQNHTVVFLGSE NOV2j
FPDDTLNFIKThPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2k FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA-
GPYQNHTVVFLGSE NOV2l
FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVR------------- --------------
NOV2m -----------------------------------------------
-------------- NOV2n
----------------------------------------------- --------------
NOV2o FPDDT------------------------------------------
-------------- NOV2p
----------------------------------------------- --------------
NOV2q FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVR-------------
-------------- NOV2r
FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2s FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA-
GPYQNHTVVFLGSE NOV2t
FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2u FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA-
GPYQNHTVVFLGSE NOV2v
FPDDTLNFIKTHPLMDEAVPSIFNRPWFLRTMVRYRLTKIAVDTAA- GPYQNHTVVFLGSE
NOV2a KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD-
GVEDKRIMGMQLDRASSSLYVAF NOV2b
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQLDRASSSLYVAF
NOV2c KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD-
GVEDKRIMGMQLDRASSSLYVAF NOV2d
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQLDRASSSLYVAF
NOV2e KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD-
GVEDKRIMGMQVDG--------- NOV2f
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQVDG---------
NOV2g KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD-
GVEDKRIMGMQVDG--------- NOV2h
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQLDRASSSLYVAF
NOV2i KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD-
GVEDKRIMGMQLDRASSSLYVAF NOV2j
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQLDRASSSLYVAF
NOV2k KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD-
GVEDKRIMGMQLDRASSSLYVAF NOV2l
---------------------------------CSYD- GVEDKRIMGMQLDRASSSLYVAF
NOV2m --------------------------------------
----------------------- NOV2n
-------------------------------------- -----------------------
NOV2o --------------------------------------
----------------------- NOV2p
-----------------------EEMSVYNSEKCSYD- GVEDKR-----------------
NOV2q ---------------------------------CSYD-
GVEDKRIMGMQLDRASSSLYVAF NOV2r
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQLDRASSSLYVAF
NOV2s KGIILKFLARIGNSGFLNDSLFLEEMSYNSEKCSYDG-
VEDKRIMGMQLDRAASSSLAAAF NOV2t
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQLDRASSSLYVAF
NOV2u KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD-
GVEDKRIMGMQLDAASSSLAAAF NOV2v
KGIILKFLARIGNSGFLNDSLFLEEMSVYNSEKCSYD- GVEDKRIMGMQLDRASSSLAAAF
NOV2a STCVIKVPLGRCERHGKCKKTCIASRDP-
YCGWIKEGGACSHLSPNSAATFEQDIERGNTD NOV2b
STCVIKVPLGRCERHGKCKKTCIASRDP- YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD
NOV2c STCVIKVPLGRCERHGKCKKTCIASRDP-
YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD NOV2d
STCVIKVPLGRCERMGKCKKTCIASRDP- YCGWIKEGGACSHLSPNSRLTFEQDIERCAAD
NOV2e -----------------------------
-------------------------------- NOV2f
----------------------------- --------------------------------
NOV2g -----------------------------
-------------------------------- NOV2h
STCVIKVPLGRCERHGKCKKTCIASRDP- YCGWIKEGOACSHLSPNSRLTFEQDIERGNTD
NOV2i STCVIKVPLGRCERHGKCKKTCIASRDP-
YCOWIKEGGACSHLSPNSRLTFEQDIERGNTD NOV2j
STCVIKVPLGRCERHGKCKKTCIASRDP- YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD
NOV2k STCVIKVPLGRCERHGKCKKTCIASRDP-
YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD NOV2l
STCVIKVPLGRCERHGKCKKTCIASRDP- YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD
NOV2m -----------------------------
-------------------------------- NOV2n
----------------------------- --------------------------------
NOV2o -----------------------------
-------------------------------- NOV2p
----------------------------- --------------------------------
NOV2q STCVIKVPLGRCERHGKCKKTCIASRDP-
YCGWIKEGGACSHLSPNSRLTFEQDIERGAAD NOV2r
STCVIKVPLGRCERHGKCKKTCIASRDP- YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD
NOV2s STCVIKVPLGRCERNGKCKKTCIASRDP-
YCGWIKEGGACSHLSPNSRLTFEQDIERCNTD NOV2t
STCVIKVPLGRCERHGKCKKTCIASRDP- YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD
NOV2u STCVIKVPLGRCERHGKCKKTCIASRDP-
YCCWIKEGGACSHLSPNSRLTFEQDIERGNTD NOV2v
STCVIKVPLGRCERHGKCKKTCIASRDP- YCGWIKEGGACSHLSPNSRLTFEQDIERGNTD
NOV2a GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2b GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2c GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2d GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGOMLDW
NOV2e ------------------------------------------------------------
NOV2f ------------------------------------------------------------
NOV2q ------------------------------------------------------------
NOV2h GLGDCHNSFVALN-----------------GHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2i GLGDCHNSFVALN-----------------------------------------------
NOV2j GLGDCHNSFVALNG-----------------HSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2k GLCDCHNSFVALN-----------------GNSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2l GLGDCHNSFVALN-----------------GHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2m ------------------------------------------------------------
NOV2n ------------------------------------------------------------
NOV2o ------------------------------------------------------------
NOV2p ------------------------------------------------------------
NOV2q GLGDCHNSFVALN-----------------CHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2r GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2s GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2t GLGDCHNSFVALN-----------------GHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2u GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGMLDW
NOV2v GLGDCHNSFVALNDISTPLPDNEMSYNTVYGHSSSLLPSTTTSDSTAQEGYESRGGAADW
NOV2a KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAFVMGAVFS
NOV2b KHLLDSPDSTDPLGAVSSMNHQDKKGVIRESYLKGHDQVDG-------------------
NOV2c KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAFVMGAVFS
NOV2d KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPAALLAIAVILAFAAGAVFS
NOV2e ------------------------------------------------------------
NOV2f ------------------------------------------------------------
NOV2g ------------------------------------------------------------
NOV2h KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAFAAGAVFS
NOV2i -------------------------GVIRESYLKGHDQLVPVTLLAIAVILAFVMGAVFS
NOV2j KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAFVMGAVFS
NOV2k KHNLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQ---------------------
NOV2l KHNLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLMAVILAFVMGAVFS
NOV2m ------------------------------------------------------------
NOV2n ------------------------------------------------------------
NOV2o ------------------------------------------------------------
NOV2p ------------------------------------------------------------
NOV2q KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAFVMGAVFS
NOV2r KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQLVPVTLLAIAVILAFVMGAVFS
NOV2s KHLLDSPDSTDPLGAVSSNNHQDKKGVIRESYLKGHDQVDG-------------------
NOV2t KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQ----------------------
NOV2u KHLLDSPDSTDPLGAVSSMNHQDKKGVIRESYLKGHDQ----------------------
NOV2v KHLLDSPDSTDPLGAVSSHNHQDKKGVIRESYLKGHDQ----------------------
NOV2a GITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQSKDPKPEA-
ILTPL NOV2b
-------------------------------------------------------- -----
NOV2c GITVYCVCDNRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQSKDPKPEA-
ILTPL NOV2d
GITVYCVCDHRRKDVAVVQRKEKELTHSRROSMSSVTKLSGLFGDTQSKDPKPEA- ILTPL
NOV2e --------------------------------------------------------
----- NOV2f
-------------------------------------------------------- -----
NOV2g --------------------------------------------------------
----- NOV2h
GITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQSKDPKPEA- ILTPL
NOV2i GITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTOSKDPKPEA-
ILTPL NOV2j
GITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQSKDPKPEA- ILTPL
NOV2k --------------------------------------------------------
----- NOV2l
GITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQ--------- -----
NOV2m --------------------------------------------------------
----- NOV2n
-------------------------------------------------------- -----
NOV2o --------------------------------------------------------
----- NOV2p
-------------------------------------------------------- -----
NOV2q GITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQSKDPKPEA-
ILTPL NOV2r
GITVYCVCDHRRKDVAVVQRKEKELTHSRRGSMSSVTKLSGLFGDTQSKDPKPEA- ILTPL
NOV2s --------------------------------------------------------
----- NOV2t
-------------------------------------------------------- -----
NOV2u --------------------------------------------------------
----- NOV2v
-------------------------------------------------------- -----
NOV2a MHNGKLATPGNTAKNLIKADQHHLDLTALPTPESTPTLQQKRKPSR-
GSREWERNQNLINA NOV2b
----------------------------------------------- --------------
NOV2c MHNGKLATPGNTAKMLIKADQHNLDLTALPTPESTPTLQQKRKPSR-
GSREWERNQNLINA NOV2d
MHNGKLATPGNTAKMLTKADQHHLDLTALPTPESTPTLQQKRKPSR- GSREWERNQNLINA
NOV2e -----------------------------------------------
-------------- NOV2f
----------------------------------------------- --------------
NOV2g -----------------------------------------------
-------------- NOV2h
MHNGKLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQKRKPSR- GSREWERNQNLINA
NOV2i MHNGKLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQKRKPSR-
GSREWERNQNLINA NOV2j
MHNGKLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQKREPSR- GTREWERNQNLINA
NOV2k -----------------------------------------------
-------------- NOV2l
----------------------------------------------- --------------
NOV2m -----------------------------------------------
-------------- NOV2n
----------------------------------------------- --------------
NOV2o -----------------------------------------------
-------------- NOV2p
----------------------------------------------- --------------
NOV2q MHNGKLATPGNTAKMLIKADQHHLDLTALPTPESTPTLQQKRKPSR-
GSREWERNQNLINA NOV2r
MHNGKLATPGNTAKMLIKADQHNLDLTALPTPESTPTLQQKRKPSR- GSREWERNQNLINA
NOV2s -----------------------------------------------
-------------- NOV2t
----------------------------------------------- --------------
NOV2u -----------------------------------------------
-------------- NQV2v
----------------------------------------------- --------------
NOV2a CTKDMPPMCSPVIPTDLPLRASPSHIPSVVVLPITQQ-
GYQHEYVDQPKMSEVAQMALEDQ NOV2b
-------------------------------------- -----------------------
NOV2c CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQ-
GYQHEYVDQPKMSEVAQMALEDQ NOV2d
CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQ- GYQHEYVDQPKMSEVAQMALEDQ
NOV2e --------------------------------------
----------------------- NOV2f
-------------------------------------- -----------------------
NOV2g --------------------------------------
----------------------- NOV2h
CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQ- CYQHEYVDQPKNSEVAQMALEDQ
NOV2i CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQ-
GYQHEYVDQPKMSEVAQMALEDQ NOV2j
CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQ- GYQHEYVDQPKMSEVAQMALEDQ
NOV2k --------------------------------------
----------------------- NOV2l
-------------------------------------- -----------------------
NOV2m --------------------------------------
----------------------- NOV2n
-------------------------------------- -----------------------
NOV2o --------------------------------------
----------------------- NOV2p
-------------------------------------- -----------------------
NOV2q CTKDMPPNGSPVIPTDLPLRASPSHIPSVVVLPITQQ-
GYQHEYVDQPKESEVAQMALEDQ NOV2r
CTKDMPPMGSPVIPTDLPLRASPSHIPSVVVLPITQQ- GYQHEYVDQPKMSEVAQMALEDQ
NOV2s --------------------------------------
----------------------- NOV2t
-------------------------------------- -----------------------
NOV2u --------------------------------------
----------------------- NOV2v
-------------------------------------- -----------------------
NOV2a AATLEYKTIKEHLSSKSPNHGVNLVENL-
DSLPPKVPQREASLGPPGASLSQTGLSKRLEM NOV2b
----------------------------- --------------------------------
NOV2c AATLEYKTIKEHLSSKSPNHGVNLVENL-
DSLPPKVPQREASLGPPGASLSQTGLSKRLEM NOV2d
AATLEYKTIKEHLSSKSPNHGVNLVENL- DSLPPKVPQREASLOPPGASLSQTGLSKRLEM
NOV2e -----------------------------
-------------------------------- NOV2f
----------------------------- --------------------------------
NOV2g -----------------------------
-------------------------------- NOV2h
AATLEYKTIKEHLSSKSPNHGVNLVENL- DSLPPKVPQREASLGPPGASLSQTGLSKRLEM
NOV2i AATLEYKTIKEHLSSKSPNHCVNLVENL-
DSLPPKVPQREASLGPPGASLSQTGLSKRLEM NOV2j
AATLEYKTIKEHLSSKSPNHGVNLVENL- DSLPPKVPQREASLGPPOASLSQTGLSKRLEM
NOV2k -----------------------------
-------------------------------- NOV2l
----------------------------- --------------------------------
NOV2m -----------------------------
-------------------------------- NOV2n
----------------------------- --------------------------------
NOV2o -----------------------------
-------------------------------- NOV2p
----------------------------- --------------------------------
NOV2q AATLEYKTIKEHLSSKSPNHGVNLVENL-
DSLPPKVPQREASLGPPGASLSQTGLSKRLEM NOV2r
AATLEYKTIKEHLSSKSPNHGVNLVENL- DSLPPKVPQREASLGPPGASLSQTOLSKRLEM
NOV2s -----------------------------
-------------------------------- NOV2t
----------------------------- --------------------------------
NOV2u -----------------------------
-------------------------------- NOV2v
----------------------------- --------------------------------
NOV2a NHSSSYGVDYKRSYPTNSLTRSHQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRV
NOV2b ------------------------------------------------------------
NOV2c NHSSSYGVDYKRSYPTNSLTRSHQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRV
NOV2d HHSSSYGVDYKRSYPTNSLTRSHQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRV
NOV2e ------------------------------------------------------------
NOV2f ------------------------------------------------------------
NOV2g ------------------------------------------------------------
NOV2h HHSSSYGVDYKRSYPTNSLTRSHQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRV
NOV2i HHSSSYGVDYKRSYPTNSLTRSNQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRV
NOV2j HHSSSYGVDYKRSYPTNSLTRSNQATTYSHQKQH--------------------------
NOV2k ------------------------------------------------------------
NOV2l ------------------------------------------------------------
NOV2m ------------------------------------------------------------
NOV2n ------------------------------------------------------------
NOV2o ------------------------------------------------------------
NOV2p ------------------------------------------------------------
NOV2q HHSSSYGVDYKRSYPTNSLTRSHQATTLKRNNTNSSNSSHLSRNQSFCRGDNPPPAPQRV
NOV2r HHSSSYGVDYKRSYPTNSLTRSHQATTLKRNNTNSSNSSHLSRNQSFGRGDNPPPAPQRV
NOV2s ------------------------------------------------------------
NOV2t ------------------------------------------------------------
NOV2u ------------------------------------------------------------
NOV2v ------------------------------------------------------------
NOV2a DSIQVHSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPN
NOV2b ------------------------------------------------------------
NOV2c DSIQVHSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPN
NOV2d DSIQVHSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPN
NOV2e ------------------------------------------------------------
NOV2f ------------------------------------------------------------
NOV2g ------------------------------------------------------------
NOV2h DSIQVHSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPN
NOV2i DSIQVHSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPN
NOV2j ------------------------------------------------------------
NOV2k ------------------------------------------------------------
NOV2l ------------------------------------------------------------
NOV2m ------------------------------------------------------------
NOV2n ------------------------------------------------------------
NOV2o ------------------------------------------------------------
NOV2p ------------------------------------------------------------
NOV2q DSIQVMSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPN
NOV2r DSIQVNSSQPSGQAVTVSRQPSLNAYNSLTRSGLKRTPSLKPDVPPKPSFAPLSTSMKPN
NOV2s ------------------------------------------------------------
NOV2t ------------------------------------------------------------
NOV2u ------------------------------------------------------------
NOV2v ------------------------------------------------------------
NOV2a DACT--- NOV2b ------- NOV2c DACTVDG NOV2d DACTVDG NOV2e
------- NOV2f ------- NOV2g ------- NOV2h DACTVDG NOV2i DACTVDG
NOV2j ------- NOV2k ------- NOV2l ------- NOV2m ------- NOV2n
------- NOV2o ------- NOV2p ------- NOV2q DACT--- NOV2r DACT---
NOV2s ------- NOV2t ------- NOV2u ------- NOV2v -------
[0366] Further analysis of the NOV2a protein yielded the following
properties shown in Table 2C.
10TABLE 2C Protein Sequence Properties NOV2a SignalP analysis:
Cleavage site between residues 19 and 20 PSORT II analysis: PSG: a
new signal peptide prediction method N-region: length 4; pos.chg 1;
neg.chg 1 H-region: length 17; peak value 9.51 PSG score: 5.11 GvH:
von Heijne's method for signal seq. recognition GvH score
(threshold: -2.1): 1.58 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 =
-11.62 Transmembrane 662-678 PERIPHERAL Likelihood = 2.28 (at 436)
ALOM score: -11.62 (number of TMSs: 1) MTOP: Prediction of membrane
topology (Hartmann et al.) Center position for calculation: 9
Charge difference: -3.5 C(-2.5) - N(1.0) N >= C: N-terminal side
will be inside >>> membrane topology: type 1a (cytoplasmic
tail 679 to 1047) MITDISC: discrimination of mitochondrial
targeting seq R content: 1 Hyd Moment(75): 3.63 Hyd Moment(95):
2.72 G content: 2 D/E content: 2 S/T content: 2 Score: -7.22 Gavel:
prediction of cleavage sites for mitochondrial preseq R-2 motif at
12 MRS.vertline.EA NUCDISC: discrimination of nuclear localization
signals pat4: HRRK (3) at 693 pat4: KRKP (4) at 784 pat7: none
bipartite: none content of basic residues: 11.4% NLS Score: -0.03
KDEL: ER retention motif in the C-terminus: none ER Membrane
Retention Signals: XXRR-like motif in the N-terminus: RSEA none
SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd
peroxisomal targeting signal: none VAC: possible vacuolar targeting
motif: none RNA-binding motif: none Actinin-type actin-binding
motif: type 1: none type 2: none NMYR: N-myristoylation pattern:
none Prenylation motif: none memYQRL: transport motif from cell
surface to Golgi: none Tyrosines in the tail: too long tail
Dileucine motif in the tail: none checking 63 PROSITE DNA binding
motifs: none checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN:
Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: nuclear Reliability: 89 COIL: Lupas's algorithm to
detect coiled-coil regions total: 0 residues Final Results (k =
9/23): 44.4%: extracellular, including cell wall 22.2%: Golgi
22.2%: endoplasmic reticulum 11.1%: plasma membrane >>
prediction for CG51896-04 is exc (k = 9)
[0367] A search of the NOV2a protein against the Geneseq database,
a proprietary database that contains sequences published in patents
and patent publication, yielded several homologous proteins shown
in Table 2D.
11TABLE 2D Geneseq Results for NOV2a NOV2a Residues/ Identities/
Geneseq Protein/Organism/Length [Patent Match Similarities for the
Expect Identifier #, Date] Residues Matched Region Value AAY71460
Human semaphorin 6A-1 - Homo 1 . . . 1047 1029/1047 (98%) 0.0
sapiens, 1030 aa. 1 . . . 1030 1029/1047 (98%) [WO200031252-A1,
02-JUN-2000] AAB23030 Human semaphorin protein-like 1 . . . 949
927/949 (97%) 0.0 splice variant, SECX 2864933-1 - 1 . . . 932
929/949 (97%) Homo sapiens, 939 aa. [WO200053742-A2, 14-SEP-2000]
AAB95139 Human protein sequence SEQ ID 332 . . . 1047 699/716 (97%)
0.0 NO: 17154 - Homo sapiens, 699 aa. 1 . . . 699 699/716 (97%)
[EP1074617-A2, 07-FEB-2001] AAB23043 Human semaphorin protein-like
17 . . . 662 627/646 (97%) 0.0 splice variant, SECX 1 . . . 629
628/646 (97%) pCR2.1-2864933 - Homo sapiens, 630 aa.
[WO200053742-A2, 14-SEP-2000] AAB90731 Human CJ145.sub.- 1 protein
sequence 1 . . . 578 575/578 (99%) 0.0 SEQ ID 161 - Homo sapiens,
975 aa. 1 . . . 578 576/578 (99%) [WO200119988-A1, 22-MAR-2001]
[0368] In a BLAST search of public sequence databases, the NOV2a
protein was found to have homology to the proteins shown in the
BLASTP data in Table 2E.
12TABLE 2E Public BLASTP Results for NOV2a NOV2a Protein Residues/
Identities/ Accession Match Similarities for the Expect Number
Protein/Organism/Length Residues Matched Portion Value Q9P2H9
Hypothetical protein KIAA1368 - 1 . . . 1047 1046/1047 (99%) 0.0
Homo sapiens (Human), 1049 aa 3 . . . 1049 1046/1047 (99%)
(fragment). Q9H2E6 Semaphorin SEMA6A1 - Homo 1 . . . 1047 1029/1047
(98%) 0.0 sapiens (Human), 1030 aa. 1 . . . 1030 1029/1047 (98%)
Q9EQ71 Axon guidance signal SEMA6A1 - 1 . . . 1047 947/1048 (90%)
0.0 Mus musculus (Mouse), 1005 aa. 1 . . . 1005 973/1048 (92%)
O35464 Semaphorin 6A precursor 1 . . . 880 815/881 (92%) 0.0
(Semaphorin VIA) (Sema VIA) 1 . . . 864 839/881 (94%) (Semaphorin
Q) (Sema Q) - Mus musculus (Mouse), 888 aa. Q96SW4 Hypothetical
protein FLJ14595 - 332 . . . 1047 699/716 (97%) 0.0 Homo sapiens
(Human), 699 aa. 1 . . . 699 699/716 (97%)
[0369] PFam analysis predicts that the NOV2a protein contains the
domains shown in the Table 2F.
13TABLE 2F Domain Analysis of NOV2a Identities/Similarities NOV2a
for the Pfam Domain Match Region Matched Region Expect Value Sema
56 . . . 491 203/497 (41%) 4.5e-212 390/497 (78%) PSI 514 . . . 557
14/68 (21%) 0.42 29/68 (43%)
Example 3
[0370] The NOV3 clone was analyzed, and the nucleotide and encoded
polypeptide sequences are shown in Table 3A.
14TABLE 3A NOV3 Sequence Analysis NOV3a, CG52324-01 SEQ ID NO:57
1932 bp DNA Sequence ORF Start: ATG at 113 ORF Stop: end of
sequence
CGCGTGCAGGTGGCAGTCCTCCCAAAGTACTTGTGTCCGGGTGGTGGACTGGATTCGCTGCGGACCCCTGCAA
GCTGCCTTTCCTTCTCCCTGTGCTTAACCAGAGGTGCCCATGGGTTGGACAATGAG-
GCTGGTCACAGCAGCAC TGTTACTGGGTCTCATGATGGTGGTCACTGGAGACGAGGA-
TGAGAACAGCCCGTGTGCCCATGAGGCCCTCTT GGACGAGGACACCCTCTTTTGCCA-
GGGCCTTGAAGTTTTCTACCCAGAGTTGGGGAACATTGGCTGCAAGGTT
GTTCCTGATTGTAACAACTACAGACAGAAGATCACCTCCTGGATGGAGCCGATAGTCAAGTTCCCGGGGGCCG
TGGACGGCGCAACCTATATCCTGGTGATGGTGGATCCAGATGCCCCTAGCAGAGCAG-
AACCCAGACAGAGATT CTGGAGACATTGGCTGGTAACAGATATCAAGGGCGCCGACC-
TGAAGGAAGGGAAGATTCAGGGCCAGGAGTTA TCAGCCTACCAGGCTCCCTCCCCAC-
CGGCACACAGTGGCTTCCATCGCTACCAGTTCTTTGTCTATCTTCAGG
AAGGAAAAGTCATCTCTCTCCTTCCCAAGGAAAACAAAACTCGAGGCTCTTGGAAAATGGACAGATTTCTGAA
CCGTTTCCACCTGGGCGAACCTGAAGCAAGCACCCAGTTCATGACCCAGAACTACCA-
GCACTCACCAACCCTC CAGGCTCCCACAGAAAGGGCCAGCGAGCCCAACCACAAAAA-
CCAGGCGGAGATAGCTGCCTGCTAGATAGCCG GCTTTGCCATCCGGGCATGTGGCCA-
CACTGCCCACCACCGACGATGTGGGTATGGAACCCCCTCTGGATACAG
AACCCCTTCTTTTCCAATTAAAAAAAAAAATCATCCAGGAAAAAAAAAAAAAAAA NOV3a,
CG52324-01 Protein Sequence SEQ ID NO:58 227 aa MW at 25734.1 kD
MGWTMRLVTAALLLGLMMVVTGDEDENSPCAHEALLDEDTLFCQGLEVFYPELGN-
IGCKVVPDCNNYRQKITS WMEPIVKFPGAVDGATYILVMVDPDAPSRAEPRQRFWR-
HWLVTDIKGADLKEGKIQGQELSAYQAPSPPAHSG
FHRYQFFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRERASEPKHK
NQAEIAAC NOV3b, 249357821 SEQ ID NO:59 706 bp DNA Sequence ORF
Start: at 2 ORF Stop: end of sequence
CACCAGATCTCCCACCATGGGTTGGACAATGAGGCTGGTCACAGCAGCACTGTTACTGGGTCTCATGATG-
GTG GTCACTCGAGACGAGGATGAGAACAGCCCGTGTGCCCATGAGGCCCTCTTGGA-
CGAGGACACCCTCTTTTGCC AGGGCCTTGAAGTTTTCTACCCAGAGTTGGGGAACAT-
TGGCTGCAAGGTTGTTCCTGATTGTAACAACTACAG
ACAGAAGATCACCTCCTGGATGGAGCCGATAGTCAAGTTCCCGGGGGCCGTGGACGGCGCAACCTATATCCTG
GTGATGGTGGATCCAGATGCCCCTAGCAGAGCAGAACCCAGACAGAGATTCTGGAGA-
CATTGGCTGGTAACAG ATATCAAGGGCGCCGACCTGAAGGAAGGGAAGATTCAGGGC-
CAGGAGTTATCAGCCTACCAGGCTCCCTCCCC ACCGGCACACAGTGGCTTCCATCGC-
TACCAGTTCTTTGTCTATCTTCAGGAAGGAAAAGTCATCTCTCTCCTT
CCCAAGGAAAACAAAACTCGAGGCTCTTGGAAAATGGACAGATTTCTGAACCGTTTCCACCTGGGCGAACCTG
AAGCAAGCACCCAGTTCATGACCCAGAACTACCAGGACTCACCAACCCTCCAGGCTC-
CCAGAGAAAGGGCCAG CGAGCCCAAGCACAAAAACCAGGCGGAGATAGCTGCCTGCG-
TCGACGGC NOV3b, 249357821 Protein Sequence SEQ ID NO:60 235 aa MW
at 26547.9 kD TRSPTMGWTMRLVTAALLLGLMMVVTGDEDENS-
PCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYR
QKITSWMEPIVKFPGAVDGATYILVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQAPSP
PAHSGFHRYQFFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQ-
NYQDSPTLQAPRERAS EPKHKNQAEIAACVDG NOV3c, 249357798 SEQ ID NO:61 706
bp DNA Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCAGATCTCCCACCATCGGTTGGACAATGAGGCTGGTCACAG-
CAGCACTGTTACTGGGTCTCATGATGGTG GTCACTGGAGACGAGGATGAGAACAGC-
CCGTGTGCCCATGAGGCCCTCTTGGACGAGGACACCCTCTTTTGCC
AGGGCCTTGAAGTTTTCTACCCAGAGTTGGGGAACATTGGCTGCAAGGTTGTTCCTGATTGTAACAACTACAG
ACAGAAGATCACCTCCTGGATGGAGCCGATAGTCAAGTTCCCGGGGGCCGTGGACGG-
CGCAACCTATATCCTG GTGATGGTGGATCCAGATGCCCCTAGCAGAGCAGAACCCAG-
ACAGAGATTCTGGAGACATTGGCTGGTAACAG ATATCAAGGGCGCCGACCTGAAGAA-
AGGGAAGATTCAGGGCCAGGAGTTATCAGCCTACCAGGCTCCCTCCCC
ACCGGCACACAGTGGCTTCCATCGCTACCAGTTCTTTGTCTATCTTCAGGAAGGAAAAGTCATCTCTCTCCTT
CCCAAGGAAAACAAAACTCGAGGCTCTTGGAAAATGGACAGATTTCTGAACCGTTTC-
CACCTGGGCGAACCTG AAGCAAGCACCCAGTTCATGACCCACAACTACCAGGACTCA-
CCAACCCTCCAGGCTCCCAGAGAAAGGGCCAG CGAGCCCAAGCACAAAAACCAGGCG-
GAGATAGCTGCCTGCGTCGACGGC NOV3c, 249357798 Protein Sequence SEQ ID
NO:62 235 aa MW at 26547.0 kD
TRSPTMGWTMRLVTAALLLGLMMVVTGDEDENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYR
QKITSWMEPIVKFPGAVDGATYILVMVDPDAPSRAEPRQRFWRIWLVTDIKGADLK-
KGKIQGQELSAYQAPSP PAHSGFHRYQFFVYLQEGKVISLLPKENKTRGSWKMDRFL-
NRFHLGEPEASTQFMTQNYQDSPTLQAPRERAS EPKHKNQAEIAACVDG NOV3d, 248644954
SEQ ID NO:63 619 bp DNA Sequence ORF Start: at 2 ORF Stop: end of
sequence CACCAGATCTCCCACCATGGGTTGGACA-
ATGAGGCTGGTCACAGCAGCACTGTTACTCGGTCTCATGATGGTG
GTCACTGGAGACGAGGATGAGAACAGCCCGTGTGCCCATGAGGCCCTCTTGGACGAGGACACCCTCTTTTGCC
AGGGCCTTGAAGTTTTCTACCCAGAGTTCGGGAACATTGGCTGCAAGGTTGTTCCTG-
ATTGTAACAACTACAG ACAGAAGATCACCTCCTGGATGGAGCCGATAGTCAAGTTCC-
CGGGGGCCGTGGACGGCGCAACCTATATCCTG GTGATGGTGGATCCAGATGCCCCTA-
GCAGAGCGGAACCCAGACAGAGATTCTGGAGACATTGGCTGGTAACAG
ATATCAAGGGCGCCGACCTGAAGGAAGGGAAGATTCAGGGCCAGGAGTTATCAGCCTACCAGGCTCCCTCCCC
ACCGGCACACAGTGGCTTCCATCGCTACCAGTTCTTTGTCTATCTTCAGGAAGGAAA-
AGTCATCTCTCTCCTT CCCAAGGAAAACAAAACTCGAGGCTCTTGGAAAATGGACAG-
ATTTCTGAACCGTTTCCACCTGGGCGAACCTG AAGCAAGCACCCAGTTCATGACCCA-
GGTCGACGCC NOV3d, 248644954 Protein Sequence SEQ ID NO:64 206 aa MW
at 23312.4 kD TRSPTMGWTMRLVTAALLLGLMMVVTGDE-
DENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYR
QKITSWMEPIVKFPGAVDGATYILVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQAPSP
PAHSGFHRYQFFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQ- VDG
NOV3e, 248644962 SEQ ID NO:65 619 bp DNA Sequence ORF Start: at 2
ORF Stop: end of sequence
CACCAGATCTCCCACCATGGGTTGGACAATGAGGCTGGTCACAGCAGCACTGTTACTGGGTCTCATGATGGTG
GTCACTGGAGACGAGGATGAGAACAGCCCGTGTGCCCATGAGGCCCTCTTGGACGA-
GGACACCCTCTTTTGCC AGGGCCTTGAAGTTTTCTACCCAGAGTTGGGGAACATTGG-
CTGCAAGGTTGTTCCTGATTGTAACAACTACAG ACAGAAGATCACCTCCTGGATGGA-
GCCGATAGTCAAGTTCCCGGGGGCCGTGGACGGCGCAACCTATATCCTG
GTGATGGTGGATCCAGATGCCCCTAGCACAGCAGAAACCCAGACAGAGATTCTGGAGACATTGGCTGTAACAG
ATATCAAGGGCGCCGACCTGAAGAAAGGGAAGATTCAGGGCCAGGAGTTATCAGCCT-
ACCAGGCTCCCTCCCC ACCGGCACACAGTGGCTTCCATCGCTACCAGTTCTTTGTCT-
ATCTTCACCAAGGAAAAGTCATCTCTCTCCTT CCCAAGGAAAACAAAACTCGAGGCT-
CTTGGAAAATGGACAGATTTCTGAACCGTTTCCACCTGGGCGAACCTG
AAGCAAGCACCCAGTTCATGACCCAGGTCGACGGC NOV3e, 248644962 Protein
Sequence SEQ ID NO:66 206 aa MW at 23311.5 kD
TRSPTMGWTMRLVTAALLLGLMMVVTGDEDENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYR
QKITSWMEPIVKFPGAVDGATYILVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLK-
KGKIQGQELSAYQAPSP PAHSGFHRYQFFVYLQEGKVISLLPKENKTRGSWKMDRFL-
NRFHLGEPEASTQFMTQVDG NOV3f, 248645004 SEQ ID NO:67 634 bp DNA
Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCAGATCTGACGAGGATCAGAACAGCCCGTGTGCCCATGAGGCCCTCTTCGACGAGGACACCCTCTTTTGC
CAGGGCCTTGAAGTTTTCTACCCAGAGTTGGGGAACATTGGCTGCAAGGTTGTTCC-
TGATTGTAACAACTACA GACAGAAGATCACCTCCTGGATGGAGCCGATAGTCAAGTT-
CCCGGGGGCCGTGGACGGCGCAACCTATATCCT GGTGATGGTGGATCCAGATGCCCC-
TAGCAGAGCAGAACCCAGACAGAGATTCTGGAGACATTGGCTGGTAACA
GATATCAAGGGCGCCGACCTGAAGGAAGGGAAGATTCAGGGCCAGGAGTTATCAGCCTACCAGGCTCCCTCCC
CACCGGCACACAGTGGCTTCCATCGCTACCAGTTCTTTGTCTATCTTCAGGAAGGAA-
AAGTCATCTCTCTCCT TCCCAACGAAAACAAAACTCGAGGCTCTTGGAAAATGGACA-
GATTTCTCAACCGTTTCCACCTGGGCGAACCT GAAGCAAGCACCCAGTTCATGACCC-
AGAACTACCAGGACTCACCAACCCTCCAGGCTCCCAGAGAAAGGGCCA
GCGAGCCCAAGCACAAAAACCAGGCGGAGATAGCTGCCTGCGTCGACGGC NOV3f, 248645004
Protein Sequence SEQ ID NO:68 211 aa MW at 24002.7 kD
TRSDEDENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYRQKITSWNEPIVKFP-
GAVDGATYIL VMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQA-
PSPPAHSGFHRYQFFVYLQEGKVISLL PKENKTRGSWKMDRFLNRFHLCEPEASTQF-
MTQNYQDSPTLQAPRERASEPKHKNQAEIAACVDG NOV3g, 249420987 SEQ ID NO:69
634 bp DNA Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCAGATCTGACGAGGATGAGAACAGCCCGTGTGCCCATGAGGCCCTCTTGGACGA-
GGACACCCTCTTTTGC CAGGGCCTTGAAGTTTTCTACCCAGAGTTGGGGAACATTG-
GCTGCAAGGTTGTTCCTGATTGTAACAACTACA GACAGAACATCACCTCCTGGATGG-
AGCCGATAGTCAAGTTCCCGGGGGCCGTGGACGGCGCAACCTATATCCT
GGTGATGGTGGATCCAGATGCCCCTAGCAGAGCAGAACCCAGACAGAGATTCTGGAGACATTGGCTGGTAACA
GATATCAAGGGCGCCGACCTGAAGAAAGGGAAGATTCAGGGCCAGGAGTTATCAGCC-
TACCAGGCTCCCTCCC CACCGGCACACAGTGCCTTCCATCGCTACCAGTTCTTTGTC-
TATCTTCAGGAAGGAAAAGTCATCTCTCTCCT TCCCAAGGAAAACAAAACTCGAGGC-
TCTTGGAAAATGGACAGATTTCTGAACCGTTTCCACCTGGGCGAACCT
GAAGCAAGCACCCAGTTCATGACCCAGAACTACCAGGACTCACCAACCCTCCAGGCTCCCAGAGAAAGGGCCA
GCGAGCCCAAGCACAAAAACCAGGCGGAGATAGCTGCCTGCGTCGACGGC NOV3g, 249420987
Protein Sequence SEQ ID NO:70 211 aa MW at 24001.8 kD
TRSDEDENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYR-
QKITSWMEPIVKFPGAVDGATYIL VNVDPDAPSRAEPRQRFWRHWLVTDIKGADLK-
KGKIQGQELSAYQAPSPPAHSGFHRYQFFVYLQEGKVISLL
PKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQMYQDSPTLQAPRERASEPKHKNQAEIAACVDG
NOV3h, 248486005 SEQ ID NO:71 547 bp DNA Sequence ORF Start: at 2
ORF Stop: end of sequence CACCAGATCTGACGAGGATGACAACAGC-
CCGTGTGCCCATGAGGCCCTCTTGGACGAGGACACCCTCTTTTGC
CAGGGCCTTGAAGTTTTCTACCCAGAGTTGGGGAACATTGCCTGCAAGGTTGTTCCTGATTGTAACAACTACA
GACAGAAGATCACCTCCTGGATGGAGCCGATAGTCAAGTTCCCGGGGGCCGTGGACG-
GCGCAACCTATATCCT GGTGATGGTGGATCCAGATGCCCCTAGCAGAGCAGAACCCA-
GACAGAGATTCTGGACACATTGGCTGGTAACA GATATCAAGGGCGCCGACCTGAAGA-
AAGGGAAGATTCAGGGCCAGGAGTTATCAGCCTACCAGGCTCCCTCCC
CACCGGCACACAGTGGCTTCCATCGCTACCAGTTCTTTGTCTATCTTCACGAAGGAAAAGTCATCTCTCTCCT
TCCCAAGGAAAACAAAACTCGAGGCTCTTGGAAAATGGACAGATTTCTGAACCGTTT-
CCACCTGGGCGAACCT GAAGCAAGCACCCAGTTCATGACCCAGGTCGACGCC NOV3h,
248486005 Protein Sequence SEQ ID NO:72 182 aa MW at 20766.3 kD
TRSDEDENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCN-
NYRQKITSWMEPIVKFPGAVDGATYIL VMVDPDAPSRAEPRQRFWRHWLVTDIKGA-
DLKKGKIQGQELSAYQAPSPPAHSGFHRYQFFVYLQEGKVISLL
PKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQVDG NOV3i, 249421046 SEQ ID NO:73
547 bp DNA Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCAGATCTGACGAGGATGAGAACAGCCCGTGTGCCCATGAGGCCCTCTTGGACGA-
GGACACCCTCTTTTGC CAGGGCCTTGAAGTTTTCTACCCAGAGTTGGGAACATTGG-
CTGCAAGGTTGTTCCTGATTGTAAACAACTACA GACAGAAGATCACCTCCTGGATGG-
AGCCGATAGTCAAGTTCCCGGGGGCCGTGGACGGCGCAACCTATATCCT
GGTGATGGTGGATCCAGATGCCCCTAGCAGAGCAGAACCCAGACAGAGATTCTGGAGACATTGGCTGGTAACA
GATATCAAGGGCGCCGACCTGAAGGAAGGGAAGATTCAGGGCCAGGAGTTATCAGCC-
TACCAGACTCCCTCCC CACCGGCACACAGTGGCTTCCATCGCTACCAGTTCTTTGTC-
TATCTTCAGGAAGGAAAAGTCATCTCTCTCCT TCCCAAGGAAAACAAAACTCGAGGC-
TCTTGGAAAATGGACAGATTTCTGAACCGTTTCCACCTGGGCGAACCT
GAAGCAAGCACCCAGTTCATGACCCAGGTCGACGGC NOV3i, 249421046 Protein
Sequence SEQ ID NO:74 182 aa MW at 20767.2 kD
TRSDEDENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYRQKITSWMEPIVKFPGAVDGATYIL
VMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQAPSPPAHSGFH-
RYQFFVYLQEGKVISLL PKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQVDG NOV3j,
CG52324-02 SEQ ID NO:75 879 bp DNA Sequence ORF Start: ATG at 108
ORF Stop: end of sequence
TCAGGTGGCAGTCCTCCCAAAGTACTTGTGTCCGGGTGGTGGACTGGATTAGCTGCGGAGCCCTGGAAGCTGC
CTTTCCTTCTCCCTGTGCTTAACCAGAGGTGCCCATGGGTTGGACAATGAGGCTGG-
TCACAGCAGCACTGTTA CTGGGTCTCATGATGGTGGTCACTGGAGACGAGGATGAGA-
ACAGCCCGTGTGCCCATGAGGCCCTCTTGGACG AGGACACCCTCTTTTGCCAGGCCT-
TGAAGTTTTCTACCCAGAGTTGGGGACATTGGCTGCAAGGTTGTTAACC
TGATTGTAACAACTACAGACAGAAGATCACCTCCTGGATGGAGCCGATAGTCAAGTTCCCGGGGGCCGTGGAC
GGCGCAACCTATATCCTGGTGATGGTGGATCCAGATGCCCCTAGCAGAGCAGAACCC-
AGACAGAGATTCTAAA GACATTGGCTGGTAACAGATATCAAGGGCGCCGACCTGAAG-
AAAGGGAAGATTCAGGGCCAGGAGTTATCAGC CTACCAGGCTCCCTCCCCACCGGCA-
CACAGTGCTTCCATCGCTACCAGTTCTTTGTCTATCTTCAAGGAAGGA
AAAGTCATCTCTCTCCTTCCCAAGGAAAACAAAACTCGAGGCTCTTGGAAAATGGACAGATTTCTGAACCGTT
TCCACCTGGGCGAACCTGAAGCAAGCACCCAGTTCATGACCCAGAACTACCAGGACT-
CACCAACCCTCCAAAC TCCCAGAGAAAGGGCCAGCGGGCCCAAGCACAAAAACCAGG-
CGGAGATAGCTGCCTGCTAGATAGCCGGCTTT GCCATCCGGGCATGTGGCCACACTG-
CCCACCACCGACGATGTGGGTATGGAACCCCCTCTGGATACAGAACCA CAT NOV3j,
CG52324-02 Protein Sequence SEQ ID NO:76 227 aa MW at 25661.1 kD
MCWTMRLVTAALLLGLMMVVTGDEDENSPCAHEALLDEDTLFCQGL-
EVFYPELGNIGCKVVPDCNNYRQKITS WNEPIVKFPGAVDGATYILVMVDPDAPSR-
AEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSG
FHRYQFFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRERASGPKHK
NQAEIAAC NOV3k, CG52324-03 SEQ ID NO:77 615 bp DNA Sequence ORF
Start: at 1 ORF Stop: end of sequence
GACGAGGATGAGAACAGCCCGTGTGCCCATGAGGCCCTCTTGGACGAGGACACCCTCTTTTGCCAGGGCC-
TTG AAGTTTTCTACCCAGAGTTGGGGAACATTGGCTGCAAGGTTGTTCCTGATTGT-
AACAACTACAGACAGAAGAT CACCTCCTGGATGGAGCCGATAGTCAAGTTCCCGGGG-
GCCGTGGACGGCGCAACCTATATCCTGGTGATGGTG
GATCCAGATGCCCCTAGCAGAGCAGAACCCAGACAGAGATTCTGGAGACATTGGCTGGTAACAGATATCAAGG
GCGCCGACCTGAAGAAAGGGAAGATTCAGGGCCAGGAGTTATCAGCCTACCAGGCTC-
CCTCCCCACCGGCACA CAGTGGCTTCCATCGCTACCAGTTCTTTGTCTATCTTCAGG-
AAGGAAAAGTCATCTCTCTCCTTCCCAAGGAA AACAAAACTCGAGGCTCTTGGAAAA-
TGGACAGATTTCTGAACCGTTTCCACCTCCGCGAACCTGAAGCAAGCA
CCCAGTTCATGACCCAGAACTACCAGGACTCACCAACCCTCCAGGCTCCCAGAGAAAGGGCCAGCGAGCCCAA
GCACAAAAACCAGGCGGAGATAGCTGCCTGC NOV3k, CG52324-03 Protein Sequence
SEQ ID NO:78 205 aa MW at 23386.2 kD
DEDENSPCAHEALLDEDTLFCQGLEVFYPELGNIGCKVVPDCNNYRQKITSWMEPIVKFPGAV-
DGATYILVMV DPDAPSRAEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSP-
PAHSGFHRYQFFVYLQEGKVISLLPKE NKTRGSWKMDRFLNRFHLGEPEASTQFMTQ-
NYQDSPTLQAPRERASEPKHKNQAEIAAC
[0371] A ClustalW comparison of the above protein sequences yields
the following sequence alignment shown in Table 3B.
15TABLE 3B Comparison of the NOV3 protein sequences. NOV3a
----MGWTMRLVTAALLLGLMMVVTG-------- ------------------DEDENSPCA (SEQ
ID NO: 58) NOV3b
TRSPTMGWTMRLVTAALLLGMMVVTG-------------------------DEDENSPCA (SEQ
ID NO: 60) NOV3c
TRSPTMGWTMRLVTAALLLGMMVVTG-------------------------DEDEN- SPCA (SEQ
ID NO: 62) NOV3d ------------------------TRSPTMGWTMRLVTAA-
LLLGLMMVVTGDEDENSPCA (SEQ ID NO: 64) NOV3e
------------------------- TRSPTMGWTMRLVTAALLLGLHMVVTGDEDENSPCA (SEQ
ID NO: 66) NOV3f
------------------------TRS------------------------DEDENSPCA (SEQ
ID NO: 68) NOV3g
------------------------TRS------------------------DEDEN- SPCA (SEQ
ID NO: 70) NOV3h -----------------------------------------
--------TRSDEDENSPCA (SEQ ID NO: 72) NOV3i
------------------------- ------------------------TRSDEDENSPCA (SEQ
ID NO: 74) NOV3j
------MGWTMRLVTAALLLGLMMVVTG-----------------------DEDENSPCA (SEQ
ID NO: 76) NOV3k
---------------------------------------------------DEDEN- SPCA (SEQ
ID NO: 78) NOV3a HEALLDEDTLFCQGLEVFYPELGNIGCKVVP-
DCNNYRQKITSWMEPIVKFPGAVDGATYI NOV3b
HEALLDEDTLFCQGLEVFYPELGNIGCKVVP- DCNNYRQKITSWMEPIVKFPGAVDGATYI
NOV3c HEALLDEDTLFCQGLEVFYPELGNIGCKVVP-
DCNNYRQKITSWMEPIVKFPGAVDGATYI NOV3d
HEALLDEDTLFCQGLEVFYPELGNIGCKVVP- DCNNYRQKITSWMEPIVKFPGAVDGATYI
NOV3e HEALLDEDTLFCQGLEVFYPELGNIGCKVVP-
DCNNYRQKITSWMEPIVKFPGAVDGATYI NOV3f
HEALLDEDTLFCQGLEVFYPELGNIGCKVVP- DCNNYRQKITSWMEPIVKFPGAVDGATYI
NOV3g HEALLDEDTLFCQGLEVFYPELGNTGCKVVP-
DCNNYRQKITSWMEPIVKFPGAVDGATYI NOV3h
HEALLDEDTLFCQGLEVFYPELGNIGCKVVP- DCNNYRQKITSWMEPIVKFPGAVDGATYI
NOV3i HEALLDEDTLFCQGLEVFYPELGNIGCKVVP-
DCNNYRQKITSWMEPIVKFPGAVDGATYI NOV3j
HEALLDEDTLFCQGLEVFYPELGNIGCKVVP- DCNNYRQKITSWMEPIVKFPGAVDGATYI
NOV3k HEALLDEDTLFCQGLEVFYPELGNIGCKVVP-
DCNNYRQKITSWMEPIVKFPGAVDGATYI NOV3a
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3b
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3c
LVMVDPDAPSRAEPRQPYWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3d
LVMVDPDAPSRAEPRQPYWRHWLVTDIKGADLKEGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3e
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3f
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3g
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3h
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3i
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKEGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3j
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3k
LVMVDPDAPSRAEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSGFHRYQ NOV3a
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRE NOV3b
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRE NOV3c
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRE NOV3d
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQVDG---------- NOV3e
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQVDG---------- NOV3f
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRE NOV3g
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRE NOV3h
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQVDG---------- NOV3i
FFVYLQEGKVISLLPKENKTRCSWKMDRFLNRFHLGEPEASTQFMTQVDG---------- NOV3j
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRE NOV3k
FFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNYQDSPTLQAPRE NOV3a
RASEPKHKNQAEIAAC--- NOV3b RASEPKHKNQAEIAACVDG NOV3c
RASEPKHKNQAEIAACVDG NOV3d ------------------- NOV3e
------------------- NOV3f RASEPKHKNQAEIAACVDG NOV3g
RASEPKHKNQAEIAACVDG NOV3h ------------------- NOV3i
------------------- NOV3j RASGPKHKNQAEIAAC--- NOV3k
RASEPKHKNQAEIAAC---
[0372] Further analysis of the NOV3a protein yielded the following
properties shown in Table 3C.
16TABLE 3C Protein Sequence Properties NOV3a SignalP analysis:
Cleavage site between residues 23 and 24 PSORT II analysis: PSG: a
new signal peptide prediction method N-region: length 6; pos.chg 1;
neg.chg 0 H-region: length 16; peak value 11.29 PSG score: 6.89
GvH: von Heijne's method for signal seq. recognition GvH score
(threshold: -2.1): 1.04 possible cleavage site: between 22 and 23
>>> Seems to have a cleavable signal peptide (1 to 22)
ALOM: Klein et al's method for TM region allocation Init position
for calculation: 23 Tentative number of TMS(s) for the threshold
0.5: 0 number of TMS(s) . . . fixed PERIPHERAL Likelihood = 2.65
(at 78) ALOM score: 2.65 (number of TMSs: 0) MTOP: Prediction of
membrane topology (Hartmann et al.) Center position for
calculation: 11 Charge difference: -7.5 C(-5.5) - N(2.0) N >= C:
N-terminal side will be inside MITDISC: discrimination of
mitochondrial targeting seq R content: 1 Hyd Moment(75): 6.09 Hyd
Moment(95): 8.77 G content: 3 D/E content: 1 S/T content: 3 Score:
-4.30 Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 16 MRL.vertline.VT NUCDISC: discrimination of nuclear
localization signals pat4: none pat7: none bipartite: none content
of basic residues: 10.6% 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 PTS2: 2nd
peroxisomal targeting signal: none VAC: possible vacuolar targeting
motif: none RNA-binding motif: none Actinin-type actin-binding
motif: type 1: none type 2: none NMYR: N-myristoylation pattern:
none Prenylation motif: none memYQRL: transport motif from cell
surface to Golgi: none Tyrosines in the tail: none Dileucine motif
in the tail: none checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none checking 33
PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's
method for Cytoplasmic/Nuclear discrimination Prediction:
cytoplasmic Reliability: 76.7 COIL: Lupas's algorithm to detect
coiled-coil regions total: 0 residues Final Results (k = 9/23):
33.3%: extracellular, including cell wall 22.2%: mitochondrial
22.2%: endoplasmic reticulum 11.1%: Golgi 11.1%: vacuolar >>
prediction for CG52324-01 is exc (k = 9)
[0373] A search of the NOV3a protein against the Geneseq database,
a proprietary database that contains sequences published in patents
and patent publication, yielded several homologous proteins shown
in Table 3D.
17TABLE 3D Geneseq Results for NOV3a NOV3a Identities/ Residues/
Similarities for Geneseq Protein/Organism/Length [Patent #, Match
the Matched Expect Identifier Date] Residues Region Value AAB48368
Human SEC1 protein sequence (clone ID 1 . . . 227 227/227 (100%)
e-135 3445452) - Homo sapiens, 227 aa. 1 . . . 227 227/227 (100%)
[WO200078802-A2, 28-DEC-2000] AAB88590 Human hydrophobic domain
containing 1 . . . 227 226/227 (99%) e-135 protein clone HP03880
#94 - Homo 1 . . . 227 227/227 (99%) sapiens, 227 aa.
[WO200112660-A2, 22-FEB-2001] AAY64647 Human 1 . . . 227 226/227
(99%) e-135 phosphatidylethanolamine-binding 1 . . . 227 227/227
(99%) protein - Homo sapiens, 227 aa. [WO9953051-A2, 21-OCT-1999]
AAG00016 Human secreted protein #4 - Homo 1 . . . 227 226/227 (99%)
e-135 sapiens, 227 aa. [EP1033401-A2, 1 . . . 227 227/227 (99%)
06-SEP-2000] AAY35976 Extended human secreted protein 1 . . . 227
226/227 (99%) e-135 sequence, SEQ ID NO. 225 - Homo 1 . . . 227
227/227 (99%) sapiens, 227 aa. [WO9931236-A2, 24-JUN-1999]
[0374] 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.
18TABLE 3E Public BLASTP Results for NOV3a NOV3a Protein Residues/
Identities/ Accession Match Similarities for the Expect Number
Protein/Organism/Length Residues Matched Portion Value Q96S96
Phosphatidylethanolamine binding 1 . . . 227 227/227 (100%) e-135
protein - Homo sapiens (Human), 1 . . . 227 227/227 (100%) 227 aa.
CAC33305 Sequence 114 from Patent 1 . . . 227 226/227 (99%) e-134
WO0112660 - Homo sapiens 1 . . . 227 227/227 (99%) (Human), 227 aa.
Q8WW74 Hypothetical protein - Homo sapiens 1 . . . 221 218/221
(98%) e-129 (Human), 223 aa. 1 . . . 221 219/221 (98%) Q9D9G2
1700081D17Rik protein - Mus 5 . . . 198 104/209 (49%) 1e-54
musculus (Mouse), 242 aa. 12 . . . 220 132/209 (62%) AAO39754
Putative antennal carrier protein A5 - 44 . . . 208 58/171 (33%)
3e-20 Anopheles gambiae (African malaria 48 . . . 205 84/171 (48%)
mosquito), 211 aa.
[0375] PFam analysis predicts that the NOV3a protein contains the
domains shown in the Table 3F.
19TABLE 3F Domain Analysis of NOV3a Identities/Similarities NOV3a
for the Pfam Domain Match Region Matched Region Expect Value PBP 60
. . . 198 51/202 (25%) 7.7e-14 100/202 (50%)
Example 4
[0376] The NOV4 clone was analyzed, and the nucleotide and encoded
polypeptide sequences are shown in Table 4A.
20TABLE 4A NOV4 Sequence Analysis NOV4a, CG53054-02 SEQ ID NO:79
1128 bp DNA Sequence ORF Start: ATG at 31 ORF Stop: end of sequence
TCCCGGCCCTCCGCGCCCTCTCG-
CGCGGCGATGGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGC
AGGCTCTGGGCAGCTACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACCCTGGA
GCCAGAGGCGGCTGCCCAGGCGCACTACAAGGCCTGCGACCGGCTGAAGCTCGAGCG-
GAAGCAGCGGCGCATG TGCCGCCGGGACCCGCGCGTGGCACAGACGCTGGTGGAGGC-
CGTGAGCATGAGTGCGCTCGAGTGCCAGTTCC AGTTCCGCTTTGAGCGCTGGAACTG-
CACGCTGGAGGGCCGCTACCGGCCCACCCTGCTCAAGCGAGGTTTCAA
CGAGACTGCCTTCCTCTATGCCATCTCCTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGC
CGCATGGAGCGCTGTACCTGCGATGAGGCACCCGACCTGCAGAACCGTGAGGCCTGG-
CAGTGGGGGGGCTGCG GAGACAACCTTAAGTACAGCAGCAAGTTCGTCAAGGAATTC-
CTGGGCAGACGGTCAAGCAAGGATCTGCGAGC CCGTGTGGACTTCCACAACAACCTC-
GTGGGTGTGAACGTGATCAAGGCTGGGGTGGAGACCACCTGCAAGTGC
CACGGCGTGTCAGGCTCATGCACGGTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGC
ATCTGAAGCACAAGTATGAGACGGCACTCAAGGTGGGCAGCACCACCAATGAAGCTG-
CCGGCGAGGCAGGTGC CATCTCCCCACCACGGGGCCGTGCCTCGGGGGCAGGTGGCA-
GCGACCCGCTGCCCCGCACTCCAGAGCTGGTG CACCTGGATGACTCGCCTAGCTTCT-
GCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTGCCACC
GTGAGAAGAACTGCGAGAGCATCTGCTGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTG
CCAGTGCCAGGTGCGTTGGTGCTGCTATGTGGAGTGCAGGCAGTGCACGCACCGTGA-
GGAGGTCTACACCTGC AAGGGCTGAGTTCCCAGGCCCTGCCAGCCCTGC NOV4a,
CG53054-02 Protein Sequence SEQ ID NO:80 357 aa MW at 39756.1 kD
MAPLGYFLLLCSLKQALGSYPIWWLTGSEPLTILPLTLEPEAAAQAHYK-
ACDRLKLERKQRRNCRRDPGVAET LVEAVSMSALECQFQFRFERWNCTLEGRYRAS-
LLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEA
PDLENREAWQWGCCGDNLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVR
TCWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLP-
RTPELVHLDDSPSFCL AGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQC-
QVRWCCYVECRQCTQREEVYTCKG NOV4b, 170251039 SEQ ID NO:81 1029 bp DNA
Sequence ORF Start: at 1 ORF Stop: end of sequence
GGATCCAGCTACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACCCTGGAG-
CCAG AGGCGGGCGCCCAGCCCCACTACAAGGCCTGCGACCGGCTGAAGCTGGAGCG-
GAAGCAGCGGCGCATGTGCCG CCGGGACCCGGGCGTGGTAGAGACGCTGGTCGAGGC-
CGTGAGCATGAGTGCGCTCGAGTGCCAGTTCCAGTTC
CGCTTTGAGCGCTGGAACTGCACGCTGGAGGGCCGCTACCGGGCCAGCCTGCTCAAGCGAGGCTTCAAGGAGA
CTGCCTTCCTCTATGCCATCTCCTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGT-
GCAGCGCGGGCCGCAT GGAGCGCTGTACCTGCGATGAGGCACCCGACCTGGAGAACC-
GTGAGGCCTGGCAGTGGGGGGGCTGCGGAGAC AACCTTAAGTACAGCAGCAAGTTCG-
TCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGGACTTCCACAACAACCTCGTGGGTGTGAAGGTGATCAAGGCTGGGGTGGAGACCACCTGCAAGTGCCACGG
CGTGTCAGGCTCATGCACGGTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGA-
GGTGGGCAAGCATCTG AAGCACAAGTATGAGACGGCACTCAAGGTGGGCAGCACCAC-
CAATGAAGCTGCCGGCGAGGCAGGTGCCATCT CCCCACCACGGGGCCGTGCCTCGGG-
GGCAGGTGGCAGCGACCCCCTGCCCCGCACTCCAGAGCTGGTGCACCT
GGATGACTCGCCTAGCTTCTGCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAG
AAGAACTGCGAGAGCATCTGCTGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTG-
ACAAGGCCCTGCCAGT GCCAGGTGCGTTGGTCCTCCTATGTGGAGTGCAGGCAGTGC-
ACGCAGCGTGAGGAGGTCTACACCTGCAAGGG CGTCGAC NOV4b, 170251039 Protein
Sequence SEQ ID NO:82 343 aa MW at 38208.1 kD
GSSYPIWWLTGSEPLTILPLTLEPEAGAQAHYKACDRLKLERKQRRMCRRDPGVV-
ETLVEAVSMSALECQFQF RFERWNCTLEGRYRASLLKRCFKETAFLYAISSAGLTH-
ALAKACSACRNERCTCDEAPDLENREAWQWGGCGD
NLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKML
KHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCL-
AGRFSPGTAGRRCHRE KNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQR-
EEVYTCKGVD NOV4c, 170251076 SEQ ID NO:83 1029 bp DNA Sequence ORF
Start: at 1 ORF Stop: end of sequence
GGATCCAGCTACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTCACCATCCTCCCGCTGACCCTGGAGCCAG
AGGCGGCCGCCCAGGCGCACTACAAGGCCTGCGACCGGCTGAAGCTGGAGCGGAAG-
CAGCGGCGCATGTGCCG CCGGGACCCGGGCGTGGCAGAGACGCTGGTGGAGGCCGTG-
AGCATGAGTGCGCTCGAGTGCCAGTTCCAGTTC CGCTTTGAGCGCTGGAACTGCACG-
CTGGAGGGCCGCTACCGGGCCAGCCTGCTCAAGCGAGGCTTCAAGGAGA
CTGCCTTCCTCTATGCCATCTCCTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGCCGCAT
GGAGCGCTGTACCTGCGATGAGGCACCCGACCTGGAGAACCGTGAGGCCTGGCAGTG-
GGGGGCCTGCGGAGAC AACCTTAAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGG-
CACACGGTCAAGCAAGGATCTGCGAGCCCGTG TGGACTTCCACAACAACCTCGTGGG-
TGTGAAGGTGATCAAGGCTGGGGTGGAGACCACCTGCAAGTGCCACGG
CGTGTCAGGCTCATGCACGGTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGCATCTG
AAGCACAAGTATGAGACGGCACTCAAGGTGGGCAGCACCACCAATGAAGCTGCCGGC-
GAGGCAGGTGCCATCT CCCCACCACGGGGCCGTGCCTCGGGGGCAGGTGGCAGCGAC-
CCGCTGCCCCGCACTCCAGAGCTGGTGCACCT GGATGACTCGCCTAGCTTCTGCCTG-
CCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAG
AAGAACTGCGAGAGCATCTGCTGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT
GCCAGGTGCGTTGTGCTGCTATGTGGAGTGCAGGCAGTCCACGCAGCGTGAGGAGGT-
CTACACCTGCAAGOG CGTCGAC NOV4c, 170251076 Protein Sequence SEQ ID
NO:84 343 aa MW at 38194.1 kD
GSSYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQF
RFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDE-
APDLENREAWQWGGCGD NLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAG-
VETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHL KHKYETALKVGSTTNEAAGEAGAI-
SPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAGRFSPGTAGRRCHRE
KNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKGVD NOV4d,
CG53054-01 SEQ ID NO:85 1085 bp DNA Sequence ORF Start: ATG at 13
ORF Stop: end of sequence TAGTGAGCCGAGATGGCACTACTATATTCC-
AGCTTGGGTGTGGTTGTGTGCACCTGTAGTCCTAGTTACTTTG
GACTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACCCTGGAGCCAGAGGCGGCTGCCCAGGCGCACTA
CAAGGCCTGCGACCGGCTGAAGCTGGAGCGGAAGCAGCGGCGCATGTGCCGCCGGGA-
CCCGGGCGTGGCAGAG ACGCTGCTGCAGGCCGTGACCATGAGTGCGCTCGAGTGCCA-
GTTCCAGTTCCGCTTTGAGCGCTGGAACTGCA CGCTGGAGGGCCGCTACCGGGCCAG-
CCTGCTCAAGCGAGGTTTCAAGGAGACTGCCTTCCTCTATGCCATCTC
CTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCACCGCGGGCCGCATGGAGCGCTGTACCTGCGATGAG
GCACCCGACCTGGAGAACCGTCAGGCCTGGAAGTGGGGTGGCTGTAGCGAGGACATC-
GAGTTTGGTGGGATGG TGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCAGAT-
GCCCGCTCAGCCATGAACCGCCACAACAACGA GGCTGGGCGCCAGGTGATCAAGGCT-
GGGGTGGAGACCACCTGCAAGTGCCACGGCGTGTCAGGCTCATGCACG
GTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGCATCTGAAGCACAAGTATGAGTCGG
CACTCAAGGTGGGCAGCACCACCAATGAAGCTGCCGGCGAGGCAGGTGCCATCTCCC-
CACCACGGGGCCGTGC CTCGGGGGCAGGTGGCAGCGACCCGCTGCCCCGCACTCCAG-
AGCTGGTGCACCTGGATGACTCGCCTAGCTTC TGCCTGGCTGGCCGCTTCTCCCCGG-
GCACCGCTGGCCGTAGGTGCCACCGTGAGAAGAACTGCGAGAGCATCT
GCTGTGGCCGCGCCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGTGCCAGGTGCGTTGGTGCTG
CTATGTGGAGTGCAGGCAGTGCACGCAGCGTGAGGAGGTCTACACCTGCAAGGGCTG- AGTTCC
NOV4d, CG53054-01 Protein Sequence SEQ ID NO:86 355 aa MW at
39194.1 kD MALLYSSLGVVVCTCSPSYFGLTGSEPLTILPL-
TLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVE
AVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDL
ENREGWKWGGCSEDIEFGGMVSREFADARENRPDARSANNRHNNEAGRQVIKAGVET-
TCKCHGVSGSCTVRTC WRQLAPFHEVGKHLKHKYESALKVGSTTNEAAGEAGAISPP-
RGRASCAGGSDPLPRTPELVHLDDSPSFCLAG RFSPGTAGRRCHREKNCESICCGRG-
HNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG NOV4e, CG53054-03 SEQ ID
NO:87 1029 bp DNA Sequence ORF Start: at 7 ORF Stop: end of
sequence GCATCCAGCTACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTGACCATCCTC-
CCGCTGACCCTGGAGCCAG AGGCGGCCGCCCAGGCGCACTACAAGGCCTGCGACCG-
GCTGAAGCTGGAGCGGAAGCAGCGGCGCATGTGCCG
CCGGGACCCGGCCCTGCCAGAGACGCTGGTGGAGGCCGTGAGCATGAGTGCGCTCGAGTGCCAGTTCCAGTTC
CGCTTTGAGCGCTGGAACTGCACGCTGGAGGGCCGCTACCGGGCCAGCCTGCTCAAG-
CGAGGCTTCAAGGAGA CTGCCTTCCTCTATGCCATCTCCTCGGCTGGCCTGACGCAC-
GCACTGGCCAAGGCGTGCAGCGCGGGCCGCAT GGAGCGCTGTACCTGCGATGAGGCA-
CCCGACCTGGAGAACCGTGAGGCCTGGCAGTGGGGGGGCTGCGGAGAC
AACCTTAAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGGACTTCCACAACAACCTCGTGGGTGTGAAGGTGATCAAGGCTGGGGTGGAGACCA-
CCTGCAAGTGCCACGG CGTGTCAGGCTCATGCACGGTGCGGACCTGCTGGCGGCAGT-
TGGCGCCTTTCCATGAGGTGGGCAAGCATCTG AAGCACAAGTATGAGACGGCACTCA-
AGGTGGGCAGCACCACCAATGAAGCTGCCGGCGAGGCAGGTGCCATCT
CCCCACCACGGGGCCGTGCCTCGGGGGCAGGTGGCAGCGACCCGCTGCCCCGCACTCCAGAGCTGGTGCACCT
GGATGACTCGCCTAGCTTCTGCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCG-
TAGGTGCCACCGTGAG AAGAACTGCGAGAGCATCTGCTGTGGCCGCGGCCATAACAC-
ACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT GCCAGGTGCGTTGGTGCTGCTATGT-
GGAGTGCAGGCAGTGCACGCAGCGTGAGGAGGTCTACACCTGCAAGGG CGTCGAC NOV4e,
CG53054-03 Protein Sequence SEQ ID NO:88 339 aa MW at 37835.8 kD
SYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQ-
RRMCRRDPGVAETLVEAVSMSALECQFQFRF ERWNCTLEGRYRASLLKRGFKETAF-
LYAISSAGLTHALAKACSAGRNERCTCDEAPDLENREAWQWGGCGDNL
KYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHLKH
KYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAG-
RFSPGTAGRRCHREKN CESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE- VYTCKG
NOV4f, CG53054-04 SEQ ID NO:89 1631 bp DNA Sequence ORF Start: ATG
at 12 ORF Stop: end of sequence
GGCGCGGCAAGATGCTGGATGGGTCCCCGCTGGCGCGCTGGCTGGCCGCGGCCTTCGGGCTGACGCTGCTGCT
CGCCGCGCTGCGCCCTTCGGCCGCCTACTTCGGGCTGACGGGCAGCGAGCCCCTGA-
CCATCCTCCCGCTGACC CTGGAGCCAGAGGCGGCCGCCCAGGCGCACTACAAGGCCT-
GCGACCGGCTGAAGCTGGAGCGGAAGCAGCGGC GCATGTGCCGCCGGGACCCGGGCG-
TGGCAOAGACGCTGGTGGAGGCCGTGAGCATGAGTGCGCTCGAGTGCCA
GTTCCAGTTCCGCTTTGAGCGCTGGAACTGCACGCTGGAGGGCCGCTACCGGGCCACCCTGCTCAAGCGAGGC
TTCAAGGAGACTGCCTTCCTCTATGCCATCTCCTCGGCTGGCCTGACGCACGCACTG-
GCCAAGGCGTGCAGCG CGGGCCGCATGGAGCGCTGTACCTGCGATGAGGCACCCGAC-
CTGGAGAACCGTGAGGCCTGGCAGTGGGGGGG CTGCGGAGACAACCTTAAGTACAGC-
AGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTG
CGAGCCCGTGTGGACTTCCACAACAACCTCGTGGGTGTGAAGGTGATCAAGGCTGGGGTGGAGACCACCTGCA
AGTGCCACGGCGTGTCAGGCTCATGCACCGTGCGGACCTGCTGGCGGCAGTTGGCGC-
CTTTCCATGACGTGGG CAAGCATCTGAAGCACAAGTATGAGACGGCACTCAAGGTGG-
GCAGCACCACCAATGAAGCTGCCGGCGAGGCA GGTGCCATCTCCCCACCACGGGGCC-
GTGCCTCGGGGGCACGTGGCAGCGACCCGCTGCCCCGCACTCCAGAGC
TGGTGCACCTGGATGACTCGCCTAGCTTCTGCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTG
CCACCGTGAGAAGAACTGCGAGAGCATCTGCTGTGGCCGCGGCCATAACACACAGAG-
CCGGGTGGTGACAAGG CCCTGCCAGTGCCAGGTGCGTTGGTGCTGCTATGTGGAGTG-
CAGGCAGTGCACGCACCGTGAGGAGGTCTACA CCTGCAAGGGCTGAGTTCCCAGGCC-
CTGCCAGCCCTGCTGCACAGGGTGCAGGCATTGCACACGGTGTGAAGG
GTCTACACCTGCACAGGCTGAGTTCCTGGGCTCGACCAGCCCAGCTGCGTGGGGTACAGGCATTGCACACAGT
GTGAATGGGTCTACACCTGCATGGGCTGAGTCCCTGGGCTCAGACCTAGCAGCGTGG-
GGTAGTCCCTGGGCTC AGTCCTAGCTGCATGGGGTGCAGGCATTGCACAGAGCATGA-
ATGGGCCTACACCTCCCAAGGCTGAATCCCTG GGCCCAGCCAGCCCTGCTGCACATG-
GCACAGGCATTGCACACGGTGTGAGGAGTGTACACCTGCAAGGGCTGA
GGCCCTGGGCCCAGTCAGCCCTGCTGCTCAGAGTGCAGGCATTGCACATGGTGTGAGAAGGTCTACACCTGCA
AGGGACGAGTCCCCGGGCCTGGCCAACCCTGCTGTGCAGGGTGAGGGCCATGCATGC-
TAGTATGAGGGGTCTA CACCTGCAAGGACTGACAGGCTTTT NOV4f, CG53054-04
Protein Sequence SEQ ID NO:90 365 aa MW at 403 19.7 kD
MLDGSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQA-
IYKACDRLKLERKQRRMCR RDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRY-
RASLLKRGFKETAFLYAISSAGLTHALAKACSAGRM
ERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHG
VSGSCTVRTCWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASG-
AGGSDPLPRTPELVHL DDSPSFCLAGRFSPGTAGRRCHREKNCESICCGRGHMTQSR-
VVTRPCQCQVRWCCYVECRQCTQREEVYTCKG
[0377] A ClustalW comparison of the above protein sequences yields
the following sequence alignment shown in Table 4B.
21TABLE 4B Comparison of the NOV4 protein sequences. NOV4a
--------MAPLGYFLLLCSLKQALGSYPIWWL- TGSEPLTILPLTLEPEAAAQAHYKACD (SEQ
ID NO:80) NOV4b
------------------------GSSYPIWWLTGSEPLTILPLTLEPEAGAQAHYKACD (SEQ
ID NO:82) NOV4c
------------------------GSSYPIWWLTGSEPLTILPLTLEPEAAAQ- AHYKACD (SEQ
ID NO:84) NOV4d -----------MALLYSSLGVVVCTCSPSYFGLTGSEP-
LTILPLTLEPEAAAQAHYKACD (SEQ ID NO:86) NOV4e
------------------------ ---SYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACD (SEQ
ID NO:88) NOV4f
MLDGSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQARYKACD (SEQ
ID NO:90) NOV4a RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNC-
TLEGRYRASLLKRGFK NOV4b
RLKLERKQRRMCRRDPGVVETLVEAVSMSALECQFQFRFERWNC- TLEGRYRASLLKRGFK
NOV4c RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNC-
TLEGRYRASLLKRGFK NOV4d
RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNC- TLEGRYRASLLKRGFK
NOV4e RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNC-
TLEGRYRASLLKRGFK NOV4f
RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNC- TLEGRYRASLLKRGFK
NOV4a ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAP-
DLENREAWQWGCCGDNLKYSSKFVK NOV4b
ETAFLYAISSAGLTHALAKACSAGRNERCTCDEAP- DLENREAWQWGGCGDNLKYSSKFVK
NOV4c ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAP-
DLENREAWQWGGCGDNLKYSSKFVK NOV4d
ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAP- DLENREGWKWGGCSEDIEFGGMVSR
NOV4e ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAP-
DLENREAWQWGGCGDNLKYSSKFVK NOV4f
ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAP- DLENREAWQWGGCGDNLKYSSKFVK
NOV4a EFLGRRSSK-DLRARVDFHNNLVGVK-
VIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV NOV4b
EFLGRRSSK-DLRARVDFHNNLVGVK- VIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV4c EFLGRRSSK-DLRARVDFHNNLVGVK-
VIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV NOV4d
EFADARENRPDARSANNRHNNEAGRQ- VIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV4e EFLGRRSSK-DLRARVDFHNNLVGVK-
VIKAGVETTCKCHCVSGSCTVRTCWRQLAPFHEV NOV4f
EFLGRRSSK-DLRARVDFHNNLVGVK- VIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV4a GKHLKHKYETALKVGSTTNEAAGEACAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV4b GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV4c CKHLKHKYETALKVGSTTNEAAGEAOAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV4d GKHLKHKYESALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV4e GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV4f GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV4a LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV4b LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV4c LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV4d LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV4e LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV4f LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV4a VYTCKG-- NOV4b VYTCKGVD NOV4c VYTCKGVD NOV4d VYTCKG-- NOV4e
VYTCKG-- NOV4f VYTCKG--
[0378] Further analysis of the NOV4a protein yielded the following
properties shown in Table 4C.
22TABLE 4C Protein Sequence Properties NOV4a 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 0;
neg.chg 0 H-region: length 13; peak value 9.00 PSG score: 4.60 GvH:
von Heijne's method for signal seq. recognition GvH score
(threshold: -2.1): 0.73 possible cleavage site: between 18 and 19
>>> Seems to have a cleavable signal peptide (1 to 18)
ALOM: Klein et al's method for TM region allocation Init position
for calculation: 19 Tentative number of TMS (s) for the threshold
0.5: 0 number of TMS (s) . . . fixed PERIPHERAL Likelihood = 3.76
(at 114) ALOM score: 3.76 (number of TMSs: 0) MTOP: Prediction of
membrane topology (Hartmann et al.) Center position for
calculation: 9 Charge difference: 0.0 C(1.0) - N(1.0) N >= C:
N-terminal side will be inside MITDISC: discrimination of
mitochondrial targeting seq R content: 0 Hyd Moment (75): 1.56 Hyd
Moment (95): 3.50 G content: 3 D/E content: 1 S/T content: 4 Score:
-6.15 Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found NUCDISC: discrimination of nuclear
localization signals pat4: none pat7: none bipartite: none content
of basic residues: 14.8% NLS Score: -0.47 KDEL: ER retention motif
in the C-terminus: none ER Membrane Retention Signals: KKXX-like
motif in the C-terminus: YTCK SKL: peroxisomal targeting signal in
the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none RNA-binding motif:
none Actinin-type actin-binding motif: type 1: none 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: 70.6 COIL: Lupas's
algorithm to detect coiled-coil regions total: 0 residues Final
Results (k = 9/23): 55.6%: extracellular, including cell wall
22.2%: mitochondrial 11.1%: vacuolar 11.1%: nuclear >>
prediction for CG53054-02 is exc (k = 9)
[0379] A search of the NOV4a protein against the Geneseq database,
a proprietary database that contains sequences published in patents
and patent publication, yielded several homologous proteins shown
in Table 4D.
23TABLE 4D Geneseq Results for NOV4a NOV4a Identities/ Residues/
Similarities for Geneseq Protein/Organism/Length [Patent #, Match
the Matched Expect Identifier Date] Residues Region Value ABG69638
Human secreted protein SCEP-18 - 2 . . . 357 311/357 (87%) 0.0 Homo
sapiens, 366 aa. 13 . . . 366 327/357 (91%) [WO200248337-A2,
20-JUN-2002] AAO18744 Human NOV8 protein - Homo sapiens, 25 . . .
357 302/334 (90%) 0.0 355 aa. [WO200257450-A2, 22 . . . 355 316/334
(94%) 25-JUL-2002] AAE17305 Human WNT15 protein, 22 . . . 356
210/338 (62%) e-124 sbg389686WNT15a #1 - Homo 14 . . . 346 257/338
(75%) sapiens, 704 aa. [WO200198342-A1, 27-DEC-2001] AAE17306 Human
WNT15 protein, 25 . . . 356 209/335 (62%) e-124 sbg389686WNT15a #2
- Homo 31 . . . 360 255/335 (75%) sapiens, 361 aa. [WO200198342-A1,
27-DEC-2001] ABB77769 Amino acid sequence of human Wnt 25 . . . 356
209/335 (62%) e-124 (Zwnt5) polypeptide variant - Homo 4 . . . 333
255/335 (75%) sapiens, 334 aa. [WO200231148-A2, 18-APR-2002]
[0380] 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.
24TABLE 4E Public BLASTP Results for NOV4a NOV4a Identities/
Protein Residues/ Similarities for Accession Match the Matched
Expect Number Protein/Organism/Length Residues Portion Value O14904
WNT-14 protein precursor - Homo 2 . . . 357 339/356 (95%) 0.0
sapiens (Human), 365 aa. 13 . . . 365 343/356 (96%) Q8R5M2 Wnt14 -
Mus musculus (Mouse), 365 2 . . . 357 333/356 (93%) 0.0 aa. 13 . .
. 365 340/356 (94%) O42280 WNT-14 protein precursor - Gallus 25 . .
. 356 283/333 (84%) e-173 gallus (Chicken), 354 aa. 24 . . . 353
310/333 (92%) Q8C718 WNT14B - Mus musculus (Mouse), 8 . . . 356
216/354 (61%) e-125 359 aa. 12 . . . 358 264/354 (74%) Q8VI90
Wnt14b (Secreted signaling protein 8 . . . 356 216/354 (61%) e-125
WNT9B) - Mus musculus (Mouse), 12 . . . 358 264/354 (74%) 359
aa.
[0381] PFam analysis predicts that the NOV4a protein contains the
domains shown in the Table 4F.
25TABLE 4F Domain Analysis of NOV4a Identities/Similarities NOV4a
for the Pfam Domain Match Region Matched Region Expect Value wnt 50
. . . 356 129/359 (36%) 4.6e-103 234/359 (65%)
Example 5
[0382] The NOV5 clone was analyzed, and the nucleotide and encoded
polypeptide sequences are shown in Table 5A.
26TABLE 5A NOV5 Sequence Analysis NOV5a, CG54818-01 SEQ ID NO:91
6474 bp DNA Sequence ORF Start: ATG at 467 ORF Stop: end of
sequence
GTTTGGCAAGTCAGTGCAAGAGGCTGACTTCTGAGAGGCTTCCAGCAGCCCGAAGAGAGGACCTCCACGGGAG
AAGGGAGTGCGTGTGCTCGGTTTTTTTTTTTTCTCTCTTTTTTTTTTTTTTTTCTG-
AATGAACAGCTTTGCCC AAGTGACTGAAAAATACAGCTTCTTCCTGAATCTACCGGC-
GTAGTTGCTGAAGAGCGCTCTAGACAGGACATG GCTCTGAAGACTCACTCTTTGGAA-
TGTCCTCTTGCTCCCGGCTTATAAACAACTGTCCCGAGGAAAGAAAGGT
TTTACATAGCCAAATACAGCCTGACAAATGGCACTTCGGAACTGTCCTTTCTGATGACAACCCGTTCGATTTC
TGACAAAGCCTCTCGCACGCTGCCCCTGGAGGGAAGTCCTAAGTAAAACTCAGACCC-
TCCTTAAAGTGAGGAG CGAGGGCTTGGACGGTGAACACGGCAGCATGGCATCCGCGG-
GGCACATTATCACCTTGCTCCTGTGGGGTTAC TTACTGGAGCTTTGGACAGGAGGTC-
ATACAGCTGATACTACCCACCCCCGGTTACGCCTGTCACATAAAGAGC
TCTTGAATCTGAACAGAACATCAATATTTCATAGCCCTTTTGGATTTCTTGATCTCCATACAATGCTGCTGGA
TGAATATCAAGAGAGGCTCTTCGTGGGAGGCAGGGACCTTGTATATTCCCTCAGCTT-
GGAGAGAATCAGTGAC GGCTATAAAGAGATACACTGGCCGAGTACAGCTCTAAAAAT-
GGAAGAATGCATAATGAAGGGAAAAGATGCGG GTGAATGTGCAAATTATGTTCGGGT-
TTTGCATCACTATAACAGGACACACCTTCTGACCTGTGGTACTGGAGC
TTTTGATCCAGTTTGTGCCTTCATCAGAGTTGGATATCATTTGGAGGATCCTCTGTTTCACCTGGAATCACCC
AGATCTGAGAGAGGAAGGGGCAGATGTCCTTTTGACCCCAGCTCCTCCTTCATCTCC-
ACTTTAATTGGTAGTG AATTGTTTGCTGGACTCTACAGTGACTACTGGAGCAGAGAC-
GCTGCGATCTTCCGCAGCATGGGGCGACTGGC CCATATCCGCACTGAGCATGACGAT-
GAGCGTCTGTTGAAAGAACCAAAATTTGTAGGTTCATACATGATTCCT
GACAATGAAGACAGAGATGACAACAAAGTATATTTCTTTTTTACTGAGAAGGCACTGGAGGCAGAAAACAATG
CTCACGCAATTTACACCAGGGTCGGGCGACTCTGTGTGAATGATGTAGGAGGGCAGA-
CAATACTGGTGAATAA GTCGAGCACTTTCCTAAAAGCGAGACTCGTTTGCTCAGTAC-
CAGGAATGAATGGAATTGACACATATTTTGAT GAATTAGAGGACGTTTTTTTGCTAC-
CTACCAGAGATCATAAGAATCCAGTGATATTTGGACTCTTTAACACTA
CCAGTAATATTTTTCGAGGGCATGCTATATGTGTCTATCACATGTCTAGCATTCGGGCAGCCTTCAACGGACC
ATATGCACATAAGGAAGGACCTGAATACCACTGGTCAGTCTATGAAGGAAAAGTCCC-
TTATCCAAGGCCTGGT TCTTGTGCCAGCAAAGTAAATGGAGGGAGATACGGAACCAC-
CAAGGACTATCCTCATGATGCCATCCGATTTG CAAGAAGTCATCCACTAATGTACCA-
GGCCATAAAACCTGCCCATAAAAAACCAATATTGGTAAAAACAGATGG
AAAATATAACCTGAAACAAATAGCAGTAGATCGAGTGGAAGCTGAGGATGGCCAATATGACGTCTTGTTTATT
GGCACAGATAATGGAATTGTGCTGAAAGTAATCACAATTTACAACCAAGAAATGGAA-
TCAATGGAAGAAGTAA TTCTAGAAGAACTTCAGATATTCAAGGATCCAGTTCCTATT-
ATTTCTATGGACATTTCTTCAAAACGGCAACA GCTGTATATTGGATCTGCTTCTGCT-
GTGGCTCAAGTCAGATTCCATCACTGTGACATGTATGGAAGTGCTTGT
GCTGACTGCTOCCTGGCTCGAGACCCTTACTGTGCCTGGGATGGCATATCCTGCTCCCGGTATTACCCAACAG
GCACACATGCAAAAAGGCGTTTCCGGAGACAACATGTTCGACATGGAAATGCAGCTC-
AGCAGTGCTTTGGACA ACAGTTTGTTGGGGATGCTTTGGATAAGACTGAAGAACATC-
TGGCTTATGGCATAGAGAACAACAGTACTTTG CTGGAATGTACCCCACGATCTTTAC-
AAGCGAAAGTTATCTGGTTTGTACAGAAAGGACGTGAGACAAGAAAAG
AGGAGGTGAAGACAGATGACAGAGTGGTTAAGATGGACCTTGGTTTACTCTTCCTAAGGTTACACAAATCAGA
TGCTGGGACCTATTTTTGCCAGACAGTAGAGCATAGCTTTGTCCATACGGTCCGTAA-
AATCACCTTGGAGGTA GTGGAAGAGGAGAAAGTCGAGGATATGTTTAACAAGGACGA-
TGAGGAGGACAGGCATCACAGGATGCCTTGTC CTGCTCAGAGTAGCATCTCGCAGGG-
AGCAAAACCATGGTACAAGGAATTCTTGCAGCTGATCGGTTATAGCAA
CTTCCAGAGAGTCGAAGAATACTGCGAGAAAGTATGGTGCACAGATAGAAAGAGGAAAAAGCTTAAAATGTCA
CCCTCCAAGTGGAAGTATGCCAACCCTCAGGAAAAGAAGCTCCGTTCCAAACCTGAG-
CATTACCGCCTGCCCA GGCACACGCTGGACTCCTGATGGGGTGAGACTATCTACTGT-
CTTTTGAAGAATTTATATTTGGAAAGTAAAAA AGTAAAAAAATAAATCATCCAACTT-
CTTTGCATTACTTAAAAGAGATTTCTGTAATACAGGAATGACTATGAA
GGTGTTATAATAAATTATTCTACATACTCATTTGACTGGATAAACTTTACATAAAATTAACTAATTTTTTAAA
TAAATGCATTCCTTAATGGTTTCTCATTATGTTTATCAAAAAACAACTGTAGCTCTT-
ATTTTCAGTACTTGGC TGCTTTTCTGTGAAAATTATTATTTTACTTTTGGAAGACAA-
GATTATTAGAATATTGAAGAAAAATTGGAGAC TTATAATCATCGTAAATATAAAACT-
AAATATGTTTTAATATTTCTGAATTTTTCTTTTCCATCACAATGTAAG
ATATGCAGAATACAAGATACTTTGGCATTCTCATGTGAACTTTCTGTACTCTTTAAGGATTATTTTATTAGTG
TTGTTTAAGCCATGAGTGTTAAGTAGCAGGTGTGTTGTGAGTGCTGTAACCCATGAA-
AGGAAAAATGTCATTC TGAGGCTTGTGCCCTTCGTAAAATATTCATTAAAGTACATT-
CACACTATTTTTGCTTTATAACACAGTCTTTA ATTTTCACTCACTGTGGAAATAAAA-
ACTAAGGTAACTTCTCAGAAAGATATCAAATCTCAGAAAGAATGTCAA
ATCAGATGAAGTTATAGTTAGGATTCTAACTACTCTAAAAGATTTTTGCTTCCCTCTTGTGGTAAAAAAAATT
ATATTCTCACACATTTCTTTTTTCTCTACAGACGGATATCTGTTTAGGAAAGATTTG-
AAAGCAGATTATCAGT AGGTACATGGATACATCAAGTTCATTTCCAGAAACAAATAA-
CTGAAATAAAAAACATGTTAATCCTTGTATCA TACTTTAATATGAAAGTATTGTTTA-
TAGATAATTTATCTCACAAGTCAAAAATGAAGATTTTGCAGCACTGAA
AATCTATTAAAGCTCCAAATTTTAAGTTTCTAAATAATCTTCGCTGAAATCTAAAATATACTATAACAACCGT
GTTTTATTTGTGAAAAAAATATTAAAGTGATTTGCTCTCAAATATCAAATTTTCTTC-
TCTCTTTTATATTAAG AGACACAAAATTGTTTCATGAGTTCACTTAACTACTGAGAT-
ATTCAGAGCATTTTTACCTCTCTCTTAAATGT TATAAAAAACAATTGTATTTTTAAG-
AATGTTTATTTATCAAAGTCTTTCCTTCTTCTATTAAATATTTAGCAA
TTACCTTTCTAAAATATGAAATTTTGTAAGATGTTTTCACCTAAATAAAAATTGAAAGCAAGTGGATTACACA
GGAGAACCATTATGAACATTTATTTAGATATTAATCTTAAACAGTGTTTATTTCAGT-
TTTCAAAGTTAGCTTA TAGGTTATACATTTAAGTTAAAGTGCTCATAATCACTTGCA-
ATTTCATTGTAAAATGAACAAATACATAAATA TTTTAAGAAAAATTTAAGTTTATTC-
AGATAAGTCACCATGCTTCAAAAGATCTAAGAAATGCAAATATACTGA
AAATTGACATCCTCTGAAAATTCCACTTGCTATTTACCCAAGAATCCACTGGAGGTCATTACTGCCATTAAAT
AATAACTGAAAAGACTATGTAGTGAAATGTATTTTTAAAAACTATATTCAGTAAAAG-
CCTGCTCAATTTGGAG AAATAGAACCACAAACACAGATCACAGGGGCCTTACAAAGT-
TTATGTCTGAACAAATAAGTCAATTAAGTACA CTTTATTGAAAATTGCCTTCCATTA-
ACACACAAGAAAGAAAGCACGATTTTCTCCTGTATCTGAATTTTAAAA
TTAAAAAGGCAGATAAGACATAAATAGTTATCATTTTAATTGCAATAACACAGACAAGTAGTTAATGATGATA
ACAATGGTGTAACTTGTAAACTAAATATTTGGTAACTGAAGCAATAGGCAGAGGAAA-
ATAGCTTTTCTATGAC ACAAGTCATAAGAAGTCCATATACTGAAGAGCGTTTGATTA-
AAATAAAGTCACTATTAACCAGAAAAGAAACA TTTTACATAAAATGCTAAAATTTAT-
TATAGCAAAATAAATCAAACCCAAAGAAAGTTTATTCAATGCTAATTT
GAAAGAAAATTGATAAGAAAACTTTGAGGGCCCAAGTCCACAATTTGGTGAGACCACTAAATTTTACATATAA
TTATACACACACATATGTACATATATATGTATATAATCTTGCTTCCCGCCTGTTTAT-
GGCAGTACTGAAGAGA AATGGGAAAGAAGAGGGAGGGAGAGAGAAAGACGAAGGGAG-
AGAGAAAGCAGTTTCCAAGGATATGTTTCATG TCCCACCATTTTCTCAGTTTCTCCC-
TCTCTCTCCCAACACACACACACACACACCCCTCACATACTATAAAAT
AAATCTTCACTGCCCTATCAAAATACAAATAAATCAATCTATGCTGTTCTGTCCTTCTTGAGAATCTAAAACA
TACCACAAAAATACATCCCCAGTCTTTTGTTCTGTCTGAGGTTAGAATTAATTCAAA-
TTCAGAATCTGTTGTG AGAAATGCCCAGGCTTTAAAAATTAAAAATGGATGGATCTT-
CTCTGAACTCAGGGAGGGCACATACTTAGATA CCTACAAGACTTGGAGGAATTAAGA-
GTTCACCCTTCATCTCACCAAATTTTCCCCATTTTTCTCTTTCTTGTA
GAAGGAGAGAAACCATGCTCTCTAGCAACATTGAGCAAAAATCATAACCACTCATCTAATTTCTAAGAGGCAC
CTCCATCGAGGGCCGGTCTCCTGCTTCTTTAGACCTCTTCTATCTTTGTTACAGGAG-
AGGACCTGTGGATAGA CTTAGTTTTCACATAAAACAATGCCCATTCACCTCCTCCTT-
CAGCACAACGTCACCCATTGGGCAAGAGATCC AGATTTGTTAACAAAAAAGATTTTA-
CTTCGTGATTCCACGTCTATAATTCTATATTGCTAATTTTTTCTTTTG
TGTGAATTACTGAATATTTCAGACCAAAGCTATCAACTTGGAGAAACAGGGATTAAAAATAAGGATAAACACT
AATAAGAGCTCTAGAAAAAAGGGAACAGAAAGTCTGCCTGTTTAGTAAGTGGCAATT-
CCATACATATTTTAGA GTTTTTTCTATCTAAAATTAGTTAAATACTTAGAATGTTTG-
TAATGAGTGTTCGATATTTGCTATAGGTTTTA GGGTTTTGTAAATCTTCATAGTAAT-
TATAAACATTTGTAAAATTTGTAAAATACTATAAGTCATTTTGAGTGT
TGGTGTTAAGCATGAAACAAACAGCAGCTGTTGTCCTTAAAAATGAATTGACCTGGCCGGGCGCGGTGGCTCA
CGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCATGAGGTCAGGAGA-
TGGAGACCATCCTGGC TAACAAGGTGAAACCCCGTCTCTACTAAAAATACAAAAAAT-
TAGCCGGGCGCGGTGGCGGGCGCCTGTAGTCC CAGCTACTTGGGAGGCTGAGGCAGG-
AGAATGGCGTGAACCCGGGAAGCGGAGCTTGCAGTGAGCCGAGATTGC
GCCACTGCAGTCCGCAGTCCGGCCTGGGCGACAGAGCGAGACTCCGTCTC NOV5a,
CG54818-01 Protein Sequence SEQ ID NO:92 775 aa MW at 89226.6 kD
MASAGHIITLLLWGYLLELWTGGHTADTTHPRLRLSHKELLNLNRTSIFHSPFGFLDLHTMLL-
DEYQERLFVG GRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKDAGECANYV-
RVLHHYNRTHLLTCGTGAFDPVCAFIR VGYHLEDPLFHLESPRSERGRGRCPFDPSS-
SFISTLIGSELFAGLYSDYWSRDAAIFRSMGRLAHIRTEHDDE
RLLKEPKFVGSYMIPDNEDRDDNKVYFFFTEKALEAENNAHAIYTRVGRLCVNDVGGQRILVNKWSTFLKARL
VCSVPGMNGIDTYFDELEDVFLLPTRDHKNPVIFGLFNTTSNIFRGHAICVYHMSSI-
RAAFNGPYAHKEGPEY HWSVYEGKVPYPRPGSCASKVNGGRYGTTKDYPDDAIRFAR-
SHPLMYQAIKPAHKKPILVKTDGKYNLKQIAV DRVEAEDGQYDVLFIGTDNGIVLKV-
ITIYNQEMESMEEVILEELQIFKDPVPIISMEISSKRQQLYIGSASAV
AQVRFHHCDMYGSACADCCLARDPYCAWDGISCSRYYPTGTHAKRRFRRQDVRHGNAAQQCFGQQFVGDALDK
TEEHLAYGIENNSTLLECTPRSLQAKVIWFVQKGRETRKEEVKTDDRVVKMDLGLLF-
LRLHKSDAGTYFCQTV EHSFVHTVRKITLEVVEEEKVEDMFNKDDEEDRHHRMPCPA-
QSSISQGAKPWYKEFLQLIGYSNFQRVEEYCE KVWCTDRKRKKLKMSPSKWKYANPQ-
EKKLRSKPEHYRLPRHTLDS NOV5b, CG54818-02 SEQ ID NO:93 2373 bp DNA
Sequence ORF Start: ATG at 23 ORF Stop: end of sequence
CTTGGACGGTGAACACGGCAGCATGGCATCCGCGGGGCACATTATCACCTTGCTCCTGTGGGGTTACTT-
ACTG GAGCTTTGGACAGGAGGTCATACAGCTGATACTACCCACCCCCGGTTACGCC-
TGTCACATAAAGAGCTCTTGA ATCTGAACAGAACATCAATATTTCATAGCCCTTTTG-
GATTTCTTGATCTCCATACAATGCTGCTGGATGAATA
TCAAGAGAGGCTCTTCGTGGGAGGCAGGGACCTTGTATATTCCCTCAGCTTGGAGAGAATCAGTGACGGCTAT
AAAGAGATACACTGGCCGAGTACAGCTCTAAAAATGGAAGAATGCATAATGAAGGGA-
AAAGATGCGGGTGAAT GTGCAAATTATGTTCGGGTTTTGCATCACTATAACAGGACA-
CACCTTCTGACCTGTGGTACTGGAGCTTTTGA TCCAGTTTGTGCCTTCATCAGAGTT-
GGATATCATTTGGAGGATCCTCTGTTTCACCTCGAATCACCCAGATCT
GAGAGAGGAAGGGGCAGATGTCCTTTTGACCCCAGCTCCTCCTTCATCTCCACTTTAATTGGTAGTGAATTGT
TTGCTGGACTCTACAGTGACTACTGGAGCAGAGACGCTGCGATCTTCCCCAGCATGG-
GGCGACTGGCCCATAT CCGCACTGAGCATGACGATGAGCGTCTGTTGAAAGAACCAA-
AATTTGTAGGTTCATACATGATTCCTGACAAT GAAGACAGAGATGACAACAAAGTAT-
ATTTCTTTTTTACTGAGAAGCCACTGGAGGCAGAAAACAATGCTCACG
CAATTTACACCAGGGTCGGGCGACTCTGTGTGAATGATGTAGGAGGGCAGAGAATACTGGTGAATAAGTGGAG
CACTTTCCTAAAAGCGAGACTCGTTTGCTCAGTACCAGGAATGAATGGAATTGACAC-
ATATTTTGATGAATTA GAGGACGTTTTTTTGCTACCTACCAGAGATCATAAGAATCC-
AGTGATATTTGGACTCTTTAACACTACCAGTA ATATTTTTCGAGGGCATGCTATATG-
TGTCTATCACATGTCTAGCATTCGGGCAGCCTTCAACGGACCATATGC
ACATAAGGAAGGACCTGAATACCACTGGTCAGTCTATGAAGGAAAACTCCCTTATCCAAGGCCTGGTTCTTGT
GCCAGCAAAGTAAATGGAGGGAGATACGGAACCACCAAGGACTATCCTGATGATGCC-
ATCCGATTTGCAAGAA GTCATCCACTAATGTACCAGGCCATAAAACCTGCCCATAAA-
AAACCAATATTAGTAAAAACAGATGGAAAATA TAACCTGAAACAAATAGCAGTAGAT-
CGAGTGGAAGCTGAGCATGGCCAATATGACGTCTTGTTTATTGGGACA
GATAATGGAATTGTGCTGAAAGTAATCACAATTTACAACCAAGAAATGGAATCAATGGAAGAAGTAATTCTAG
AAGAACTTCAGATATTCAAGGATCCAGTTCCTATTATTTCTATGGAGATTTCTTCAA-
AGCGGCAACAGCTGTA TATTGGATCTGCTTCTGCTGTGGCTCAAGTCAGATTCCATC-
ACTGTGACATGTATGGAAGTGCTTGTGCTGAC TGCTGCCTGGCTCGAGACCCTTACT-
GTGCCTGGGATGGCATATCCTGCTCCCGGTATTACCCAACAGGCACAC
ATGCAAAAAGGCGTTTCCGGAGACAAGATGTTCGACATGCAAATGCAGCTCAGCAGTGCTTTGGACAACAGTT
TGTTGGGGATCCTTTGGATAAGACTGAAGAACATCTGGCTTATGGCATAGAGAACAA-
CAGTACTTTGCTGGAA TGTACCCCACGATCTTTACAAGCGAAAGTTATCTGGTTTGT-
ACAGAAAGGACGTGAGACAAGAAAAGAGGAGG TGAAGACAGATGACAGAGTGGTTAA-
GATGGACCTTGGTTTACTCTTCCTAAGGTTACACAAATCAGATGCTGG
GACCTATTTTTGCCAGACAGTAGAGCATAGCTTTGTCCATACGGTCCGTAAAATCACCTTGGAGGTAGTGGAA
GAGGAGAAAGTCGAGGATATGTTTAACAAGGACGATGAGGAGGACAGGCATCACAGG-
ATGCCTTGTCCTGCTC AGAGTAGCATCTCGCAGGGAGCAAAACCATGGTACAAGGAA-
TTCTTGCAGCTGATCGGTTATAGCAACTTCCA GAGAGTGGAAGAATACTGCGAGAAA-
GTATGGTGCACAGATAGAAAGAGGAAAAAGCTTAAAATGTCACCCTCC
AAGTGGAAGTATGCCAACCCTCAGGAAAAGAAGCTCCGTTCCAAACCTGAGCATTACCGCCTGCCCAGGCACA
CGCTGGACTCCTGATGGGGTGAGACTATCTACTGTCT NOV5b, CG54818-02 Protein
Sequence SEQ ID NO:94 755 aa MW at 89226.6 kD
MASAGHIITLLLWGYLLELWTGGHTADTTHPRLRLSHKELLNLNRTSIFHSPFGF-
LDLHTMLLDEYQERLFVG GRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKD-
AGECANYVRVLHHYIRTHLLTCGTGAFDPVCAFIR
VGYHLEDPLFHLESPRSERGRGRCPFDPSSSFISTLIGSELFAGLYSDYWSRDAAIFRSMGRLAHIRTEHDDE
RLLKEPKFVGSYMIPDNEDRDDNKVYFFFTEKALEAENNAHAIYTRVGRLCVNDVGG-
QRILVNKWSTFLKARL VCSVPGMNGIDTYFDELEDVFLLPTRDHKNPVIFGLFNTTS-
NIFRGHAICVYHMSSIRAAFNGPYAHKEGPEY HWSVYEGKVPYPRPGSCASKVNGGR-
YGTTKDYPDDAIRFARSHPLMYQAIKPAHKKPILVKTDGKYNLKQIAV
DRVEAEDGQYDVLFIGTDNGIVLKVITIYNQEMESMEEVILEELQIFKDPVPIISMEISSKRQQLYIGSASAV
AQVRFHHCDMYGSACADCCLARDPYCANDGISCSRYYPTGTHAKRRFRRQDVRHGNA-
AQQCFGQQFVGDALDK TEEHLAYGIENNSTLLECTPRSLQAKVIWFVQKGRETRKEE-
VKTDDRVVKMDLGLLFLRLHKSDAGTYFCQTV EHSFVIITVRKITLEVVEEEKVEDM-
FNKDDEEDRHRNPCPAQSSISQGAKPWYKEFLQLIGYSNFQRVEEYCE
KVWCTDRKRKKLKMSPSKWKYANPQEKKLRSKPEHYRLPRHTLDS NOV5c, CG54818-03 SEQ
ID NO:95 2347 bp DNA Sequence ORF Start: ATG at 14 ORF Stop: end of
sequence CACCGGTACCACCATGGCATCCGCGGGGCACATTATCACC-
TTGCTCCTGTGGGGTTACTTACTGGAGCTTTAA ACAGGAGGTCATACAGCTGATAC-
TACCCACCCCCGGTTACGCCTGTCACATAAAGAGCTCTTGAATCTGAACA
GAACATCAATATTTCATAGCCCTTTTGGATTTCTTGATCTCCATACAATGCTGCTGGATGAATATCAAGAGAG
GCTCTTCGTGGGAGGCAGCGACCTTGTATATTCCCTCAGCTTGGAGAGAATCAGTGA-
CGGCTATAAAGAGATA CACTGGCCGAGTACAGCTCTAAAAATGGAAGAATGCATAAT-
GAAGGGAAAAGATGCGGGTGAATGTGCAAATT ATGTTCGGGTTTTGCATCACTATAA-
CAGGACACACCTTCTGACCTGTGGTACTGGAGCTTTTGATCCAGTTTG
TGCCTTCATCAGAGTTGGATATCATTTGGAGGATCCTCTGTTTCACCTGGAATCACCCAGATCTGAGAGAGGA
AGCGGCAGATGTCCTTTTGACCCCAGCTCCTCCTTCATCTCCACTTTAATTGGTAGT-
GAATTGTTTGCTGGAC TCTACAGTGACTACTGGAGCAGAGACGCTGCGATCTTCCGC-
AGCATGGGGCGACTGGCCCATATCCGCACTGA GCATGACGATGAGCGTCTGTTGAAA-
GAACCAAAATTTGTAGGTTCATACATGATTCCTGACAATGAAGACAGA
GATGACAACAAAGTATATTTCTTTTTTACTGAGAAGGCACTGGAGGCAGAAAACAATGCTCACGCAATTTACA
CCAGGGTCGGGCGACTCTGTGTGAATGATGTAGGAGGGCAGAGAATACTGGTGAATA-
AGTGGAGCACTTTCCT AAAAGCGAGACTCGTTTGCTCAGTACCAGGAATGAATGGAA-
TTGACACATATTTTGATGAATTAGAGGACGTT TTTTTGCTACCTACCAGAGATCATA-
AGAATCCAGTGATATTTGGACTCTTTAACACTACCAGTAATATTTTTC
GAGGGCATGCTATATGTGTCTATCACATGTCTACCATTCGGGCAGCCTTCAACGGACCATATGCACATAAGGA
AGGACCTGAATACCACTGGTCAGTCTATGAAGGAAAAGTCCCTTATCCAAGGCCTGG-
TTCTTGTGCCAGCAAA GTAAATGGAGGGAGATACGGAACCACCAAGGACTATCCTGA-
TGATGCCATCCGATTTGCAAGAAGTAATCCAC TAATGTACCAGGCCATAAAACCTGC-
CCATAAAAAACCAATATTAGTAAAAACAGATGGAAAATATAACCTGAA
ACAAATAGCAGTAGATCGAGTGGAAGCTGAGGATGGCCAATATGACGTCTTGTTTATTGGGACAGATAATAAA
ATTGTGCTGAAAGTAATCACAATTTACAAACCAAGAATGGAATCAATGGAAGAAGTA-
ATTCTAGAAGAACTTC AGATATTCAAGGATCCAGTTCCTATTATTTCTATGCAGATT-
TCTTCAAAGCGGCAACAGCTGTATATTGGATC TGCTTCTGCTGTGGCTCAAGTCAGA-
TTCCATCACTGTGACATGTATGGAAGTGCTTGTGCTGACTGCTGCCTG
GCTCGAGACCCTTACTGTGCCTGGGATGGCATATCCTGCTCCCGGTATTACCCAACAGGCACACATGCAAAAA
GGCGTTTCCGGAGACAAGATGTTCGACATGGAAATGCAGCTCAGCAGTGCTTTGGAC-
AACAGTTTGTTGGGGA TGCTTTGGATAAGACTGAAGAACATCTGGCTTATGGCATAG-
AGAACAACAGTACTTTGCTGGAATGTACCCCA CGATCTTTACAAGCGAAAGTTATCT-
GGTTTGTACAGAAAGGACGTGAGACAAGAAAAGAGGAGGTGAAGACAG
ATGACAGAGTGGTTAAGATGGACCTTGGTTTACTCTTCCTAAGGTTACACAAATCAGATGCTGGCACCTATTT
TTGCCAGACAGTAGAGCATAGCTTTGTCCATACGGTCCGTAAAATCACCTTGGAGGT-
AGTGGAAGAGGAGAAA GTCGAGGATATGTTTAACAAGGACGATGAGGAGGACAGGCA-
TCACAGGATGCCTTGTCCTGCTCAGAGTAGCA TCTCGCAGGGAGCAAAACCATGGTA-
CAAGGAATTCTTGCAGCTGATCGGTTATAGCAACTTCCAGAGAGTGGA
AGAATACTGCGAGAAAGTATGGTGCACAGATAGAAAGAGGAAAAAGCTTAAAATGTCACCCTCCAAGTGGAAG
TATGCCAACCCTCAGGAAAAGAAGCTCCGTTCCAAACCTGAGCATTACCGCCTGCCC-
AGGCACACGCTGGACT CCGTCGACCGC NOV5c, CG54818-03 Protein Sequence SEQ
ID NO:96 775 aa MW at 89226.6 kD
MASAGHIITLLLWGYLLELWTGGHTADTTHPRLRLSHKELLNLNRTSIFHSPFGFLDLHTMLL-
DEYQERLFVG GRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKDAGECANYV-
RVLHHYNRTHLLTCGTGAFDPVCAFIR VGYHLEDPLFHLESPRSERGRGRCPFDPSS-
SFISTLIGSELFAGLYSDYWSRDAAIFRSMGRLAHIRTEHDDE
RLLKEPKFVGSYMIPDNEDRDDNKVYFFFTEKALEAENNAHAIYTRVGRLCVNDVGGQRILVNKWSTFLKAAA
VCSVPGMNGIDTYFDELEDVFLLPTRDHAAPVIFGLFNTTSNIFRGHAICVYHMSSI-
RAAFNGPYAAAAGPEY HWSVYEGKVPYPRPGSCASKVNGGRYGTTKDYPDDAIRFAR-
SHPLMYQAIKPAHKKPILVKTDGKYNLKQIAV DRVEAEDGQYDVLFIGTDNGIVLKV-
ITIYNQEMESMEEVILEELQIFKDPVPIISMEISSKRQQLYIGSASAV
AQVRFHHCDMYGSACADCCLARDPYCAWDGISCSRYYPTGTHAKRRFRRQDVRHGNAAQQCFGQQFVGDALDK
TEEHLAYGIENNSTLLECTPRSLQAKVIWFVQKGRETRKEEVKTDDRVVKMDLGLLF-
LRLHKSDAGTYFCQTV EHSFVHTVRKITLEVVEEEKEDMFNKDDEEDRHHRMPCPAQ-
SSISQGAKPWYKEFLQLIGYSNFQRVEEYACE KVWCTDRKRKKLKMSPSKWKYANPQ-
EKKLRSKPEHYRLPRHTLDS NOV5d, CG54818-04 SEQ ID NO:97 2347 bp DNA
Sequence ORF Start: ATG at 14 ORF Stop: end of sequence
CACCTCGCGAACCATGGCATCCGCGCGGCACATTATCACCTTGCTCCTGTGGGGTTACTTACTGGACCT-
TTAA ACAGGAGGTCATACAGCTGATACTACCCACCCCCGGTTACGCCTGTCACATA-
AAGAGCTCTTGAATCTGAACA GAACATCAATATTTCATAGCCCTTTTGGATTTCTTG-
ATCTCCATACAATGCTGCTGGATGAATATCAAGAGAG
GCTCTTCGTGGGAGGCAGGGACCTTGTATATTCCTCAGCTTGGAGAGAATCAGTGACGGCTATAAAAGAGATA
CACTGGCCGAGTACAGCTCTAAAAATGGAAGAATGCATAATGAAGGGAAAAGATGCG-
GGTGAATGTGCAAATT ATGTTCGGGTTTTGCATCACTATAACAGGACACACCTTCTG-
ACCTGTGGTACTGGAGCTTTTGATCCAGTTTG TGCCTTCATCAGAGTTGGATATCAT-
TTGGAGGATCCTCTGTTTCACCTGGAATCACCCAGATCTGAGAGAGGA
AGGGGCAGATGTCCTTTTGACCCCAGCTCCTCCTTCATCTCCACTTTAATTGGTAGTGAATTGTTTGCTGGAC
TCTACAGTGACTACTGGAGCAGAGACGCTGCGATCTTCCGCAGCATGGAACGACTGG-
CCCATATCCGCACTGA GCATGACGATGAGCGTCTGTTGAAAGAACCAAAATTTGTAG-
GTTCATACATGATTCCTGACAATGAAGACAGA GATGACAACAAAGTATATTTCTTTT-
TTACTGAGAAGGCACTGGAGGCAGAAAACAATGCTCACGCAATTTACA
CCAGGGTCGGGCGACTCTGTGTGAATGATGTAGGAGGGCAGAGAATACTGGTGAATAAGTGGAGCACTTTCCT
AAAGCGAGACTCGTTTGCTCAGTACCAGGAATGAATGGAATTGACACATATTTTGAT-
GAAATTAGAAAACGTT TTTTTGCTACCTACCAGAGATCATAAGAATCCAGTGATATT-
TGGACTCTTTAACACTACCAGTAATATTTTTC GAGGGCATGCTATATGTGTCTATCA-
CATGTCTAGCATTCAAGCAGCCTTCAACAAACCATATGCACATAAGGA
AGGACCTGAATACCACTGGTCAGTCTATGAAGGAAAAGTCCCTTATCCAAGGCCTGGTTCTTGTGCCAGCAAA
GTAAATGGAGGGAGATACGGAACCACCAAGGACTATCCTGATGATGCCATCCGATTT-
GCAAGAAGTCATCCAC TAATGTACCAGGCCATAAAACCTGCCCATAAAAAACCAATA-
TTAGTAAAAACAGATGGAAAATATAACCTGAA ACAAATAGCAGTAGATCGAGTGGAA-
GCTGAGGATGGCCAATATGACGTCTTGTTTATTGAAACAGATAATAAA
ATTGTGCTGAAAGTAATCACAATTTACAACCAAGAAATGGAATCAATGGAAGAAGTAATTCTAGAAGAACTTC
AGATATTCAAGGATCCAGTTCCTATTATTTCTATGGAGATTTCTTCAAAGCGGCAAC-
AGCTGTATATTGGATC TGCTTCTGCTGTGGCTCAAGTCAGATTCCATCACTGTGACA-
TGTATGGAAGTGCTTGTGCTGACTGCTGCCTG GCTCGAGACCCTTACTGTGCCTGGG-
ATGGCATATCCTGCTCCCGGTATTACCCAACAAACACACATGCAAAAA
GGCGTTTCCGGAGACAAGATGTTCGACATGGAAATGCAGCTCAGCAGTGCTTTGGACAACAGTTTGTTGGGGA
TGCTTTGGATAAGACTGAAGAACATCTCGCTTATGGCATAGAGAACAACAGTACTTT-
GCTGGAATGTACCCCA CGATCTTTACAAGCGAAAGTTATCTGGTTTGTACAGAAAGG-
ACGTGAGACAAGAAAAGAGGAGGTGAAGACAG ATGACAGAGTGGTTAAGATGGACCT-
TGGTTTACTCTTCCTAAGGTTACACAAATCAGATGCTAAGACCTATTT
TTGCCAGACAGTAGAGCATAGCTTTGTCCATACGGTCCGTAAAATCACCTTGGAGGTAGTGGAAGAGGAGAAA
GTCGAGGATATGTTTAACAAGGACGATGAGCAGGACAGGCATCACAGGATGCCTTGT-
CCTGCTCAGAGTAGCA TCTCGCACGGAGCAAAACCATGGTACAAGGAATTCTTGCAG-
CTGATCGGTTATAGCAACTTCCAGAGAGTAAA AGAATACTGCGAGAAAGTATGGTGC-
ACAGATAGAAAGAGGAAAAAGCTTAAAATGTCACCCTCCAAGTGGAAG
TATGCCAACCCTCAGGAAAAGAAGCTCCGTTCCAAACCTGAGCATTACCGCCTGCCCAGGCACACGCTGGACT
CCCCCGGGGGC NOV5d, CG54818-04 Protein Sequence SEQ ID NO:98 775 aa
MW at 89226.6 kD
MASAGHIITLLLWGYLLELWTGGHTADTTHPRLRLSHKELLNLNRTSIFHSPFGFLDLHTMLLDEYQERLAAG
GRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKDAGECANYVRVLHHYNRTH-
LLTCGTGAFDPVCAFIR VGYHLEDPLFHLESPRSERGRGRCPFDPSSSFISTLIGSE-
LFAGLYSDYWSRDAAIFRSMGRLARIRTEHDDE RLLKEPKFVGSYMIPDNEDRDDNK-
VYFFFTEKALEAENNAHAIYTRVGRLCVNDVGGQRILVNKWSTFLKARL
VCSVPGMNGIDTYFDELEDVFLLPTRDHKNPVIFGLFNTTSNIFRGHAICVYHMSSIRAAFNCPYAHKEGPEY
HWSVYEGKVPYPRPGSCASKVNGGRYGTTKDYPDDAIRFARSHPLMYQAIKPAHKKP-
ILVKTDGKYNLKQIAV DRVEAEDGQYDVLFIGTDNGIVLKVITIYNQEMESMEEVIL-
EELQIFKDPVPIISMEISSKRQQLYIGSASAV AGVRFHHCDMYGSACADCCLARDPY-
CAWDGISCSRYYPTGHAKRRFRRQDVRHGNAAQQCRFGQQFVGDALDK
TEEHLAYGIENNSTLLECTPRSLQAKVIWFVQKGRETRKEEVKTDDRVVKMDLGLLFLRLHKSDAGTYFCQAA
EHSFVHTVRKITLEVVEEEKVEDMFNKDDEEDRHHRMPCPAQSSISQGAKPWYKEFL-
QLIGYSNFQRVEEYCE KVWCTDRKRKKLKMSPSKWKYANPQEKKLRSKPEHYRLPRH-
TLDS
[0383] A ClustalW comparison of the above protein sequences yields
the following sequence alignment shown in Table 5B.
27TABLE 5B Comparison of the NOV5 protein sequences. NOV5a
MASAGHIITLLLWGYLLELWTGGHTADTTHPRL- RLSHKELLHLNRTSIFHSPFGFLDLHT (SEQ
ID NO: 92) NOV5b
MASAGHIITLLLWGYLLELWTGGHTADTTHPRLRLSHKELLNLMRTSIFHSPFGFLDLHT (SEQ
ID NO: 94) NOV5c
MASAGHIITLLLWGYLLELWTGGHTADTTHPRLRLSHKELLNLNRTSIFHSPFGFL- DLHT (SEQ
ID NO: 96) NOV5d MASAGHIITLLLWGYLLELWTGGHTADTTHPRLRLSHKEL-
LNLNRTSIFHSPFGFLDLHT (SEQ ID NO: 98) NOV5a
MLLDEYQERLFVGGRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKDAGECAAYAA NOV5b
MLLDEYQERLFVGGRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKDAGECANYVR NOV5c
MLLDEYQERLFVGGRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKDAGECANYVR NOV5d
MLLDEYQERLFVGGRDLVYSLSLERISDGYKEIHWPSTALKMEECIMKGKDAGECANYVR NOV5a
VLHHYNRTHLLTCGTGAFDPVCAFIRVGYHLEDPLFHLESPRSERGRGRCPFDPSSSFIS NOV5b
VLHHYNRTHLLTCGTGAFDPVCAFIRVGYHLEDPLFHLESPRSERGRGRCPFDPSSSFIS NOV5c
VLHHYNRTHLLTCGTGAFDPVCAFIRVGYHLEDPLFHLESPRSERGRGRCPFDPSSSFIS NOV5d
VLHHYNRTHLLTCGTGAFDPVCAFIRVGYHLEDPLFHLESPRSERGRGRCPFDPSSSFIS NOV5a
TLIGSELFAGLYSDYWSRDAAIFRSMGRLAHIRTEHDDERLLKEPKFVGSYMIPD- NEDRD
NOV5b TLIGSELFAGLYSDYWSRDAAIFRSMGRLAHIRTEHDDERLLKEPKFVGSYMIPD-
NEDRD NOV5c
TLIGSELFAGLYSDYWSRDAAIFRSMGRLAHIRTEHDDERLLKEPKFVGSYMIPD- NEDRD
NOV5d TLIGSELFAGLYSDYWSRDAAIFRSMGRLAHIRTEHDDERLLKEPKFVGSYMIPD-
NEDRD NOV5a DNKWYFFFTEKALEAENNAHAIYTRVGRLCVNDVGGQRILVNKWST-
FLKARLVCSVPGAA NOV5b
DNKVYFFFTEKALEAENNAHAIYTRVGRLCVNDVGGQRILVNKWST- FLKAALVCSVPGAA
NOV5c DNKVYFFFTEKALEAENNAHAIYTRVGRLCVNDVGGQRILVNKWST-
FLAAVCVCSVPGAA NOV5d
DNKVYFFFTEKALEAENNAHAIYTRVGRLCVNDVGGQRILVNKWST- FLKARLVCSVPGAA
NOV5a GIDTYFDELEDVFLLPTRDHKNPVIFGLFNTTSNIFR-
GHAIGVYHMSSIRAAFNGPYAAK NOV5b
GIDTYFDELEDVFLLPTRDHKNPVIFGLFNTTSNIFR- GHAIGVYHMSSIRAAFNGPYAAK
NOV5c GIDTYFDELEDVFLLPTRDHAAPVIFGLFNTTSNIFR-
GHAICVYHMSSIRAAFNGPYAAK NOV5d
GIDTYFDELEDVFLLPTRDHAAPVIFGLFNTTSNIFR- GHAICVYHMSSIAAAFNGPYAAK
NOV5a EGPEYHWSVYEGKVPYPRPGSCASKVNG-
GRYGTTKDYPDDAIRFARSHPLMYQAIKPAHK NOV5b
EGPEYHWSWYEGKVPYPRPGSCASKVNG- GRYGTTKDYPDDAIRFARSHPLMYQAIKPAAK
NOV5c EGPEYHWSVYEGKVPYPRPGSCASKMGG-
RYGTTKDYPDDAIRFARSHPIAAWQAIKPAAK NOV5d
EGPEYHWSVYEGKVPYPRPGSCASKVNG- GRYGTTKDYPDDAIRFARSHPLMYQAIKPAAK
NOV5a KPILVKTDGKYNLKQIAVDRVEAEDGQYDVLFIGTDNGIVLKVITIYNQEMESMEEVILE
NOV5b KPILVKTDGKYNLKQIAVDRVEAEDGQYDVLFIGTDNGIVLKVITIYMQEMESMEEVILE
NOV5c KPILVKTDGKYNLKQIAVDRVEAEDGQYDVLFIGTDNGIVLKVITIYMQEMESMEEVILE
NOV5d KPILVKTDGKYNLKOIAVDRVEAEDGQYDVLFIGTDNGIVLKVITIYNQEMESMEEVILE
NOV5a ELQIFKDPVPIISMEISSKRQQLYIGSASAVAQVRFHHCDMYGSACADCCLARDPYCAWD
NOV5b ELQIFKDPVPIISMEISSKRQQLYIGSASAVAQVRFHHCDMYGSACADCCLARDPYCAWD
NOV5c ELQTFKDPVPIISMEISSKRQQLYIGSASAVAQVRFHHCDMYGSACADCCLARDPYCAWD
NOV5d ELQIFKDPVPIISMEISSKROQLYIGSASAVAQVRFHHCDMYGSACADCCLARDPYCAWD
NOV5a GISCSRYYPTGTHAKRRFRRQDVRHGNAAQQCFGQQFVGDAIDKTEEHLAYGIEN-
NSTLL NOV5b
GISCSRYYPTGTHAKRRFRRQDVRHGNAAQQCFGQQFVGDALDKTEEHLAYGIEA- ASTLL
NOV5c GISCSRYYPTGTHAKRRFRRQDVRHGNAAQQCFGQQFVGDALDKTEEHLAYGIEA-
ASTLL NOV5d
GISCSRYYPTGTHAKRRFRRQDVRHGNAAQQCFGQQFVGDALDKTEEHLAYGIEA- ASTLL
NOV5a ECTPRSLQAKVIWFVQKGRETRKEEVKTDDRVVKMDLGLLFLRLHK-
SDAGTYFCQTVEHS NOV5b
ECTPRSLQAKVIWFVQKGRETRKEEVKTDDRVVKMDLGLLFLRLHK- SDAGTYFCQTVEHS
NOV5c ECTPRSLQAKVIWFVQKGRETRKEEVKTDDRVVKMDLGLLFLRLHK-
SDAGTYFCQTVEHS NOV5d
ECTPRSLQAKVIWFVQKGRETRKEEVKTDDRVVKMDLGLLELRLHK- SDAGTYFCQTAAHS
NOV5a FVHTVRKITLEVVEEEKVEDNFNKDDEEDRHHRMPCP-
AQSSISQGAKPWYKEFLQLIGYS NOV5b
FVMTVRKITLEVVEEEKVEDMFNKDDEEDRHHRMPCP- AQSSISQGAKPWYKEFLQLIGYS
NOV5c FVHTVRKITLEVVEEEKVEDMFNKDDEEDRHHRMPCP-
AQSSISQGAKPWYKEFLQLIGYS NOV5d
FVHTVRKITLEVVEEEKVEDMFNKDDEEDRHHRMPCP- AQSSISQGAKPWYKEFLQLIGYS
NOV5a NFQRVEEYCEKVWCTDRKRKKLKMSPSK- WKYANPQEKKLRSKPEHYRLPRHTLDS
NOV5b NFQRVEEYCEKVWCTDRKRKKLKMSPSKWKYAN- PQEKKLRSKPEHYRLPRHTLDS
NOV5c NFQRVEEYCEKVWCTDRKRKKLKMSPSKWKYANPQEKK- LRSKPEHYRLPRHTLDS
NOV5d NFQRVEEYCEKVWCTDRKRKKLKMSPSKWKYANPQEKKLRSKP- EHYRLPRHTLDS
[0384] Further analysis of the NOV5a protein yielded the following
properties shown in Table 5C.
28TABLE 5C Protein Sequence Properties NOV5a SignalP analysis:
Cleavage site between residues 27 and 28 PSORT II analysis: PSG: a
new signal peptide prediction method N-region: length 0; pos.chg 0;
neg.chg 0 H-region: length 17; peak value 9.78 PSG score: 5.38 GvH:
von Heijne's method for signal seq. recognition GvH score
(threshold: -2.1): -3.54 possible cleavage site: between 23 and 24
>>> Seems to have no N-terminal signal peptide ALOM: Klein
et al's method for TM region allocation Init position for
calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 1
Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 0.58
(at 1) ALOM score: -0.53 (number of TMSs: 0) MTOP: Prediction of
membrane topology (Hartmann et al.) Center position for
calculation: 6 Charge difference: -1.5 C(0.0) - N(1.5) N >= C:
N-terminal side will be inside MITDISC: discrimination of
mitochondrial targeting seq R content: 0 Hyd Moment(75): 2.30 Hyd
Moment(95): 4.12 G content: 2 D/E content: 1 S/T content: 2 Score:
-6.11 Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found NUCDISC: discrimination of nuclear
localization signals pat4: RKRK (5) at 737 pat4: KRKK (5) at 738
pat7: PQEKKLR (3) at 754 bipartite: none content of basic residues:
13.2% NLS Score: 0.41 KDEL: ER retention motif in the C-terminus:
none ER Membrane Retention Signals: none SKL: peroxisomal targeting
signal in the C-terminus: none PTS2: 2nd peroxisomal targeting
signal: none VAC: possible vacuolar targeting motif: none
RNA-binding motif: none Actinin-type actin-binding motif: type 1:
none type 2: none NMYR: N-myristoylation pattern: none Prenylation
motif: none 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 *** LDLHTMLLDEYQERLFVGGRDL at 56 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 = 9/23): 39.1%:
cytoplasmic 30.4%: mitochondrial 17.4%: nuclear 4.3%:
extracellular, including cell wall 4.3%: vacuolar 4.3%: endoplasmic
reticulum >> prediction for CG54818-01 is cyt (k = 23)
[0385] A search of the NOV5a protein against the Geneseq database,
a proprietary database that contains sequences published in patents
and patent publication, yielded several homologous proteins shown
in Table 5D.
29TABLE 5D Geneseq Results for NOV5a NOV5a Identities/ Residues/
Similarities for Geneseq Protein/Organism/Length [Patent #, Match
the Matched Expect Identifier Date] Residues Region Value AAW63748
Human semaphorin - Homo sapiens, 1 . . . 773 698/773 (90%) 0.0 775
aa. [WO9822504-A1, 1 . . . 773 738/773 (95%) 28-MAY-1998] AAY43090
Mouse semaphorin H (Sema H) amino 1 . . . 773 696/773 (90%) 0.0
acid sequence - Mus sp, 775 aa. 1 . . . 773 736/773 (95%)
[WO9947671-A2, 23-SEP-1999] AAG62730 Amino acid sequence of mouse 1
. . . 773 677/775 (87%) 0.0 semaphorin Sema3E - Mus sp, 777 aa. 1 .
. . 775 721/775 (92%) [WO200138491-A2, 31-MAY-2001] AAY43091 Mouse
semaphorin H variant (Sema 1 . . . 773 677/775 (87%) 0.0 Hv) amino
acid sequence - Mus sp, 1 . . . 775 721/775 (92%) 777 aa.
[WO9947671-A2, 23-SEP-1999] ABG96413 Human ovarian cancer marker
M473 - 8 . . . 770 373/770 (48%) 0.0 Homo sapiens, 771 aa. 7 . . .
769 519/770 (66%) [WO200271928-A2, 19-SEP-2002]
[0386] In a BLAST search of public sequence databases, the NOV5a
protein was found to have homology to the proteins shown in the
BLASTP data in Table 5E.
30TABLE 5E Public BLASTP Results for NOV5a NOV5a Protein Residues/
Identities/ Accession Match Similarities for the Expect Number
Protein/Organism/Length Residues Matched Portion Value O15041
Semaphorin 3E precursor - Homo 1 . . . 775 775/775 (100%) 0.0
sapiens (Human), 775 aa. 1 . . . 775 775/775 (100%) Q9QX23
Semaphorin M-SemaK - Mus 1 . . . 773 698/773 (90%) 0.0 musculus
(Mouse), 775 aa. 1 . . . 773 738/773 (95%) P70275 Semaphorin 3E
precursor 1 . . . 773 697/773 (90%) 0.0 (Semaphorin H) (Sema H) -
Mus 1 . . . 773 737/773 (95%) musculus (Mouse), 775 aa. O42237
Semaphorin 3E precursor 1 . . . 775 632/781 (80%) 0.0 (Collapsin-5)
(COLL-5) - Gallus 5 . . . 785 697/781 (88%) gallus (Chicken), 785
aa. Q63548 Semaphorin 3A precursor 8 . . . 770 374/771 (48%) 0.0
(Semaphorin III) (Sema III) - Rattus 7 . . . 770 523/771 (67%)
norvegicus (Rat), 772 aa.
[0387] PFam analysis predicts that the NOV5a protein contains the
domains shown in the Table 5F.
31TABLE 5F Domain Analysis of NOV5a Identities/Similarities NOV5a
for the Pfam Domain Match Region Matched Region Expect Value Sema
58 . . . 500 240/491 (49%) 3.8e-250 417/491 (85%) ig 595 . . . 656
12/66 (18%) 2.3e-05 46/66 (70%)
Example 6
[0388] The NOV6 clone was analyzed, and the nucleotide and encoded
polypeptide sequences are shown in Table 6A.
32TABLE 6A NOV6 Sequence Analysis NOV6a, CG55023-01 SEQ ID NO:99
527 bp DNA Sequence ORF Start: ATG at 49 ORF Stop: end of sequence
CCGTCAGTCTAGAAGGATAAGAG-
AAAGAAAGTTAAGCAACTACAGGAAATGGCTTTGGCAGTTCCAATATCAG
TCTATCTTTTATTCAACGCAATGACAGCACTGACCGAAGAGGCAGCCGTGACTGTAACACCTCCAATCACAGC
CCAGCAAGGTAACTGGACAGTTAACAAAACAGAAGCTCACAACATAGAAGGACCCAT-
AGCCTTGAAGTTCTCA CACCTTTGCCTGGAAGATCATAACAGTTACTGCATCAACGG-
TGCTTGTGCATTCCACCATGACCTAGAGAAAG CCATCTGCAGGTGTTTTACTGGTTA-
TACTGCAGAAAGGTGTGAGCACTTGACTTTAACTTCATATGCTGTGGA
TTCTTATGAAAAATACATTGCAATTGGGATTGGTGTTGGATTACTATTAAGTGGTTTTCTTGTTATTTTTTAC
TGCTATATAAGAAAGAGGTGTCTAAAATTGAAATCGCCTTACAATGTCTGTTCTGGA-
GAAAGACGACCACTGT GAGGCCTTTGTGAAGA NOV6a, CG55023-01 Protein
Sequence SEQ ID NO:100 154 aa MW at 17112.6 kD
MALGVPISVYLLFNAMTALTEEAAVTVTPPITAQQGNWTVNKTEAHNIEGPIALKFSHLCLED-
HNSYCINGAC AFHHELEKAICRCFTGYTGERCEHLTLTSYAVDSYEKYIAIGIGVG-
LLLSGFLVIFYCYIRKRCLKLKSPYNV CSGERRPL NOV6b, 248209521 SEQ ID NO:101
286 bp DNA Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCGGATCCGAAGAGGCAGCCGTGACTGTAACACCTC-
CAATCACAGCCCAGCAAGGTAACTGGACAGTTAAC
AAAACAGAAGCTGACAACATAGAAGGACCCATAGCCTTGAAGTTCTCACACCTTTGCCTGGAAGATCATAACA
GTTACTGCATCAACGGTGCTTGTGCATTCCACCATGAGCTAGAGAAAGCCATCTGCA-
GGTGTTTTACTGGTTA TACTGGAGAAACGTGTGAGCACTTGACTTTAACTTCATATG-
CTGTGGATTCTTATGAACTCCAGGGC NOV6b, 248209521 Protein Sequence SEQ ID
NO:102 95 aa MW at 10423.4 kD
TGSEEAAVTVTPPITAQQGNWTVNKTEADNIEGPIALKFSHLCLEDHNSYCINGACAFHHELEKAICRCFTGY
TGERCEHLTLTSYAVDSYELEC NOV6c, 317459649 SEQ ID NO:103 331 bp DNA
Sequence ORF Start: at 2 ORF Stop: end of sequence
CACCGGATCCACCATGGCTTTGGGAGTTCCAATATCAGTCTATCTTTTATTCAA-
CGCAATGACAGCACTGACC GAAGAGGCAGCCGTGACTGTAACACCTCCAATCACAG-
CCCAGCAAGGTAACTGGACAGTTAACAAAACAGAAG
CTGACAACATAGAAGGACCCATAGCCTTGAAGTTCTCACACCTTTGCCTGGAAGATCATAACAGTTACTGCAT
CAACGGTGCTTGTGCATTCCACCATGAGCTAGAGAAAGCCATCTGCAGGTGTCTAAA-
ATTGAAATCGCCTTAC AATGTCTGTTCTGCAGAAAGACGACCACTGCTCGAGCGC NOV6c,
317459649 Protein Sequence SEQ ID NO:104 110 aa MW at 11949.6 kD
TGSTMALGVPISVYLLFNAMTALTEEAAVTVTPPITAQQGNW-
TVNKTEADNIEGPIALKFSHLCLEDHNSYCI INGACAFUHELEKAICRCLKLKSPY-
NVCSGERRPLLEG NOV6d, 317459665 SEQ ID NO:105 358 bp DNA Sequence
ORF Start: at 2 ORF Stop: end of sequence
CACCGGATCCACCATGGCTTTGGGAGTTCCAATATCAGTCTATCTTTTATTCAACGCAATGACAGCACTGACC
GAAGAGGCAGCCGTGACTGTAACACCTCCAATCACAGCCCAGCAAGGTAACTGGAC-
AGTTAACAAAACAGAAG CTGACAACATAGAAGGACCCATAGCCTTGAAGTTCTCACA-
CCTTTGCCTGGAAGATCATAACAGTTACTGCAT CAACGGTGCTTGTGCATTCCACCA-
TGAGCTAGAGAAAGCCATCTGCAGGTGTTTTACTGGTTATACTGGAGAA
AGGTGTCTAAAATTGAAATCGCCTTACAATGTCTGTTCTGGAGAAAGACGACCACTGCTCGAGGGC
NOV6d, 317459665 Protein Sequence SEQ ID NO:106 119 aa MW at
12964.7 kD TGSTMALGVPISVYLLFNAMTALTEEAAVTVTPPITAQQGNWTVNK-
TEADNIEGPIALKFSHLCLEDHNSYCI NGACAFHHELEKAICRCFTGYTGERCLKL-
KSPYNVCSGERRPLLEG NOV6e,317459901 SEQ ID NO:107 331 bp DNA Sequence
ORF Start: at 2 ORF Stop: end of sequence
CACCGGATCCACCATGGCTTTGGGAGTTCCAATATCAGTCTATCTTTTATTCAACGCAATGACAGCACTGACC
GAAGAGGCAGCCGTGACTGTAACACCTCCAATCACAGCCCAGCAAGCTGACAACAT-
AGAAGGACCCATAGCCT TGAAGTTCTCACACCTTTGCCTGGAAGATCATAACAGTTA-
CTGCATCAACGGTGCTTGTGCATTCCACCATGA GCTAGAGAAACCCATCTGCAGGTG-
TTTTACTGGTTATACTGGAGAAAGGTGTCTAAAATTGAAATCGCCTTAC
AATGTCTGTTCTGGAGAAAGACGACCACTGCTCGAGGGC NOV6e, 317459901 Protein
Sequence SEQ ID NO:108 110 aa MW at 119346 kD
TGSTMALGVPISVYLLFNAMTALTEEAAVTVTPPITAQQADNIEGPIALKFSHLCLEDHNSYCINGACAFHHE
LEKAICRCFTGYTGERCLKLKSPYNVCSGERRPLLEG NOV6f, 317459910 SEQ ID
NO:109 268 bp DNA Sequence ORF Start: at 2 ORF Stop: end of
sequence CACCGGATCCGAAGAGGCAGCCGTGACTGTAACACCTC-
CAATCACAGCCCAGCAAGCTGACAACATACAAGGA
CCCATAGCCTTGAAGTTCTCACACCTTTGCCTGGAAGATCATAACAGTTACTGCATCAACGGTGCTTGTGCAT
TCCACCATGAGCTAGAGAAAGCCATCTGCAGGTGTTTTACTGGTTATACTCGAGAAA-
GGTGTCTAAAATTGAA ATCGCCTTACAATGTCTGTTCTGGAGAAAGACGACCACTGC-
TCGAGGGC NOV6f, 317459910 Protein Sequence SEQ ID NO:110 89 aa MW
at 9725.9 kD TGSEEAAVTVTPPITAQQADNIEGPIALKFSHLC-
LEDHNSYCINGACAFHHELEKAICRCFTGYTGERCLKLK SPYNVCSGERRPLLEG NOV6g,
CG55023-02 SEQ ID NO:111 274 bp DNA Sequence ORF Start: at 18 ORF
Stop: end of sequence
GAATTCGGCTTGGATCCGAAGAGGCAGCCGTGACTGTAACACCTCCAATCACAGCCCAGCAAGCTGACAACAT
AGAAGGACCCATAGCCTTGAAGTTCTCACACCTTTGCCTGGAAGATCATAACAGTT-
ACTGCATCAACGGTGCT TGTGCATTCCACCATGAGCTAGAGAAAGCCATCTGCAGGT-
GTTTTACTGGTTATACTGGAGAAAGGTGTGAGC ACTTGACTTTAACTTCATATGCTG-
TGGATTCTTATGAACTCGAGAAGCCGAATTC NOV6g, CG55023-02 Protein Sequence
SEQ ID NO:112 80 aa MW at 88483 kD
EEAAVTVTPPITAQQADNTEGPIALKFSHLCLEDHNSYCINGACAFHIIELEKAICRCFTGYTGERCEHLTLT-
S YAVDSYE NOV6h, CG55023-03 SEQ ID NO:113 249 bp DNA Sequence ORF
Start: at 1 ORF Stop: end of sequence
GAAGAGGCAGCCGTGACTGTAACACCTCCAATCACAGCCCAGCAAGGTAACTGGACAGTTAACAAAACAG-
AAG CTGACAACATAGAAGGACCCATAGCCTTGAAGTTCTCACACCTTTGCCTGGAA-
GATCATAACACTTACTGCAT CAACCGTGCTTGTGCATTCCACCATGAGCTAGAGAAA-
GCCATCTGCAGGTGTCTAAAATTGAAATCGCCTTAC AATGTCTGTTCTGGAGAAAGACGACCACTG
NOV6h, CG55023-03 Protein Sequence SEQ ID NO:114 83 aa MW at 9196.3
kD
EEAAVTVTPPITAQQGNWTVNKTEADNIEGPIALKFSHLCLEDHNSYCINGACAFHHELEKAICRCLKLKSPY
NVCSGERRPL NOV6i, CG55023-04 SEQ ID NO:115 249 bp DNA Sequence ORF
Start: at 1 ORF Stop: end of sequence
GAAGAGGCAGCCGTGACTGTAACACCTCCAATCACAGCCCAGCAAGCTGACAACATA-
GAAGGACCCATAGCCT TGAAGTTCTCACACCTTTGCCTGGAAGATCATAACAGTTA-
CTGCATCAACGGTGCTTGTGCATTCCACCATGA GCTAGAGAAAGCCATCTGCAGGTG-
TTTTACTGGTTATACTGGAGAAAGGTGTCTAPAATTGAAATCGCCTTAC
AATGTCTGTTCTGGAGAAAGACGACCACTG NOV6i, CG55023-04 Protein Sequence
SEQ ID NO:116 83 aa MW at 9181.3 kD
EEAAVTVTPPITAQQADNIEGPIALKFSHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCLKLKSPY
NVCSGERRPL NOV6j, CG55023-05 SEQ ID NO:117 267 bp DNA Sequence ORF
Start: at 1 ORF Stop: end of sequence
GAAGAGGCAGCCCTCACTGTAACACCTCCAATCACAGCCCAGCAAGGTAACTGGACA-
GTTAACAAAACAGAAG CTCACAACATAGAAGGACCCATAGCCTTGAAGTTCTCACA-
CCTTTGCCTGGAAGATCATAACAGTTACTGCAT CAACGGTGCTTGTGCATTCCACCA-
TGAGCTAGAGAAAGCCATCTGCAGGTGTTTTACTGGTTATACTGGAGAA
AGGTGTGAGCACTTGACTTTAACTTCATATGCTGTGGATTCTTATGAA NOV6j,CG55023-05
Protein Sequence SEQ ID NO:118 89 aa MW at 9878.8 kD
EEAAVTVTPPITAQQGNWTVNKTEADNIEGPIALKFSHLCLEDHNSYCINGACAFH-
HELEKAICRCFTGYTGE RCEHLTLTSYAVDSYE NOV6k, CG55023-06 SEQ ID NO:119
286 bp DNA Sequence ORF Start: at 11 ORF Stop: end of sequence
CACCGGATCCGAAGAGGCAGCCGTGACTGTAACACCTCCAATCA-
CAGCCCAGCAAGGTAACTGGACAGTTAAC AAAACAGAAGCTGACAACATAGAAGGA-
CCCATAGCCTTGAAGTTCTCACACCTTTGCCTGGAAGATCATAACA
GTTACTCCATCAACGGTGCTTGTGCATTCCACCATGAGCTAGAGAAAGCCATCTGCAGGTGTTTTACTGGTTA
TACTGGAGAAAGGTGTGAGCACTTGACTTTAACTTCATATGCTGTGGATTCTTATGA-
ACTCGAGGGC NOV6k, CG55023-06 Protein Sequence SEQ ID NO:120 89 aa
MW at 9878.8 kD EEAAVTVTPPITAQQGNWTVNKTEADNIEG-
PIALKFSHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGE RCEHLTLTSYAVDSYE
[0389] A ClustalW comparison of the above protein sequences yields
the following sequence alignment shown in Table 6B.
33TABLE 6B Comparison of the NOV6 protein sequences. NOV6a
----MALGVPISVYLLFNAMTALTEEAAVTVTP- PITAQQGNWTVNKTEAHNIEGPIALKF (SEQ
ID NO: 100) NOV6b
---------------------TGSEEAAVTVTPPITAQQONWTVNKTEADNIEGPIALKF (SEQ
ID NO: 102) NOV6c
TGSTMALGVPISVYLLFNAMTALTEEAAVTVTPPITAQQGNWTVNKTEADNIEGP- IALKF (SEQ
ID NO: 104) NOV6d TGSTMALGVPISVYLLFNANTALTEEAAVTVTPPITAQ-
QGNWTVNKTEADNIEGPIALKF (SEQ ID NO: 106) NOV6e
TGSTMALGVPISVYLLFNAMTALTEEAAVTVTPPITAQQ---------ADNIEGPIALKF (SEQ
ID NO: 108) NOV6f
---------------------TGSEEAAVTVTPPITAQQ---------ADNIEGP- IALKF (SEQ
ID NO: 110) NOV6g ------------------------EEAAVTVTPPITAQ-
Q---------ADNIEGPIALKF (SEQ ID NO: 112) NOV6h
------------------------EEAAVTVTPPITAQQGNWTVNKTEADNIECPIALKF (SEQ
ID NO: 114) NOV6i
------------------------EEAAVTVTPPITAQQ---------ADNIEGP- TALKF (SEQ
ID NO: 116) NOV6j ------------------------EEAAVTVTPPITAQ-
QGNWTVNKTEADNIEGPIALKF (SEQ ID NO: 118) NOV6k
------------------------EEAAVTVTPPITAQQGNWTVNKTEADNIEGPIALKF (SEQ
ID NO: 120) NOV6a SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCEHLTLTS-
YAVDSYEKYIAIGI NOV6b
SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCEHLTLTS- YAVDSYELEG----
NOV6c SHLCLEDHNSYCINGACAFHHELEKAICR---------CLKLKSPY-
NVCSGERRPLLEG- NOV6d
SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCLKLKSPY- NVCSGERRPLLEG-
NOV6e SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCLKLKSPY-
NVCSGERRPLLEG- NOV6f
SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCLKLKSPY- NVCSGERRPLLEG-
NOV6g SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCEHLTLTS-
YAVDSYE------- NOV6h
SHLCLEDHNSYCINGACAFHHELEKAICR---------CLKLKSPY- NVCSGERRPL----
NOV6i SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCLKLKSPY-
AACSGERRPL---- NOV6j
SHLCLEDHNSYCINGACAFHHELEKAICRCFTGYTGERCEHLTLTS- YAAASYE-------
NOV6k SHLCLEDHNSYCINGACAFMHELEKAICRCFTGYTGERCEHLTLTS-
YAAASYE------- NOV6a GVGLLLSGFLVIFYCYIRKRCLKLKSPYNVCSGERRP- L NOV6b
-------------------------------------- NOV6c
-------------------------------------- NOV6d
-------------------------------------- NOV6e
-------------------------------------- NOV6f
-------------------------------------- NOV6g
-------------------------------------- NOV6h
-------------------------------------- NOV6i
-------------------------------------- NOV6j
-------------------------------------- NOV6k
--------------------------------------
[0390] Further analysis of the NOV6a protein yielded the following
properties shown in Table 6C.
34TABLE 6C Protein Sequence Properties NOV6a SignalP analysis:
Cleavage site between residues 21 and 22 PSORT II analysis: PSG: a
new signal peptide prediction method N-region: length 0; pos.chg 0;
neg.chg 0 H-region: length 20; peak value 7.80 PSG Score: 3.40 GvH:
von Heijne's method for signal seq. recognition GvH score
(threshold: -2.1): -4.78 possible cleavage site: between 20 and 21
>>> 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 = -8.97
Transmenbrane 112-128 PERIPHERAL Likelihood = 9.39 (at 45) ALOM
score: -8.97 (number of TMSs: 1) MTOP: Prediction of membrane
topology (Hartmann et al.) Center position for calculation: 119
Charge difference: 6.5 C(5.0) - N(-1.5) C > N: C-terminal side
will be inside >>>Caution: Inconsistent mtop result with
signal peptide >>> membrane topology: type 1b (cytoplasmic
tail 112 to 154) MITDISC: discrimination of mitochondrial targeting
seq R content: 0 Hyd Moment(75) : 1.79 Hyd Moment(95): 0.79 G
content: 1 D/E content: 1 S/T content: 3 Score: -5.79 Gavel:
prediction of cleavage sites for mitochondrial preseq cleavage site
motif not found NUCDISC: discrimination of nuclear localization
signals pat4: none pat7: none bipartite: none content of basic
residues: 8.4% NLS Score: -0.47 KDEL: ER retention motif in the
C-terminus: none ER Membrane Retention Signals: none SKL:
peroxisomal targeting signal in the C-terminus: none PTS2: 2nd
peroxisomal targeting signal: none VAC: possible vacuolar targeting
motif: none RNA-binding motif: none Actinin-type actin-binding
motif: type 1: none type 2: none NMYR: N-myristoylation pattern:
none Prenylation motif: none memYQRL: transport motif from cell
surface to Golgi: none Tyrosines in the tail: 129,131,143 Dileucine
motif in the tail: found LL at 120 checking 63 PROSITE DNA binding
motifs: none checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN:
Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm
to detect coiled-coil regions total: 0 residues Final Results (k =
9/23): 30.4%: nuclear 26.1%: mitochondrial 21.7%: cytoplasmic 8.7%:
vesicles of secretory system 4.3%: vacuolar 4.3%: peroxisomal 4.3%:
endoplasmic reticulum >> prediction for CG55023-01 is nuc (k
= 23)
[0391] A search of the NOV1a protein against the Geneseq database,
a proprietary database that contains sequences published in patents
and patent publication, yielded several homologous proteins shown
in Table 6D.
35TABLE 6D Geneseq Results for NOV6a NOV6a Identities/ Residues/
Similarities for Geneseq Protein/Organism/Length [Patent #, Match
the Matched Expect Identifier Date] Residues Region Value ABB72157
Human protein isolated from skin cells 1 . . . 154 154/154 (100%)
4e-89 SEQ ID NO: 196 - Homo sapiens, 154 1 . . . 154 154/154 (100%)
aa. [WO200190357-A1, 29-NOV-2001] ABG76940 Human protein,
designated SEC4 - 1 . . . 154 154/154 (100%) 4e-89 Homo sapiens,
154 aa. 1 . . . 154 154/154 (100%) [WO200255705-A2, 18-JUL-2002]
AAB55957 Skin cell protein, SEQ ID NO: 196 - 1 . . . 154 154/154
(100%) 4e-89 Homo sapiens, 154 aa. 1 . . . 154 154/154 (100%)
[WO200069884-A2, 23-NOV-2000] AAE06704 Human transforming growth
factor 1 . . . 154 154/154 (100%) 4e-89 (TGF) alpha homologue huTR1
- 1 . . . 154 154/154 (100%) Homo sapiens, 154 aa. [WO200155333-A2,
02-AUG-2001] AAY76018 Human TGF-alpha homologue huTR1, 1 . . . 154
154/154 (100%) 4e-89 SEQ ID NO: 196 - Homo sapiens, 154 1 . . . 154
154/154 (100%) aa. [WO9955865-A1, 04-NOV-1999]
[0392] In a BLAST search of public sequence databases, the NOV6a
protein was found to have homology to the proteins shown in the
BLASTP data in Table 6E.
36TABLE 6E Public BLASTP Results for NOV6a NOV6a Identities/
Protein Residues/ Similarities for Accession Match the Matched
Expect Number Protein/Organism/Length Residues Portion Value Q924X1
Epigen protein precursor - Mus 1 . . . 149 119/150 (79%) 1e-64
musculus (Mouse), 152 aa. 1 . . . 148 129/150 (85%) Q8CEX5
Epithelial mitogen - Mus musculus 18 . . . 149 107/132 (81%) 3e-60
(Mouse), 136 aa (fragment). 3 . . . 132 116/132 (87%) Q06922
Transforming growth factor alpha 41 . . . 137 33/106 (31%) 2e-08
precursor (TGF-alpha) (EGF-like TGF) 25 . . . 130 52/106 (48%)
(ETGF) (TGF type 1) - Sus scrofa (Pig), 160 aa. Q8BRP7
Transmembrane protein with EGF-like 57 . . . 139 32/87 (36%) 3e-08
and two follistatin-like domains 1 - 147 . . . 233 48/87 (54%) Mus
musculus (Mouse), 256 aa (fragment). Q8C536 Transmembrane protein
with EGF-like 57 . . . 139 32/87 (36%) 3e-08 and two
follistatin-like domains 1 - 138 . . . 224 48/87 (54%) Mus musculus
(Mouse), 247 aa (fragment).
[0393] PFam analysis predicts that the NOV6a protein contains the
domains shown in the Table 6F.
37TABLE 6F Domain Analysis of NOV6a Identities/Similarities NOV6a
for the Pfam Domain Match Region Matched Region Expect Value EGF 60
. . . 95 13/47 (28%) 0.39 23/47 (49%)
Example 7
[0394] The NOV7 clone was analyzed, and the nucleotide and encoded
polypeptide sequences are shown in Table 7A.
38TABLE 7A NOV7 Sequence Analysis NOV7a, CG56136-01 SEQ ID NO:121
477 bp DNA Sequence ORF Start: ATG at 1 ORF Stop: end of sequence
ATGGAAAAAGCATTGAAAATTGAC-
ACACCTCAGCAGGGGAGCATTCAGGATATCAATCATCGGGTGTCGGTTC
TTCAGGACCAGACGCTCATAGCAGTCCCGACGAAGGACCGTATGTCTCCAGTCACTATTGCCTTAATCTCATG
CCGACATGTGGAGACCCTTGAGAAAGACAGAGGGAACCCCATCTACCTGGGCCTGAA-
TGGACTCAATCTCTGC CTGATGTGTGCTAAAGTCGGGGACCAGCCCACACTGCAGCT-
GAAGGAAAAGGATATAATGGATTTGTACAACC AACCCGAGCCTGTGAAGTCCTTTCT-
CTTCTACCACAGCCAGAGTGGCAGGAACTCCACCTTCGAGTCTGTGGC
TTTCCCTGGCTGGTTCATCGCTGTCAGCTCTGAAGGAGGCTGTCCTCTCATCCTTACCCAAGAACTGGGAAAA
GCCAACACTACTGACTTTGGGTTAACTATGCTGTTTTAA NOV7a, CG56136-01 Protein
Sequence SEQ ID NO:122 158 aa MW at 17684.2 kD
MEKALKIDTPQQGSIQDINHRVNVLQDQTLIAVPRKDRMSPVTIALISCRHVETL-
EKDRGNPIYLGLNGLNLC LMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQ-
SGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGK ANTTDFGLTMLF NOV7b, CG56136-02
SEQ ID NO:123 411 bp DNA Sequence ORF Start: ATG at 1 Stop: end of
sequence ATGGAAAAAGCATTGAAAGTTGACACAC-
CTCAGCGGGGGAGCATTCAGGATATCAATCATCGGGTGTGGGTTC
TTCAGGACCAGACGCTCATAGCAGTCCCGAGGAAGGACCGTATGTCTCCAGTCACTATTGCCTTAATCTCATG
CCCACATGTGGAGACCCTTGAGAAAGACAGAGGGAACCCCATCTACCTGGGCCTGAA-
TGGACTCAATCTCTGC CTGATGTGTGTTCAAGTCGGGGACCAGCCCACACTGCAGAT-
GAACCAGAGTGGCAGGAACTCCACCTTCGAGT CTGTGGCTTTCCCTGGCTGGTTGAT-
CGCTGTCAGCTCTGAAGGAGGCTGTCCTCTCATCCTTACCCAAGAACT
GGGGAAAGCCAACACTACTGACTTTGGGTTAACTATGCTCTTTTAA NOV7b, CG56136-02
Protein Sequence SEQ ID NO:124 136 aa MW at 15013.2 kD
MEKALKVDTPQRGSIQDINHRVWVLQDQTLIAVPRKDRNSPVTIALISCRHVETLEKDRGNPI-
YLGLNGLNC LMCVQVGDQPTLQMNQSGRNSTFESVAFPGWLIAVSSEGGCPLILTQ-
ELGKANTTDFGLTMLF NOV7c, CG56136-03 SEQ ID NO:125 474 bp DNA
Sequence ORF Start: ATG at 1 ORF Stop: end of sequence
ATGGAAAAAGCATTGAAAATTGACACACCTCAGCAGGGGAGCATTCAGGATATCAATCATCGGGTGTGGGTTC
TTCAGGACCAGACGCTCATAGCAGTCCCGAGGAAGGACCGTATGTCTCCAGTCACT-
ATTGCCTTAATCTCATG CCGACATGTGGAGACCCTTGAGAAAAGACAGAGGGAACCC-
CATCTACCTGGGCCTGAATGGACTCAATCTCTC CTCATGTGTGCTAAAGTCGGGGAC-
CAGCCCACACTGCAGCTGAAGGAAAAGGATATAATGGATTTGTACAACC
AACCCGACCCTGTCAAGTCCTTTCTCTTCTACCACAGCCAGAGTGGCAGGAACTCCACCTTCGAGTCTGTGGC
TTTCCCTGGCTGGTTCATCGCTGTCAGCTCTGAAGGAGGCTGTCCTCTCATCCTTAC-
CCAAGAACTGGGGAAA GCCAACACTACTGACTTTGGGTTAACTATGCTGTTT NOV7c,
CG56136-03 Protein Sequence SEQ ID NO:126 158 aa MW at 17684.2 kD
MEKALKIDTPQQGSIQDINHRVWVLQDQTLIAVPRKDRNSPV-
TIALISCRHVETLEKDRGNPIYLGLNGLNLC LMCAKVGDQPTLQLKEKDIMDLYNQ-
PEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGK ANTTDFGLTMLF
NOV7d,CG56136-04 SEQ ID NO:127 99 bp DNA Sequence ORF Start: at 1
ORF Stop: at 100
TGTGTTCAAGTCGGGGACCAGCCCACACTGCAGATGAACCAGAGTGGCAGGAACTCCACCTTCCAGTCTGTGG
CTTTCCCTGGCTGGTTGATCGCTGTC NOV7d, CG56136-04 Protein Sequence SEQ
ID NO:128 33 aa MW at 3581.0 kD
CVQVGDQPTLQMNQSGRNSTFESVAFPCWLIAV
[0395] A ClustalW comparison of the above protein sequences yields
the following sequence alignment shown in Table 7B.
39TABLE 7B Comparison of the NOV7 protein sequences. NOV7a
MEKALKIDTPQQGSIQDINHRVWVLQDQTLIAV- PRKDRMSPVTIALISCRHVETLEKDRG (SEQ
ID NO: 122) NOV7b
MEKALKVDTPQRGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRG (SEQ
ID NO: 124) NOV7c
MEKALKIDTPQQGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETL- EKDRC (SEQ
ID NO: 126) NOV7d ---------------------------------------
---------------------- (SEQ ID NO: 128) NOV7a
NPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFES NOV7b
NPIYLGLNGLNLCLMCVQVGDQPTLQMN----------------------QSGRNSTFES NOV7c
NPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFES NOV7d
---------------CVQVGDQPTLQMN----------------------QSGRNSTFES NOV7a
VAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLTMLF NOV7b
VAFPGWLIAVSSECGCPLILTQELGKANTTDFGLTMLF NOV7c
VAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLTMLF NOV7d VAFPGWLIAV
[0396] Further analysis of the NOV7a protein yielded the following
properties shown in Table 7C.
40TABLE 7C Protein Sequence Properties NOV7a SignalP analysis: No
Known Signal Sequence Predicted PSORT II analysis: PSG: a new
signal peptide prediction method N-region: length 8; pos.chg 2;
neg.chg 2 H-region: length 8; peak value -3.46 PSG score: -7.86
GvH: von Heijne's method for signal seq. recognition GvH score
(threshold: -2.1): -5.60 possible cleavage site: between 54 and 55
>>> Seems to have no N-terminal signal peptide ALOM: Klein
et al's method for TM region allocation Init position for
calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) . . . fixed PERIPHERAL Likelihood = 1.85 (at 63)
ALOM score: 1.85 (number of TMSs: 0) MITDISC: discrimination of
mitochondrial targeting seq R content: 0 Hyd Moment (75): 10.39 Hyd
Moment (95): 7.73 G content: 0 D/E content: 2 S/T content: 0 Score:
-6.26 Gavel: prediction of cleavage sites for mitochondrial preseq
cleavage site motif not found NUCDISC: discrimination of nuclear
localization signals pat4: none pat7: PRKDRMS (5) at 34 bipartite:
none content of basic residues: 9.5% NLS Score: -0.04 KDEL: ER
retention motif in the C-terminus: none ER Membrane Retention
Signals: none SKL: peroxisomal targeting signal in the C-terminus:
none PTS2: 2nd peroxisomal targeting signal: none VAC: possible
vacuolar targeting motif: none RNA-binding motif: none Actinin-type
actin-binding motif: type 1: none type 2: none NMYR:
N-myristoylation pattern: none Prenylation motif: none memYQRL:
transport motif from cell surface to Golgi: none Tyrosines in the
tail: none Dileucine motif in the tail: none checking 63 PROSITE
DNA binding motifs: none checking 71 PROSITE ribosomal protein
motifs: none checking 33 PROSITE prokaryotic DNA binding motifs:
none NNCN: Reinhardt's method for Cytoplasmic/Nuclear
discrimination Prediction: cytoplasmic Reliability: 76.7 COIL:
Lupas's algorithm to detect coiled-coil regions total: 0 residues
Final Results (k = 9/23): 39.1%: cytoplasmic 34.8%: mitochondrial
21.7%: nuclear 4.3%: vacuolar >> prediction for CG56136-01 is
cyt (k = 23)
[0397] A search of the NOV7a protein against the Geneseq database,
a proprietary database that contains sequences published in patents
and patent publication, yielded several homologous proteins shown
in Table 7D.
41TABLE 7D Geneseq Results for NOV7a NOV7a Identities/ Residues/
Similarities for Geneseq Protein/Organism/Length [Patent #, Match
the Matched Expect Identifier Date] Residues Region Value AAY70217
Human Interleukin-1 epsilon protein - 1 . . . 158 158/158 (100%)
2e-89 Homo sapiens, 158 aa. 1 . . . 158 158/158 (100%)
[WO200011174-A1, 02-MAR-2000] AAY70218 Human Interleukin-1 epsilon
1 . . . 158 157/158 (99%) 7e-89 polymorphic variant - Homo sapiens,
1 . . . 158 158/158 (99%) 158 aa. [WO200011174-A1, 02-MAR-2000]
AAE03417 Human interleukin-1 receptor 5 . . . 158 152/154 (98%)
5e-86 antagonist-like (IL-1ra-L) - Homo 120 . . . 273 154/154 (99%)
sapiens, 273 aa. [WO200141792-A1, 14-JUN-2001] ABP52038 NOVINTRA C
homologous amino 17 . . . 146 130/130 (100%) 8e-73 acid sequence
SEQ ID NO: 87 - Homo 1 . . . 130 130/130 (100%) sapiens, 130 aa.
[US2002068279-A1, 06-JUN-2002] ABP52037 NOVINTRA C homologous amino
17 . . . 146 126/130 (96%) 1e-68 acid sequence SEQ ID NO: 86 - Homo
1 . . . 126 126/130 (96%) sapiens, 126 aa. [US2002068279-A1,
06-JUN-2002]
[0398] In a BLAST search of public sequence databases, the NOV7a
protein was found to have homology to the proteins shown in the
BLASTP data in Table 7E.
42TABLE 7E Public BLASTP Results for NOV7a NOV7a Identities/
Protein Residues/ Similarities for Accession Match the Matched
Expect Number Protein/Organism/Length Residues Portion Value Q9UHA7
Interleukin 1 family member 6 1 . . . 158 158/158 (100%) 7e-89
(IL-1F6) (Interleukin-1 epsilon) (IL-1 1 . . . 158 158/158 (100%)
epsilon) (FIL1 epsilon) - Homo sapiens (Human), 158 aa. Q9NZH8
Interleukin 1 family member 9 10 . . . 156 88/147 (59%) 4e-46
(IL-1F9) (Interleukin-1 homolog 1) 22 . . . 167 112/147 (75%)
(IL-1H1) (Interleukin-1 epsilon) (IL-1 epsilon) (IL-1 related
protein 2) (IL-1RP2) - Homo sapiens (Human), 169 aa. Q9JLA2
Interleukin 1 family member 6 2 . . . 157 85/156 (54%) 7e-46
(IL-1F6) (Interleukin-1 epsilon) (IL-1 4 . . . 159 115/156 (73%)
epsilon) (FIL1 epsilon) (Interleukin-1 homolog 1) (IL-1H1) - Mus
musculus (Mouse), 160 aa. CAC21807 Sequence 1 from Patent WO0071720
- 10 . . . 154 71/145 (48%) 1e-36 Homo sapiens (Human), 157 aa. 9 .
. . 153 100/145 (68%) Q8R460 Interleukin 1 family member 9 8 . . .
154 69/147 (46%) 2e-36 (IL-1F9) - Mus musculus (Mouse), 164 15 . .
. 160 105/147 (70%) aa.
[0399] PFam analysis predicts that the NOV7a protein contains the
domains shown in the Table 7F.
43TABLE 7F Domain Analysis of NOV7a Identities/Similarities NOV7a
for the Pfam Domain Match Region Matched Region Expect Value IL1 15
. . . 158 44/147(30%) 4.5e-18 90/147 (61%)
Example B
Sequencing Methodology and Identification of NOVX Clones
[0400] 1. GeneCalling.TM. 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 additional, gene-specific competitive PCR or by
isolation and sequencing of the gene fragment.
[0401] 2. SeqCalling.TM. Technology: cDNA was derived from various
human samples representing multiple tissue types, normal and
diseased states, physiological states, and developmental states
from different donors. Samples were obtained as whole tissue,
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 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.
[0402] 3. PathCalling.TM. 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:
[0403] 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, Calif.) were
then transferred from E. coli into a CuraGen Corporation
proprietary yeast strain (disclosed in U.S. Pat. Nos. 6,057,101 and
6,083,693, incorporated herein by reference in their
entireties).
[0404] 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 forms single
nucleotide polymorphisms (SNPs), insertions, deletions and other
sequence variations.
[0405] 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. Pat. Nos. 6,057,101
and 6,083,693).
[0406] 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.
[0407] 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. 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.
[0408] 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.
[0409] The PCR product derived by exon linking, covering the entire
open reading frame, was cloned into the pCR2.1 vector from
Invitrogen to provide clones used for expression and screening
purposes.
Example C
Quantitative Expression Analysis of Clones in Various Cells and
Tissues
[0410] The quantitative expression of various clones was assessed
using microtiter plates containing RNA samples from a variety of
normal and pathology-derived cells, cell lines and tissues using
real time quantitative PCR (RTQ PCR). RTQ PCR was performed on an
Applied Biosystems ABI PRISM.RTM. 7700 or an ABI PRISM.RTM. 7900 HT
Sequence Detection System. Various collections of samples are
assembled on the plates, and referred to as Panel 1 (containing
normal tissues and cancer cell lines), Panel 2 (containing samples
derived from tissues from normal and cancer sources), Panel 3
(containing cancer cell lines), Panel 4 (containing cells and cell
lines from normal tissues and cells related to inflammatory
conditions), Panel 5D/5I (containing human tissues and cell lines
with an emphasis on metabolic diseases), AI_comprehensive_panel
(containing normal tissue and samples from autoinflammatory
diseases), Panel CNSD.01 (containing samples from normal and
diseased brains) and CNS_neurodegeneration_panel (containing
samples from normal and Alzheimer's diseased brains).
[0411] RNA integrity from all samples is controlled for quality by
visual assessment of agarose gel electropherograms using 28S and
18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1
28s:18s) and the absence of low molecular weight RNAs that would be
indicative of degradation products. Samples are controlled against
genomic DNA contamination by RTQ PCR reactions run in the absence
of reverse transcriptase using probe and primer sets designed to
amplify across the span of a single exon.
[0412] First, the RNA samples were normalized to reference nucleic
acids such as constitutively expressed genes (for example,
.beta.-actin and GAPDH). Normalized RNA (5 ul) was converted to
cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix
Reagents (Applied Biosystems; Catalog No. 4309169) and
gene-specific primers according to the manufacturer's
instructions.
[0413] In other cases, non-normalized RNA samples were converted to
single strand cDNA (sscDNA) using Superscript II (Invitrogen
Corporation; Catalog No. 18064-147) and random hexamers according
to the manufacturer's instructions. Reactions containing up to 10
.mu.g of total RNA were performed in a volume of 20 .mu.l and
incubated for 60 minutes at 42.degree. C. This reaction can be
scaled up to 50 .mu.g of total RNA in a final volume of 100 .mu.l.
sscDNA samples are then normalized to reference nucleic acids as
described previously, using 1.times. TaqMan.RTM. Universal Master
mix (Applied Biosystems; catalog No. 4324020), following the
manufacturer's instructions.
[0414] Probes and primers were designed for each assay according to
Applied Biosystems Primer Express Software package (version I for
Apple Computer's Macintosh Power PC) or a similar algorithm using
the target sequence as input. Default settings were used for
reaction conditions and the following parameters were set before
selecting primers: primer concentration=250 nM, primer melting
temperature (Tm) range=58.degree.-60.degree. C., primer optimal
Tm=59.degree. C., maximum primer difference=2.degree. C., probe
does not have 5'G, probe Tm must be 10.degree. C. greater than
primer Tm, amplicon size 75 bp to 100 bp. The probes and primers
selected (see below) were synthesized by Synthegen (Houston, Tex.,
USA). Probes were double purified by HPLC to remove uncoupled dye
and evaluated by mass spectroscopy to verify coupling of reporter
and quencher dyes to the 5' and 3' ends of the probe, respectively.
Their final concentrations were: forward and reverse primers, 900
nM each, and probe, 200 nM.
[0415] PCR conditions: When working with RNA samples, normalized
RNA from each tissue and each cell line was spotted in each well of
either a 96 well or a 384-well PCR plate (Applied Biosystems). PCR
cocktails included either a single gene specific probe and primers
set, or two multiplexed probe and primers sets (a set specific for
the target clone and another gene-specific set multiplexed with the
target probe). PCR reactions were set up using TaqMan.RTM. One-Step
RT-PCR Master Mix (Applied Biosystems, Catalog No. 4313803)
following manufacturer's instructions. Reverse transcription was
performed at 48.degree. C. for 30 minutes followed by
amplification/PCR cycles as follows: 95.degree. C. 10 min, then 40
cycles of 95.degree. C. for 15 seconds, 60.degree. C. for 1 minute.
Results were recorded as CT values (cycle at which a given sample
crosses a threshold level of fluorescence) using a log scale, with
the difference in RNA concentration between a given sample and the
sample with the lowest CT value being represented as 2 to the power
of delta CT. The percent relative expression is then obtained by
taking the reciprocal of this RNA difference and multiplying by
100. Expression with CT values below 28 is considered as high
expression, CT values between 28 and 32 is considered moderate and
CT value between 32 to 35 is considered as low expression. All the
relative expression with CT values above 35 is not considered as
significant expression.
[0416] When working with sscDNA samples, normalized sscDNA was used
as described previously for RNA samples. PCR reactions containing
one or two sets of probe and primers were set up as described
previously, using 1.times. TaqMan.RTM. Universal Master mix
(Applied Biosystems; catalog No. 4324020), following the
manufacturer's instructions. PCR amplification was performed as
follows: 95.degree. C. 10 min, then 40 cycles of 95.degree. C. for
15 seconds, 60.degree. C. for 1 minute. Results were analyzed and
processed as described previously.
[0417] Panels 1, 1.1, 1.2, and 1.3D
[0418] The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control
wells (genomic DNA control and chemistry control) and 94 wells
containing cDNA from various samples. The samples in these panels
are broken into 2 classes: samples derived from cultured cell lines
and samples derived from primary normal tissues. The cell lines are
derived from cancers of the following types: lung cancer, breast
cancer, melanoma, colon cancer, prostate cancer, CNS cancer,
squamous cell carcinoma, ovarian cancer, liver cancer, renal
cancer, gastric cancer and pancreatic cancer. Cell lines used in
these panels are widely available through the American Type Culture
Collection (ATCC), a repository for cultured cell lines, and were
cultured using the conditions recommended by the ATCC. The normal
tissues found on these panels are comprised of samples derived from
all major organ systems from single adult individuals or fetuses.
These samples are derived from the following organs: adult skeletal
muscle, fetal skeletal muscle, adult heart, fetal heart, adult
kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal
lung, various regions of the brain, the spleen, bone marrow, lymph
node, pancreas, salivary gland, pituitary gland, adrenal gland,
spinal cord, thymus, stomach, small intestine, colon, bladder,
trachea, breast, ovary, uterus, placenta, prostate, testis and
adipose.
[0419] In the results for Panels 1, 1.1, 1.2 and 1.3D, the
following abbreviations are used:
[0420] ca.=carcinoma,
[0421] *=established from metastasis,
[0422] met=metastasis,
[0423] s cell var=small cell variant,
[0424] non-s=non-sm=non-small,
[0425] squam=squamous,
[0426] pl. eff=pl effusion=pleural effusion,
[0427] glio=glioma,
[0428] astro=astrocytoma, and
[0429] neuro=neuroblastoma.
[0430] General_Screening_Panel_v1.4, v1.5, v1.6 and 1.7
[0431] The plates for Panels 1.4, 1.5, 1.6 and 1.7 include 2
control wells (genomic DNA control and chemistry control) and 88 to
94 wells containing cDNA from various samples. The samples in
Panels 1.4, 1.5, 1.6 and 1.7 are broken into 2 classes: samples
derived from cultured cell lines and samples derived from primary
normal tissues. The cell lines are derived from cancers of the
following types: lung cancer, breast cancer, melanoma, colon
cancer, prostate cancer, CNS cancer, squamous cell carcinoma,
ovarian cancer, liver cancer, renal cancer, gastric cancer and
pancreatic cancer. Cell lines used in Panels 1.4, 1.5, 1.6 and 1.7
are widely available through the American Type Culture Collection
(ATCC), a repository for cultured cell lines, and were cultured
using the conditions recommended by the ATCC. The normal tissues
found on Panels 1.4, 1.5, 1.6 and 1.7 are comprised of pools of
samples derived from all major organ systems from 2 to 5 different
adult individuals or fetuses. These samples are derived from the
following organs: adult skeletal muscle, fetal skeletal muscle,
adult heart, fetal heart, adult kidney, fetal kidney, adult liver,
fetal liver, adult lung, fetal lung, various regions of the brain,
the spleen, bone marrow, lymph node, pancreas, salivary gland,
pituitary gland, adrenal gland, spinal cord, thymus, stomach, small
intestine, colon, bladder, trachea, breast, ovary, uterus,
placenta, prostate, testis and adipose. Abbreviations are as
described for Panels 1, 1.1, 1.2, and 1.3D.
[0432] Panels 2D, 2.2, 2.3 and 2.4
[0433] The plates for Panels 2D, 2.2, 2.3 and 2.4 generally include
2 control wells and 94 test samples composed of RNA or cDNA
isolated from human tissue procured by surgeons working in close
cooperation with the National Cancer Institute's Cooperative Human
Tissue Network (CHTN) or the National Disease Research Initiative
(NDRI) or from Ardais or Clinomics). The tissues are derived from
human malignancies and in cases where indicated many malignant
tissues have "matched margins" obtained from noncancerous tissue
just adjacent to the tumor. These are termed normal adjacent
tissues and are denoted "NAT" in the results below. The tumor
tissue and the "matched margins" are evaluated by two independent
pathologists (the surgical pathologists and again by a pathologist
at NDRI/CHTN/Ardais/Clinomics). Unmatched RNA samples from tissues
without malignancy (normal tissues) were also obtained from Ardais
or Clinomics. This analysis provides a gross histopathological
assessment of tumor differentiation grade. Moreover, most samples
include the original surgical pathology report that provides
information regarding the clinical stage of the patient. These
matched margins are taken from the tissue surrounding (i.e.
immediately proximal) to the zone of surgery (designated "NAT", for
normal adjacent tissue, in Table RR). In addition, RNA and cDNA
samples were obtained from various human tissues derived from
autopsies performed on elderly people or sudden death victims
(accidents, etc.). These tissues were ascertained to be free of
disease and were purchased from various commercial sources such as
Clontech (Palo Alto, Calif.), Research Genetics, and
Invitrogen.
[0434] HASS Panel v 1.0
[0435] The HASS panel v 1.0 plates are comprised of 93 cDNA samples
and two controls. Specifically, 81 of these samples are derived
from cultured human cancer cell lines that had been subjected to
serum starvation, acidosis and anoxia for different time periods as
well as controls for these treatments, 3 samples of human primary
cells, 9 samples of malignant brain cancer (4 medulloblastomas and
5 glioblastomas) and 2 controls. The human cancer cell lines are
obtained from ATCC (American Type Culture Collection) and fall into
the following tissue groups: breast cancer, prostate cancer,
bladder carcinomas, pancreatic cancers and CNS cancer cell lines.
These cancer cells are all cultured under standard recommended
conditions. The treatments used (serum starvation, acidosis and
anoxia) have been previously published in the scientific
literature. The primary human cells were obtained from Clonetics
(Walkersville, Md.) and were grown in the media and conditions
recommended by Clonetics. The malignant brain cancer samples are
obtained as part of a collaboration (Henry Ford Cancer Center) and
are evaluated by a pathologist prior to CuraGen receiving the
samples. RNA was prepared from these samples using the standard
procedures. The genomic and chemistry control wells have been
described previously.
[0436] ARDAIS Panel v 1.0
[0437] The plates for ARDAIS panel v 1.0 generally include 2
control wells and 22 test samples composed of RNA isolated from
human tissue procured by surgeons working in close cooperation with
Ardais Corporation. The tissues are derived from human lung
malignancies (lung adenocarcinoma or lung squamous cell carcinoma)
and in cases where indicated many malignant samples have "matched
margins" obtained from noncancerous lung tissue just adjacent to
the tumor. These matched margins are taken from the tissue
surrounding (i.e. immediately proximal) to the zone of surgery
(designated "NAT", for normal adjacent tissue) in the results
below. The tumor tissue and the "matched margins" are evaluated by
independent pathologists (the surgical pathologists and again by a
pathologist at Ardais). Unmatched malignant and non-malignant RNA
samples from lungs were also obtained from Ardais. Additional
information from Ardais provides a gross histopathological
assessment of tumor differentiation grade and stage. Moreover, most
samples include the original surgical pathology report that
provides information regarding the clinical state of the
patient.
[0438] ARDAIS Prostate v 1.0
[0439] The plates for ARDAIS prostate 1.0 generally include 2
control wells and 68 test samples composed of RNA isolated from
human tissue procured by surgeons working in close cooperation with
Ardais Corporation. The tissues are derived from human prostate
malignancies and in cases where indicated malignant samples have
"matched margins" obtained from noncancerous prostate tissue just
adjacent to the tumor. These matched margins are taken from the
tissue surrounding (i.e. immediately proximal) to the zone of
surgery (designated "NAT", for normal adjacent tissue) in the
results below. The tumor tissue and the "matched margins" are
evaluated by independent pathologists (the surgical pathologists
and again by a pathologist at Ardais). RNA from unmatched malignant
and non-malignant prostate samples were also obtained from Ardais.
Additional information from Ardais provides a gross
histopathological assessment of tumor differentiation grade and
stage. Moreover, most samples include the original surgical
pathology report that provides information regarding the clinical
state of the patient.
[0440] Panel 3D, 3.1 and 3.2
[0441] The plates of Panel 3D, 3.1, and 3.2 are comprised of 94
cDNA samples and two control samples. Specifically, 92 of these
samples are derived from cultured human cancer cell lines, 2
samples of human primary cerebellar tissue and 2 controls. The
human cell lines are generally obtained from ATCC (American Type
Culture Collection), NCI or the German tumor cell bank and fall
into the following tissue groups: Squamous cell carcinoma of the
tongue, breast cancer, prostate cancer, melanoma, epidermoid
carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney
cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric,
colon, lung and CNS cancer cell lines. In addition, there are two
independent samples of cerebellum. These cells are all cultured
under standard recommended conditions and RNA extracted using the
standard procedures. The cell lines in panel 3D, 3.1, 3.2, 1, 1.1.,
1.2, 1.3D, 1.4, 1.5, and 1.6 are of the most common cell lines used
in the scientific literature.
[0442] Panels 4D, 4R, and 4.1D
[0443] Panel 4 includes samples on a 96 well plate (2 control
wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels
4D/4.1D) isolated from various human cell lines or tissues related
to inflammatory conditions. Total RNA from control normal tissues
such as colon and lung (Stratagene, La Jolla, Calif.) and thymus
and kidney (Clontech) was employed. Total RNA from liver tissue
from cirrhosis patients and kidney from lupus patients was obtained
from BioChain (Biochain Institute, Inc., Hayward, Calif.).
Intestinal tissue for RNA preparation from patients diagnosed as
having Crohn's disease and ulcerative colitis was obtained from the
National Disease Research Interchange (NDRI) (Philadelphia,
Pa.).
[0444] Astrocytes, lung fibroblasts, dermal fibroblasts, coronary
artery smooth muscle cells, small airway epithelium, bronchial
epithelium, microvascular dermal endothelial cells, microvascular
lung endothelial cells, human pulmonary aortic endothelial cells,
human umbilical vein endothelial cells were all purchased from
Clonetics (Walkersville, Md.) and grown in the media supplied for
these cell types by Clonetics. These primary cell types were
activated with various cytokines or combinations of cytokines for 6
and/or 12-14 hours, as indicated. The following cytokines were
used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at
approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml,
IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml,
IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes
starved for various times by culture in the basal media from
Clonetics with 0.1% serum.
[0445] Mononuclear cells were prepared from blood of employees at
CuraGen Corporation, using Ficoll. LAK cells were prepared from
these cells by culture in DMEM 5% FCS (Hyclone), 100 .mu.M non
essential amino acids (Gibco/Life Technologies, Rockville, Md.), 1
mM sodium pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M
(Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days.
Cells were then either activated with 10-20 ng/ml PMA and 1-2
.mu.g/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml
and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear
cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and 10 mM
Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed
mitogen) at approximately 5 .mu.g/ml. Samples were taken at 24, 48
and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction)
samples were obtained by taking blood from two donors, isolating
the mononuclear cells using Ficoll and mixing the isolated
mononuclear cells 1:1 at a final concentration of approximately
2.times.10.sup.6 cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non
essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco),
mercaptoethanol (5.5.times.10.sup.-5M) (Gibco), and 10 mM Hepes
(Gibco). The MLR was cultured and samples taken at various time
points ranging from 1-7 days for RNA preparation.
[0446] Monocytes were isolated from mononuclear cells using CD14
Miltenyi Beads, +ve VS selection columns and a Vario Magnet
according to the manufacturer's instructions. Monocytes were
differentiated into dendritic cells by culture in DMEM 5% fetal
calf serum (FCS) (Hyclone, Logan, Utah), 100 .mu.M non essential
amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml
GMCSF and 5 ng/ml IL-4 for 5-7 days. Macrophages were prepared by
culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), 10 mM Hepes
(Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml.
Monocytes, macrophages and dendritic cells were stimulated for 6
and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml.
Dendritic cells were also stimulated with anti-CD40 monoclonal
antibody (Pharmingen) at 10 .mu.g/ml for 6 and 12-14 hours.
[0447] CD4 lymphocytes, CD8 lymphocytes and NK cells were also
isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi
beads, positive VS selection columns and a Vario Magnet according
to the manufacturer's instructions. CD45RA and CD45RO CD4
lymphocytes were isolated by depleting mononuclear cells of CD8,
CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi
beads and positive selection. CD45RO beads were then used to
isolate the CD45RO CD4 lymphocytes with the remaining cells being
CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes
were placed in DMEM 5% FCS (Hyclone), 100 .mu.M non essential amino
acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), and 10 mM Hepes (Gibco) and plated at
10.sup.6 cells/ml onto Falcon 6 well tissue culture plates that had
been coated overnight with 0.5 .mu.g/ml anti-CD28 (Pharmingen) and
3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the
cells were harvested for RNA preparation. To prepare chronically
activated CD8 lymphocytes, we activated the isolated CD8
lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and
then harvested the cells and expanded them in DMEM 5% FCS
(Hyclone), 100 .mu.M non essential amino acids (Gibco), 1 mM sodium
pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and
10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then
activated again with plate bound anti-CD3 and anti-CD28 for 4 days
and expanded as before. RNA was isolated 6 and 24 hours after the
second activation and after 4 days of the second expansion culture.
The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and 10 mM
Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
[0448] To obtain B cells, tonsils were procured from NDRI. The
tonsil was cut up with sterile dissecting scissors and then passed
through a sieve. Tonsil cells were then spun down and resupended at
10.sup.6 cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non essential
amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), and 10 mM Hepes (Gibco). To activate
the cells, we used PWM at 5 .mu.g/ml or anti-CD40 (Pharmingen) at
approximately 10 .mu.g/ml and IL-4 at 5-10 ng/ml. Cells were
harvested for RNA preparation at 24, 48 and 72 hours.
[0449] To prepare the primary and secondary Th1/Th2 and Tr1 cells,
six-well Falcon plates were coated overnight with 10 .mu.g/ml
anti-CD28 (Pharmingen) and 2 .mu.g/ml OKT3 (ATCC), and then washed
twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic
Systems, German Town, Md.) were cultured at 10.sup.5-10.sup.6
cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non essential amino
acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4
ng/ml). IL-12 (5 ng/ml) and anti-IL4 (1 .mu.g/ml) were used to
direct to Th1, while IL-4 (5 ng/ml) and anti-IFN gamma (1 .mu.g/ml)
were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct
to Tr1. After 4-5 days, the activated Th1, Th2 and Tr1 lymphocytes
were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS
(Hyclone), 100 .mu.M non essential amino acids (Gibco), 1 mM sodium
pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), 10
mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated
Th1, Th2 and Tr1 lymphocytes were re-stimulated for 5 days with
anti-CD28/OKT3 and cytokines as described above, but with the
addition of anti-CD95L (1 .mu.g/ml) to prevent apoptosis. After 4-5
days, the Th1, Th2 and Tr1 lymphocytes were washed and then
expanded again with IL-2 for 4-7 days. Activated Th1 and Th2
lymphocytes were maintained in this way for a maximum of three
cycles. RNA was prepared from primary and secondary Th1, Th2 and
Tr1 after 6 and 24 hours following the second and third activations
with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the
second and third expansion cultures in Interleukin 2.
[0450] The following leukocyte cells lines were obtained from the
ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated
by culture in 0.1 mM dbcAMP at 5.times.10.sup.5 cells/ml for 8
days, changing the media every 3 days and adjusting the cell
concentration to 5.times.10.sup.5 cells/ml. For the culture of
these cells, we used DMEM or RPMI (as recommended by the ATCC),
with the addition of 5% FCS (Hyclone), 100 .mu.M non essential
amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5M (Gibco), 10 mM Hepes (Gibco). RNA was either
prepared from resting cells or cells activated with PMA at 10 ng/ml
and ionomycin at 1 .mu.g/ml for 6 and 14 hours. Keratinocyte line
CCD106 and an airway epithelial tumor line NCI-H292 were also
obtained from the ATCC. Both were cultured in DMEM 5% FCS
(Hyclone), 100 .mu.M non essential amino acids (Gibco), 1 mM sodium
pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5M (Gibco), and
10 mM Hepes (Gibco). CCD1106 cells were activated for 6 and 14
hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta,
while NCI-H292 cells were activated for 6 and 14 hours with the
following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and
25 ng/ml IFN gamma.
[0451] For these cell lines and blood cells, RNA was prepared by
lysing approximately 10.sup.7 cells/ml using Trizol (Gibco BRL).
Briefly, {fraction (1/10)} volume of bromochloropropane (Molecular
Research Corporation) was added to the RNA sample, vortexed and
after 10 minutes at room temperature, the tubes were spun at 14,000
rpm in a Sorvall SS34 rotor. The aqueous phase was removed and
placed in a 15 ml Falcon Tube. An equal volume of isopropanol was
added and left at -20.degree. C. overnight. The precipitated RNA
was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and
washed in 70% ethanol. The pellet was redissolved in 300 .mu.l of
RNAse-free water and 35 .mu.l buffer (Promega) 5 .mu.l DTT, 7 .mu.l
RNAsin and 8 .mu.l DNAse were added. The tube was incubated at
37.degree. C. for 30 minutes to remove contaminating genomic DNA,
extracted once with phenol chloroform and re-precipitated with
{fraction (1/10)} volume of 3M sodium acetate and 2 volumes of 100%
ethanol. The RNA was spun down and placed in RNAse free water. RNA
was stored at -80.degree. C.
[0452] AI_Comprehensive Panel_v1.0
[0453] The plates for AI_comprehensive panel_v1.0 include two
control wells and 89 test samples comprised of cDNA isolated from
surgical and postmortem human tissues obtained from the Backus
Hospital and Clinomics (Frederick, Md.). Total RNA was extracted
from tissue samples from the Backus Hospital in the Facility at
CuraGen. Total RNA from other tissues was obtained from
Clinomics.
[0454] Joint tissues including synovial fluid, synovium, bone and
cartilage were obtained from patients undergoing total knee or hip
replacement surgery at the Backus Hospital. Tissue samples were
immediately snap frozen in liquid nitrogen to ensure that isolated
RNA was of optimal quality and not degraded. Additional samples of
osteoarthritis and rheumatoid arthritis joint tissues were obtained
from Clinomics. Normal control tissues were supplied by Clinomics
and were obtained during autopsy of trauma victims.
[0455] Surgical specimens of psoriatic tissues and adjacent matched
tissues were provided as total RNA by Clinomics. Two male and two
female patients were selected between the ages of 25 and 47. None
of the patients were taking prescription drugs at the time samples
were isolated.
[0456] Surgical specimens of diseased colon from patients with
ulcerative colitis and Crohns disease and adjacent matched tissues
were obtained from Clinomics. Bowel tissue from three female and
three male Crohn's patients between the ages of 41-69 were used.
Two patients were not on prescription medication while the others
were taking dexamethasone, phenobarbital, or tylenol. Ulcerative
colitis tissue was from three male and four female patients. Four
of the patients were taking lebvid and two were on
phenobarbital.
[0457] Total RNA from post mortem lung tissue from trauma victims
with no disease or with emphysema, asthma or COPD was purchased
from Clinomics. Emphysema patients ranged in age from 40-70 and all
were smokers, this age range was chosen to focus on patients with
cigarette-linked emphysema and to avoid those patients with
alpha-lanti-trypsin deficiencies. Asthma patients ranged in age
from 36-75, and excluded smokers to prevent those patients that
could also have COPD. COPD patients ranged in age from 35-80 and
included both smokers and non-smokers. Most patients were taking
corticosteroids, and bronchodilators.
[0458] In the labels employed to identify tissues in the
AI_comprehensive panel_v1.0 panel, the following abbreviations are
used:
[0459] AI=Autoimmunity
[0460] Syn=Synovial
[0461] Normal=No apparent disease
[0462] Rep22/Rep20=individual patients
[0463] RA=Rheumatoid arthritis
[0464] Backus=From Backus Hospital
[0465] OA=Osteoarthritis
[0466] (SS) (BA) (MF)=Individual patients
[0467] Adj=Adjacent tissue
[0468] Match control=adjacent tissues
[0469] -M=Male
[0470] -F=Female
[0471] COPD=Chronic obstructive pulmonary disease
[0472] AI.05 Chondrosarcoma
[0473] The AI.05 chondrosarcoma plates are comprised of SW1353
cells that had been subjected to serum starvation and treatment
with cytokines that are known to induce MMP (1, 3 and 13) synthesis
(eg. IL1beta). These treatments include: IL-1beta (10 ng/ml),
IL-1beta+TNF-alpha (50 ng/ml), IL-1beta+Oncostatin (50 ng/ml) and
PMA (100 ng/ml). The SW1353 cells were obtained from the ATCC
(American Type Culture Collection) and were all cultured under
standard recommended conditions. The SW1353 cells were plated at
3.times.10.sup.5 cells/ml (in DMEM medium-10% FBS) in 6-well
plates. The treatment was done in triplicate, for 6 and 18 h. The
supernatants were collected for analysis of MMP 1, 3 and 13
production and for RNA extraction. RNA was prepared from these
samples using the standard procedures.
[0474] Panels 5D and 5I
[0475] The plates for Panel 5D and 5I include two control wells and
a variety of cDNAs isolated from human tissues and cell lines with
an emphasis on metabolic diseases. Metabolic tissues were obtained
from patients enrolled in the Gestational Diabetes study. Cells
were obtained during different stages in the differentiation of
adipocytes from human mesenchymal stem cells. Human pancreatic
islets were also obtained.
[0476] In the Gestational Diabetes study subjects are young (18-40
years), otherwise healthy women with and without gestational
diabetes undergoing routine (elective) Caesarean section. After
delivery of the infant, when the surgical incisions were being
repaired/closed, the obstetrician removed a small sample (less than
1 cc) of the exposed metabolic tissues during the closure of each
surgical level. The biopsy material was rinsed in sterile saline,
blotted and fast frozen within 5 minutes from the time of removal.
The tissue was then flash frozen in liquid nitrogen and stored,
individually, in sterile screw-top tubes and kept on dry ice for
shipment to or to be picked up by CuraGen. The metabolic tissues of
interest include uterine wall (smooth muscle), visceral adipose,
skeletal muscle (rectus) and subcutaneous adipose. Patient
descriptions are as follows:
[0477] Patient 2: Diabetic Hispanic, overweight, not on insulin
[0478] Patient 7-9: Nondiabetic Caucasian and obese (BMI>30)
[0479] Patient 10: Diabetic Hispanic, overweight, on insulin
[0480] Patient 11: Nondiabetic African American and overweight
[0481] Patient 12: Diabetic Hispanic on insulin
[0482] Adiocyte differentiation was induced in donor progenitor
cells obtained from Osirus (a division of Clonetics/BioWhittaker)
in triplicate, except for Donor 3U which had only two replicates.
Scientists at Clonetics isolated, grew and differentiated human
mesenchymal stem cells (HuMSCs) for CuraGen based on the published
protocol found in Mark F. Pittenger, et al., Multilineage Potential
of Adult Human Mesenchymal Stem Cells Science Apr. 2, 1999:
143-147. Clonetics provided Trizol lysates or frozen pellets
suitable for mRNA isolation and ds cDNA production. A general
description of each donor is as follows:
[0483] Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated
Adipose
[0484] Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated
[0485] Donor 2 and 3 AD: Adipose, Adipose Differentiated
[0486] Human cell lines were generally obtained from ATCC (American
Type Culture Collection), NCI or the German tumor cell bank and
fall into the following tissue groups: kidney proximal convoluted
tubule, uterine smooth muscle cells, small intestine, liver HepG2
cancer cells, heart primary stromal cells, and adrenal cortical
adenoma cells. These cells are all cultured under standard
recommended conditions and RNA extracted using the standard
procedures. All samples were processed at CuraGen to produce single
stranded cDNA.
[0487] Panel 5I contains all samples previously described with the
addition of pancreatic islets from a 58 year old female patient
obtained from the Diabetes Research Institute at the University of
Miami School of Medicine. Islet tissue was processed to total RNA
at an outside source and delivered to CuraGen for addition to panel
5I.
[0488] In the labels employed to identify tissues in the 5D and 5I
panels, the following abbreviations are used:
[0489] GO Adipose=Greater Omentum Adipose
[0490] SK=Skeletal Muscle
[0491] UT=Uterus
[0492] PL=Placenta
[0493] AD=Adipose Differentiated
[0494] AM=Adipose Midway Differentiated
[0495] U=Undifferentiated Stem Cells
[0496] Human Metabolic RTQ-PCR Panel
[0497] The plates for the Human Metabolic RTQ-PCR Panel include two
control wells (genomic DNA control and chemistry control) and 211
cDNAs isolated from human tissues and cell lines with an emphasis
on metabolic diseases. This panel is useful for establishing the
tissue and cellular expression profiles for genes believed to play
a role in the etiology and pathogenesis of obesity and/or diabetes
and to confirm differential expression of such genes derived from
other methods. Metabolic tissues were obtained from patients
enrolled in the CuraGen Gestational Diabetes study and from autopsy
tissues from Type II diabetics and age, sex and race-matched
control patients. One or more of the following were used to
characterize the patients: body mass index [BMI=wt (kg)/ht
(m.sup.2)], serum glucose, HgbA1c. Cell lines used in this panel
are widely available through the American Type Culture Collection
(ATCC), a repository for cultured cell lines. RNA from human
Pancreatic Islets was also obtained.
[0498] In the Gestational Diabetes study, subjects are young (18-40
years), otherwise healthy women with and without gestational
diabetes undergoing routine (elective) Caesarian section. After
delivery of the infant, when the surgical incisions were being
repaired/closed, the obstetrician removed a small sample (less than
1 cc) of the exposed metabolic tissues during the closure of each
surgical level. The biopsy material was rinsed in sterile saline,
blotted, and then flash frozen in liquid nitrogen and stored,
individually, in sterile screw-top tubes and kept on dry ice for
shipment to or to be picked up by CuraGen. The metabolic tissues of
interest include uterine wall (smooth muscle), visceral adipose,
skeletal muscle (rectus), and subcutaneous adipose. Patient
descriptions are as follows:
[0499] Patient 7--Non-diabetic Caucasian and obese
[0500] Patient 8--Non-diabetic Caucasian and obese
[0501] Patient 12--Diabetic Caucasian with unknown BMI and on
insulin
[0502] Patient 13--Diabetic Caucasian, overweight, not on
insulin
[0503] Patient 15--Diabetic Caucasian, obese, not on insulin
[0504] Patient 17--Diabetic Caucasian, normal weight, not on
insulin
[0505] Patient 18--Diabetic Hispanic, obese, not on insulin
[0506] Patient 19--Non-diabetic Caucasian and normal weight
[0507] Patient 20--Diabetic Caucasian, overweight, and on
insulin
[0508] Patient 21--Non-diabetic Caucasian and overweight
[0509] Patient 22--Diabetic Caucasian, normal weight, on
insulin
[0510] Patient 23--Non-diabetic Caucasian and overweight
[0511] Patient 25--Diabetic Caucasian, normal weight, not on
insulin
[0512] Patient 26--Diabetic Caucasian, obese, on insulin
[0513] Patient 27--Diabetic Caucasian, obese, on insulin
[0514] Total RNA was isolated from metabolic tissues of 12 Type II
diabetic patients and 12 matched control patients included
hypothalamus, liver, pancreas, small intestine, psoas muscle,
diaphragm muscle, visceral adipose, and subcutaneous adipose. The
diabetics and non-diabetics were matched for age, sex, ethnicity,
and BMI where possible.
[0515] The panel also contains pancreatic islets from a 22 year old
male patient (with a BMI of 35) obtained from the Diabetes Research
Institute at the University of Miami School of Medicine. Islet
tissue was processed to total RNA at CuraGen.
[0516] Cell lines used in this panel are widely available through
the American Type Culture Collection (ATCC), a repository for
cultured cell lines, and were cultured at an outside facility. The
RNA was extracted at CuraGen according to CuraGen protocols. All
samples were then processed at CuraGen to produce single stranded
cDNA.
[0517] In the labels used to identify tissues in the Human
Metabolic panel, the following abbreviations are used:
[0518] Pl=placenta
[0519] Go=greater omentum
[0520] Sk=skeletal muscle
[0521] Ut=uterus
[0522] CC=Caucasian
[0523] HI=Hispanic
[0524] AA=African American
[0525] AS=Asian
[0526] Diab=Type II diabetic
[0527] Norm=Non-diabetic
[0528] Overwt=Overweight; med BMI
[0529] Obese=Hi BMI
[0530] Low BM=20-25
[0531] Med BM=26-30
[0532] Hi BMI=Greater than 30
[0533] M=Male
[0534] #=Patient identifier
[0535] Vis.=Visceral
[0536] SubQ=Subcutaneous
[0537] Panel CNSD.01
[0538] The plates for Panel CNSD.01 include two control wells and
94 test samples comprised of cDNA isolated from postmortem human
brain tissue obtained from the Harvard Brain Tissue Resource
Center. Brains are removed from calvaria of donors between 4 and 24
hours after death, sectioned by neuroanatomists, and frozen at
-80.degree. C. in liquid nitrogen vapor. All brains are sectioned
and examined by neuropathologists to confirm diagnoses with clear
associated neuropathology.
[0539] Disease diagnoses are taken from patient records. The panel
contains two brains from each of the following diagnoses:
Alzheimer's disease, Parkinson's disease, Huntington's disease,
Progressive Supernuclear Palsy, Depression, and "Normal controls".
Within each of these brains, the following regions are represented:
cingulate gyrus, temporal pole, globus palladus, substantia nigra,
Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal
cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17
(occipital cortex). Not all brain regions are represented in all
cases; e.g., Huntington's disease is characterized in part by
neurodegeneration in the globus palladus, thus this region is
impossible to obtain from confirmed Huntington's cases. Likewise
Parkinson's disease is characterized by degeneration of the
substantia nigra making this region more difficult to obtain.
Normal control brains were examined for neuropathology and found to
be free of any pathology consistent with neurodegeneration.
[0540] In the labels employed to identify tissues in the CNS panel,
the following abbreviations are used:
[0541] PSP=Progressive supranuclear palsy
[0542] Sub Nigra=Substantia nigra
[0543] Glob Palladus=Globus palladus
[0544] Temp Pole=Temporal pole
[0545] Cing Gyr=Cingulate gyrus
[0546] BA 4=Brodman Area 4
[0547] Panel CNS_Neurodegeneration_V1.0
[0548] The plates for Panel CNS_Neurodegeneration_V1.0 include two
control wells and 47 test samples comprised of cDNA isolated from
postmortem human brain tissue obtained from the Harvard Brain
Tissue Resource Center (McLean Hospital) and the Human Brain and
Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare
System). Brains are removed from calvaria of donors between 4 and
24 hours after death, sectioned by neuroanatomists, and frozen at
-80.degree. C. in liquid nitrogen vapor. All brains are sectioned
and examined by neuropathologists to confirm diagnoses with clear
associated neuropathology.
[0549] Disease diagnoses are taken from patient records. The panel
contains six brains from Alzheimer's disease (AD) patients, and
eight brains from "Normal controls" who showed no evidence of
dementia prior to death. The eight normal control brains are
divided into two categories: Controls with no dementia and no
Alzheimer's like pathology (Controls) and controls with no dementia
but evidence of severe Alzheimer's like pathology, (specifically
senile plaque load rated as level 3 on a scale of 0-3; 0=no
evidence of plaques, 3=severe AD senile plaque load). Within each
of these brains, the following regions are represented:
hippocampus, temporal cortex (Brodman Area 21), parietal cortex
(Brodman area 7), and occipital cortex (Brodman area 17). These
regions were chosen to encompass all levels of neurodegeneration in
AD. The hippocampus is a region of early and severe neuronal loss
in AD; the temporal cortex is known to show neurodegeneration in AD
after the hippocampus; the parietal cortex shows moderate neuronal
death in the late stages of the disease; the occipital cortex is
spared in AD and therefore acts as a "control" region within AD
patients. Not all brain regions are represented in all cases.
[0550] In the labels employed to identify tissues in the
CNS_Neurodegeneration_V1.0 panel, the following abbreviations are
used:
[0551] AD=Alzheimer's disease brain; patient was demented and
showed AD-like pathology upon autopsy
[0552] Control=Control brains; patient not demented, showing no
neuropathology
[0553] Control (Path)=Control brains; pateint not demented but
showing sever AD-like pathology
[0554] SupTemporal Ctx=Superior Temporal Cortex
[0555] Inf Temporal Ctx=Inferior Temporal Cortex
[0556] Panel CNS_Neurodegeneration_V2.0
[0557] The plates for Panel CNS_Neurodegeneration_V2.0 include two
control wells and 47 test samples comprised of cDNA isolated from
postmortem human brain tissue obtained from the Harvard Brain
Tissue Resource Center (McLean Hospital) and the Human Brain and
Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare
System). Brains are removed from calvaria of donors between 4 and
24 hours after death, sectioned by neuroanatomists, and frozen at
-80.degree. C. in liquid nitrogen vapor. All brains are sectioned
and examined by neuropathologists to confirm diagnoses with clear
associated neuropathology.
[0558] Disease diagnoses are taken from patient records. The panel
contains sixteen brains from Alzheimer's disease (AD) patients, and
twenty-nine brains from "Normal controls" who showed no evidence of
dementia prior to death. The twenty-nine normal control brains are
divided into two categories: Fourteen controls with no dementia and
no Alzheimer's like pathology (Controls) and fifteen controls with
no dementia but evidence of severe Alzheimer's like pathology,
(specifically senile plaque load rated as level 3 on a scale of
0-3; 0=no evidence of plaques, 3=severe AD senile plaque load).
Tissue from the temporal cotex (Broddmann Area 21) was selected for
all samples from the Harvard Brain Tissue Resource Center; from the
two sample from the Human Brain and Spinal Fluid Resource Center
(samples 1 and 2) tissue from the inferior and superior temporal
cortex was used; each sample on the panel represents a pool of
inferior and superior temporal cortex from an individual patient.
The temporal cortex was chosen as it shows a loss of neurons in the
intermediate stages of the disease. Selection of a region which is
affected in the early stages of Alzheimer's disease (e.g.,
hippocampus or entorhinal cortex) could potentially result in the
examination of gene expression after vulnerable neurons are lost,
and missing genes involved in the actual neurodegeneration
process.
[0559] In the labels employed to identify tissues in the
CNS_Neurodegeneration_V2.0 panel, the following abbreviations are
used:
[0560] AD=Alzheimer's disease brain; patient was demented and
showed AD-like pathology upon autopsy
[0561] Control=Control brains; patient not demented, showing no
neuropathology
[0562] AH3=Control brains; pateint not demented but showing sever
AD-like pathology
[0563] Inf & Sup Temp Ctx Pool=Pool of inferior and superior
temporal cortex for a given individual
[0564] A. CG50907-03 and CG50907-04: Sema4C.
[0565] Expression of gene CG50907-03 and CG50907-04 was assessed
using the primer-probe sets Ag1215, Ag1382, Ag37 and Ag7452,
described in Tables AA, AB, AC, and AD a. Results of the RTQ-PCR
runs are shown in Tables AE, AF, AG, AH, AI, AJ, AK, AL, AM, AN,
AO, AP, AQ, and AR. Please note that CG50907-03 is a physical clone
for the extracellular domain of this gene. Also, probe-primer set
Ag7452 specifically recognizes the splice variant, CG50907-04.
44TABLE AA Probe Name Ag1215 Start SEQ ID Primers Sequences Length
Position No Forward 5'-aacccattatcctgcgtaacat-3' 22 569 129 Probe
TET-5'ccccaccactccatgaagacagagta-3'-TAMRA 26 595 130 Reverse
5'-cctacaaagtgaggttcgttga-3' 22 635 131
[0566]
45TABLE AB Probe Name Ag1382 Start SEQ ID Primers Sequences Length
Position No Forward 5'-aacccattatcctgcgtaacat-3' 22 569 132 Probe
TET-5'-ccccaccactccatgaagacagagta-3'-TAMRA 26 595 133 Reverse
5'-cctacaaagtgaggttcgttga-3' 22 635 134
[0567]
46TABLE AC Probe Name Ag37 Start SEQ ID Primers Sequences Length
Position No Forward 5'-ggcttcctcatggtactcctta-3' 22 1041 135 Probe
TET-5'-ccgctggatctcttccaactggtact-3'-TAMRA 26 998 136 Reverse
5'-acagtggggtgacatgtacct-3' 21 963 137
[0568]
47TABLE AD Probe Name Ag7452 Start SEQ ID Primers Sequences Length
Position No Forward 5'-atgagaagcctggtgctatctc-3' 22 1420 138 Probe
TET-5'-agagcaagaccctccaagctgtgct-3'-TAMRA 25 1442 139 Reverse
5'-gcatggtcagggaagag-3' 18 1484 140
[0569]
48TABLE AE Ardais Panel v.1.0 Tissue Name A Tissue Name A
136799_Lung cancer(362) 100.0 136787_lung cancer(356) 51.4
136800_Lung NAT(363) 44.4 136788_lung NAT(357) 42.9 136813_Lung
cancer(372) 72.2 136804_Lung cancer(369) 82.4 136814_Lung NAT(373)
13.6 136805_Lung NAT(36A) 25.5 136815_Lung cancer(374) 35.8
136806_Lung cancer(36B) 98.6 136816_Lung NAT(375) 77.4 136807_Lung
NAT(36C) 16.3 136791_Lung cancer(35A) 42.9 136789_lung cancer(358)
85.9 136795_Lung cancer(35E) 91.4 136802_Lung cancer(365) 49.7
136797_Lung cancer(360) 19.6 136803_Lung cancer(368) 69.3
136794_lung NAT(35D) 34.6 136811_Lung cancer(370) 25.0 136818_Lung
NAT(377) 20.7 136810_Lung NAT(36F) 63.7 Column A - Rel. Exp.(%)
Ag1215, Run 67680187
[0570]
49TABLE AF CNS_neurodegeneration_v1.0 Tissue Name A B Tissue Name A
B AD 1 Hippo 12.2 0.0 Control (Path) 3 Temporal Ctx 5.1 13.8 AD 2
Hippo 23.7 27.4 Control (Path) 4 Temporal Ctx 24.3 29.9 AD 3 Hippo
6.0 0.0 AD 1 Occipital Ctx 15.8 10.7 AD 4 Hippo 8.3 9.0 AD 2
Occipital Ctx (Missing) 0.0 0.0 AD 5 Hippo 97.9 26.2 AD 3 Occipital
Ctx 5.6 0.0 AD 6 Hippo 48.0 54.0 AD 4 Occipital Ctx 16.6 32.5
Control 2 Hippo 24.1 60.7 AD 5 Occipital Ctx 44.8 40.3 Control 4
Hippo 11.4 13.8 AD 6 Occipital Ctx 33.0 36.3 Control (Path) 3 Hippo
5.4 16.4 Control 1 Occipital Ctx 7.5 19.2 AD 1 Temporal Ctx 20.7
17.0 Control 2 Occipital Ctx 66.4 54.7 AD 2 Temporal Ctx 29.7 9.9
Control 3 Occipital Ctx 16.8 20.4 AD 3 Temporal Ctx 12.9 0.0
Control 4 Occipital Ctx 12.2 14.7 AD 4 Temporal Ctx 23.2 34.6
Control (Path) 1 Occipital Ctx 81.8 55.1 AD 5 Inf Temporal Ctx
100.0 76.3 Control (Path) 2 Occipital Ctx 10.7 7.9 AD 5 Sup
Temporal Ctx 35.8 46.0 Control (Path) 3 Occipital Ctx 6.6 0.0 AD 6
Inf Temporal Ctx 65.5 100.0 Control (Path) 4 Occipital Ctx 16.6
17.1 AD 6 Sup Temporal Ctx 52.1 55.5 Control 1 Parietal Ctx 8.7
21.0 Control 1 Temporal Ctx 6.4 5.4 Control 2 Parietal Ctx 49.3
42.0 Control 2 Temporal Ctx 34.9 40.1 Control 3 Parietal Ctx 14.9
12.8 Control 3 Temporal Ctx 14.3 17.3 Control (Path) 1 Parietal Ctx
56.3 43.2 Control 3 Temporal Ctx 8.0 0.0 Control (Path) 2 Parietal
Ctx 19.9 20.7 Control (Path) 1 Temporal Ctx 39.2 40.6 Control
(Path) 3 Parietal Ctx 4.1 5.1 Control (Path) 2 Temporal Ctx 19.8
35.6 Control (Path) 4 Parietal Ctx 28.7 28.3 Column A - Rel. Exp.(%
Ag1215, Run 206228023 Column B - Rel. Exp.(%) Ag7452, Run
306518756
[0571]
50TABLE AG General_screening_panel_v1.4 Tissue Name A B Tissue Name
A B Adipose 4.1 15.3 Renal ca. TK-10 10.0 20.0 Melanoma* Hs688(A).T
8.1 13.6 Bladder 9.5 19.9 Melanoma* Hs688(B).T 6.1 14.7 Gastric ca.
(liver met.) NCI-N87 11.7 19.8 Melanoma* M14 40.3 52.1 Gastric ca.
KATO III 0.0 0.3 Melanoma* LOXIMVI 12.0 25.2 Colon ca. SW-948 2.4
8.7 Melanoma* SK-MEL-5 22.4 41.5 Colon ca. SW480 13.7 25.9 Squamous
Cell carcinoma SCC-4 6.1 30.6 Colon ca.* (SW480 met) SW620 7.9 17.1
Testis Pool 3.1 3.5 Colon ca. HT29 1.6 3.6 Prostate ca.* (bone met)
PC-3 12.7 21.3 Colon ca. HCT-116 11.6 20.2 Prostate Pool 4.6 11.8
Colon ca. CaCo-2 10.6 29.3 Placenta 26.2 19.8 Colon cancer tissue
7.1 31.6 Uterus Pool 3.8 13.2 Colon ca. SW1116 2.5 5.4 Ovarian ca.
OVCAR-3 20.2 27.5 Colon ca. Colo-205 0.0 0.1 Ovarian ca. SK-OV-3
8.8 23.8 Colon ca. SW-48 0.4 0.9 Ovarian ca. OVCAR-4 3.9 11.5 Colon
Pool 9.9 36.6 Ovarian ca. OVCAR-5 21.3 37.6 Small Intestine Pool
20.2 33.9 Ovarian ca. IGROV-1 9.7 30.4 Stomach Pool 7.6 13.1
Ovarian ca. OVCAR-8 8.2 23.3 Bone Marrow Pool 5.2 12.9 Ovary 7.2
2.1 Fetal Heart 6.7 12.7 Breast ca. MCF-7 100.0 100.0 Heart Pool
4.2 11.4 Breast ca. MDA-MB-231 11.0 17.6 Lymph Node Pool 14.3 32.3
Breast ca. BT 549 17.2 23.2 Fetal Skeletal Muscle 5.6 6.4 Breast
ca. T47D 33.2 62.9 Skeletal Muscle Pool 3.4 5.3 Breast ca. MDA-N
8.0 10.9 Spleen Pool 10.4 20.6 Breast Pool 13.2 43.5 Thymus pool
6.5 15.8 Trachea 9.0 18.4 CNS cancer (glio/astro) U87-MG 9.2 14.6
Lung 1.4 2.2 CNS cancer (glio/astro) U-118-MG 12.4 21.3 Fetal Lung
21.0 34.9 CNS cancer (neuro; met) SK-N-AS 18.8 33.4 Lung ca.
NCI-N417 0.9 2.6 CNS cancer (astro) SF-539 17.9 35.4 Lung ca. LX-1
12.9 21.8 CNS cancer (astro) SNB-75 42.9 74.2 Lung ca. NCI-H146 4.4
6.1 CNS cancer (glio) SNB-19 7.2 16.6 Lung ca. SHP-77 8.2 11.8 CNS
cancer (glio) SF-295 14.9 33.7 Lung ca. A549 9.4 11.3 Brain
(Amygdala) Pool 11.0 21.2 Lung ca. NCI-H526 3.0 6.5 Brain
(cerebellum) 47.0 65.1 Lung ca. NCI-H23 33.9 50.0 Brain (fetal)
33.2 43.8 Lung ca. NCI-H460 8.8 16.6 Brain (Hippocampus) Pool 9.4
14.2 Lung ca. HOP-62 11.3 21.6 Cerebral Cortex Pool 7.5 23.8 Lung
ca. NCI-H522 18.3 30.6 Brain (Substantia nigra) Pool 9.4 26.2 Liver
0.5 1.7 Brain (Thalamus) Pool 11.7 18.6 Fetal Liver 6.8 5.1 Brain
(whole) 16.2 27.9 Liver ca. HepG2 9.5 12.8 Spinal Cord Pool 12.6
20.9 Kidney Pool 18.8 62.0 Adrenal Gland 8.6 14.4 Fetal Kidney 5.0
12.8 Pituitary gland Pool 1.0 3.1 Renal ca. 786-0 8.0 15.8 Salivary
Gland 5.2 4.7 Renal ca. A498 6.8 8.2 Thyroid (female) 3.5 6.7 Renal
ca. ACHN 7.8 14.2 Pancreatic ca. CAPAN2 5.0 14.3 Renal ca. UO-31
8.7 14.8 Pancreas Pool 18.2 26.4 Column A - Rel. Exp.(%) Ag1215,
Run 212696257 Column B - Rel. Exp.(%) Ag37, Run 216861491
[0572]
51TABLE AH General_screening_panel_v1.7 Tissue Name A Tissue Name A
Adipose 17.8 Gastric ca. (liver met.) NCI-N87 0.7 HUVEC 6.6 Stomach
0.8 Melanoma* Hs688(A).T 0.0 Colon ca. SW-948 5.4 Melanoma*
Hs688(B).T 10.2 Colon ca. SW480 5.2 Melanoma (met) SK-MEL-5 33.7
Colon ca. (SW480 met) SW620 25.2 Testis 2.0 Colon ca. HT29 6.7
Prostate ca. (bone met) PC-3 0.0 Colon ca. HCT-116 9.0 Prostate ca.
DU145 9.2 Colon cancer tissue 0.6 Prostate pool 2.4 Colon ca.
SW1116 2.3 Uterus pool 2.2 Colon ca. Colo-205 0.0 Ovarian ca.
OVCAR-3 11.8 Colon ca. SW-48 0.0 Ovarian ca. (ascites) SK-OV-3 0.1
Colon 10.5 Ovarian ca. OVCAR-4 13.4 Small Intestine 3.9 Ovarian ca.
OVCAR-5 17.3 Fetal Heart 1.4 Ovarian ca. IGROV-1 12.2 Heart 2.5
Ovarian ca. OVCAR-8 11.9 Lymph Node pool 1 0.8 Ovary 4.1 Lymph Node
pool 2 12.9 Breast ca. MCF-7 100.0 Fetal Skeletal Muscle 0.4 Breast
ca. MDA-MB-231 21.0 Skeletal Muscle pool 0.3 Breast ca. BT-549 4.5
Skeletal Muscle 3.3 Breast ca. T47D 5.6 Spleen 3.5 Breast pool 3.5
Thymus 2.1 Trachea 17.1 CNS cancer (glio/astro) SF-268 2.4 Lung
20.9 CNS cancer (glio/astro) T98G 6.7 Fetal Lung 16.2 CNS cancer
(neuro; met) SK-N-AS 5.3 Lung ca. NCI-N417 0.7 CNS cancer (astro)
SF-539 58.2 Lung ca. LX-1 6.7 CNS cancer (astro) SNB-75 17.6 Lung
ca. NCI-H146 18.7 CNS cancer (glio) SNB-19 5.8 Lung ca. SHP-77 14.0
CNS cancer (glio) SF-295 1.5 Lung ca. NCI-H23 46.7 Brain (Amygdala)
4.5 Lung ca. NCI-H460 5.0 Brain (Cerebellum) 8.6 Lung ca. HOP-62
31.4 Brain (Fetal) 11.3 Lung ca. NCI-H522 16.8 Brain (Hippocampus)
4.6 Lung ca. DMS-114 9.3 Cerebral Cortex pool 2.6 Liver 0.0 Brain
(Substantia nigra) 1.6 Fetal Liver 3.8 Brain (Thalamus) 4.8 Kidney
pool 4.8 Brain (Whole) 29.3 Fetal Kidney 0.5 Spinal Cord 2.3 Renal
ca. 786-0 14.0 Adrenal Gland 8.4 Renal ca. A498 6.2 Pituitary Gland
2.6 Renal ca. ACHN 5.0 Salivary Gland 3.0 Renal ca. UO-31 5.0
Thyroid 7.0 Renal ca. TK-10 7.5 Pancreatic ca. PANC-1 5.4 Bladder
9.5 Pancreas pool 2.3 Column A - Rel. Exp.(%) Ag7452, Run
318350229
[0573]
52TABLE AI HASS Panel v1.0 Tissue Name A Tissue Name A MCF-7 C1
50.3 U87-MG F1 (B) 3.9 MCF-7 C2 58.2 U87-MG F2 1.8 MCF-7 C3 42.0
U87-MG F3 2.9 MCF-7 C4 59.5 U87-MG F4 4.2 MCF-7 C5 57.0 U87-MG F5
7.9 MCF-7 C6 73.2 U87-MG F6 8.5 MCF-7 C7 29.7 U87-MG F7 3.5 MCF-7
C9 32.3 U87-MG F8 5.1 MCF-7 C10 73.7 U87-MG F9 2.6 MCF-7 C11 12.8
U87-MG F10 5.2 MCF-7 C12 46.7 U87-MG F11 5.6 MCF-7 C13 35.6 U87-MG
F12 5.2 MCF-7 C15 18.4 U87-MG F13 3.5 MCF-7 C16 100.0 U87-MG F14
5.9 MCF-7 C17 60.7 U87-MG F15 3.8 T24 D1 10.4 U87-MG F16 6.6 T24 D2
5.8 U87-MG F17 5.7 T24 D3 9.4 LnCAP A1 3.6 T24 D4 11.9 LnCAP A2 3.1
T24 D5 8.5 LnCAP A3 3.5 T24 D6 11.7 LnCAP A4 3.4 T24 D7 3.1 LnCAP
A5 2.7 T24 D9 3.2 LnCAP A6 2.6 T24 D10 4.9 LnCAP A7 4.3 T24 D11 4.8
LnCAP A8 3.5 T24 D12 6.6 LnCAP A9 3.4 T24 D13 2.5 LnCAP A10 3.5 T24
D15 3.2 LnCAP A11 7.5 T24 D16 3.2 LnCAP A12 0.7 T24 D17 4.6 LnCAP
A13 0.8 CAPaN B1 6.3 LnCAP A14 0.5 CAPaN B2 4.0 LnCAP A15 0.3 CAPaN
B3 1.4 LnCAP A16 5.9 CAPaN B4 6.9 LnCAP A17 3.9 CAPaN B5 4.7
Primary Astrocytes 6.0 CAPaN B6 6.1 Primary Renal Proximal Tubule
Epithelial 3.2 cell A2 CAPaN B7 3.7 Primary melanocytes A5 4.8
CAPaN B8 5.3 126443 - 341 medullo 0.8 CAPaN B9 5.0 126444 - 487
medullo 19.3 CAPaN B10 8.1 126445 - 425 medullo 1.8 CAPaN B11 5.6
126446 - 690 medullo 9.0 CAPaN B12 4.3 126447 - 54 adult glioma 3.8
CAPaN B13 6.6 126448 - 245 adult glioma 7.7 CAPaN B14 5.6 126449 -
317 adult glioma 8.3 CAPaN B15 3.3 126450 - 212 glioma 7.4 CAPaN
B16 7.0 126451 - 456 glioma 8.4 CAPaN B17 7.4 Column A - Rel.
Exp.(%) Ag1215, Run 267674603
[0574]
53TABLE AJ Oncology_cell_line_screening_panel_v3.2 Tissue Name A
Tissue Name A 94905_Daoy_Medulloblast- oma/Cerebellum_ssc 2.5
94954_Ca Ski_Cervical epidermoid 14.9 DNA carcinoma
(metastasis)_sscDNA 94906_TE671_Medulloblastom/Cerebellu- m_ssc 7.2
94955_ES-2_Ovarian clear cell 6.6 DNA carcinoma_sscDNA 94907_D283
8.8 94957_Ramos/6 h stim_Stimulated with 0.0
Med_Medulloblastoma/Cerebellum_sscDNA PMA/ionomycin 6 h_sscDNA
94908_PFSK-1_Primitive 9.3 94958_Ramos/14 h stim_Stimulated 0.0
Neuroectodermal/Cerebellum_sscDNA with PMA/ionomycin 14 h_sscDNA
94909_XF-498_CNS_sscDNA 18.8 94962_MEG-01_Chronic myelogenous 12.5
leukemia (megokaryoblast)_sscDNA 94910_SNB-78_CNS/glioma_sscDNA 7.4
94963_Raji_Burkitt's 0.0 lymphoma_sscDNA
94911_SF-268_CNS/glioblastoma_sscDNA 7.4 94964_Daudi_Burkitt's 0.0
lymphoma_sscDNA 94912_T98G_Glioblastoma_sscDNA 11.7
94965_U266_B-cell 0.3 plasmacytoma/myeloma_sscDNA
96776_SK-N-SH_Neuroblastoma 8.5 94968_CA46_Burkitt's 0.0
(metastasis)_sscDNA lymphoma_sscDNA 94913_SF-295_CNS/glioblastoma-
_sscDNA 6.4 94970_RL_non-Hodgkin's B-cell 0.0 lymphoma_sscDNA
132565_NT2 pool_sscDNA 22.8 94972_JM1_pre-B-cell 0.0
lymphoma/leukemia_sscDNA 94914_Cerebellum_sscDNA 20.9
94973_Jurkat_T cell leukemia_sscDNA 1.2 96777_Cerebellum_sscDNA
20.2 94974_TF-1_Erythroleukemia_sscDNA 16.5
94916_NCI-H292_Mucoepidermoid lung 7.2 94975_HUT 78_T-cell 4.3
carcinoma_sscDNA lymphoma_sscDNA 94917_DMS-114_Small cell lung 13.6
94977_U937_Histiocytic 10.8 cancer_sscDNA lymphoma_sscDNA
94918_DMS-79_Small cell lung 100.0 94980_KU-812_Myelogenous 14.0
cancer/neuroendocrine_sscDNA leukemia_sscDNA 94919_NCI-H146_Small
cell lung 11.0 94981_769-P_Clear cell renal 6.3
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94920_NCI-H526_Small
cell lung 14.8 94983_Caki-2_Clear cell renal 6.3
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94921_NCI-N417_Small
cell lung 2.5 94984_SW 839_Clear cell renal 5.7
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94923_NCI-H82_Small
cell lung 6.3 94986_G401_Wilms' tumor_sscDNA 3.4
cancer/neuroendocrine_sscDNA 94924_NCI-H157_Squamous cell lung
cancer 8.9 126768_293 cells_sscDNA 2.9 (metastasis)_sscDNA
94925_NCI-H1155_Large cell lung 10.1 94987_Hs766T_Pancreatic
carcinoma 7.1 cancer/neuroendocrine_sscDNA (LN metastasis)_sscDNA
94926_NCI-H1299_Large cell lung 14.8 94988_CAPAN-1_Pancreatic 5.4
cancer/neuroendocrine_sscDNA adenocarcinoma (liver
metastasis)_sscDNA 94927_NCI-H727_Lung carcinoid_sscDNA 22.5
94989_SU86.86_Pancreatic carcinoma 13.6 (liver metastasis)_sscDNA
94928_NCI-UMC-11_Lung carcinoid_sscDNA 11.3 94990_BxPC-3_Pancreatic
6.3 adenocarcinoma_sscDNA 94929_LX-1_Small cell lung cancer_sscDNA
7.1 94991_HPAC_Pancreatic 18.4 adenocarcinoma_sscDNA
94930_Colo-205_Colon cancer_sscDNA 0.0 94992_MIA PaCa-2_Pancreatic
2.1 carcinoma_sscDNA 94931_KM12_Colon cancer_sscDNA 3.1
94993_CFPAC-1_Pancreatic ductal 16.6 adenocarcinoma_sscDNA
94932_KM20L2_Colon cancer_sscDNA 1.0 94994_PANC-1_Pancreatic
epithelioid 9.5 ductal carcinoma_sscDNA 94933_NCI-H716_Colon
cancer_sscDNA 4.0 94996_T24_Bladder carcinma 5.0 (transitional
cell)_sscDNA 94935_SW-48_Colon adenocarcinoma_sscDNA 0.6
94997_5637_Bladder 9.0 carcinoma_sscDNA 94936_SW1116_Colon 3.7
94998_HT-1197_Bladder 11.0 adenocarcinoma_sscDNA carcinoma_sscDNA
94937_LS 174T_Colon 5.4 94999_UM-UC-3_Bladder carcinma 3.5
adenocarcinoma_sscDNA (transitional cell)_sscDNA 94938_SW-948_Colon
0.8 95000_A204_Rhabdomyosarcoma_ssc 6.3 adenocarcinoma_sscDNA DNA
94939_SW-480_Colon 5.9 95001_HT-1080_Fibrosarcoma_sscDNA 7.7
adenocarcinoma_sscDNA 94940_NCI-SNU-5_Gastric carcinoma_sscDNA 7.3
95002_MG-63_Osteosarcoma 5.6 (bone)_sscDNA 112197_KATO
III_Stomach_sscDNA 0.0 95003_SK-LMS-1_Leiomyosarcoma 9.7
(vulva)_sscDNA 94943_NCI-SNU-16_Gastric 2.9
95004_SJRH30_Rhabdomyosarcoma 11.0 carcinoma_sscDNA (met to bone
marrow)_sscDNA 94944_NCI-SNU-1_Gastric carcinoma_sscDNA 2.4
95005_A431_Epidermoid 3.1 carcinoma_sscDNA 94946_RF-1_Gastric
adenocarcinoma_sscDNA 3.3 95007_WM266-4_Melanoma_sscDN- A 5.3
94947_RF-48_Gastric 3.6 112195_DU 145_Prostate_sscDNA 6.8
adenocarcinoma_sscDNA 96778_MKN-45_Gastric carcinoma_sscDNA 10.4
95012_MDA-MB-468_Breast 1.9 adenocarcinoma_sscDNA
94949_NCI-N87_Gastric carcinoma_sscDNA 10.4
112196_SSC-4_Tongue_sscDNA 5.1 94951_OVCAR-5_Ovarian
carcinoma_sscDNA 2.7 112194_SSC-9_Tongue_sscDNA 2.6
94952_RL95-2_Uterine carcinoma_sscDNA 3.9
112191_SSC-15_Tongue_sscDNA 4.1 94953_HelaS3_Cervical 4.4 95017_CAL
27_Squamous cell 7.8 adenocarcinoma_sscDNA carcinoma of
tongue_sscDNA Column A - Rel. Exp.(%) Ag121, Run 267673682
[0575]
54TABLE AK Panel 1 Tissue Name A Tissue Name A Endothelial cells
1.1 Renal ca. 786-0 4.6 Endothelial cells (treated) 2.2 Renal ca.
A498 2.7 Pancreas 4.9 Renal ca. RXF 393 3.2 Pancreatic ca. CAPAN 2
2.0 Renal ca. ACHN 2.4 Adrenal gland 9.2 Renal ca. UO-31 1.7
Thyroid 6.3 Renal ca.TK-10 4.3 Salivary gland 4.6 Liver 8.5
Pituitary gland 9.1 Liver (fetal) 4.5 Brain (fetal) 12.1 Liver ca.
(hepatoblast) HepG2 7.1 Brain (whole) 24.0 Lung 4.5 Brain
(amygdala) 6.4 Lung (fetal) 15.1 Brain (cerebellum) 37.9 Lung ca.
(small cell) LX-1 3.7 Brain (hippocampus) 28.7 Lung ca. (small
cell) NCI-H69 2.1 Brain (substantia nigra) 15.1 Lung ca. (s.cell
var.) SHP-77 0.0 Brain (thalamus) 14.5 Lung ca. (large
cell)NCI-H460 0.0 Brain (hypothalamus) 4.0 Lung ca. (non-sm. cell)
A549 3.0 Spinal cord 32.3 Lung ca. (non-s.cell) NCI-H23 12.1
glio/astro U87-MG 1.9 Lung ca. (non-s.cell) HOP-62 4.7 glio/astro
U-118-MG 2.4 Lung ca. (non-s.cl) NCI-H522 14.1 astrocytoma SW1783
0.7 Lung ca. (squam.) SW 900 13.9 neuro*; met SK-N-AS 11.0 Lung ca.
(squam.) NCI-H596 2.0 astrocytoma SF-539 9.3 Mammary gland 16.4
astrocytoma SNB-75 10.5 Breast ca.* (pl.ef) MCF-7 100.0 glioma
SNB-19 5.3 Breast ca.* (pl.ef) MDA-MB-231 1.4 glioma U251 1.6
Breast ca.* (pl.ef) T47D 2.6 glioma SF-295 3.7 Breast ca. BT-549
0.0 Heart 8.3 Breast ca. MDA-N 7.3 Skeletal muscle 2.9 Ovary 18.8
Bone marrow 1.9 Ovarian ca. OVCAR-3 6.1 Thymus 5.8 Ovarian ca.
OVCAR-4 1.5 Spleen 18.8 Ovarian ca. OVCAR-5 5.5 Lymph node 13.0
Ovarian ca. OVCAR-8 6.9 Colon (ascending) 2.0 Ovarian ca. IGROV-1
2.6 Stomach 7.2 Ovarian ca. (ascites) SK-OV-3 2.5 Small intestine
6.5 Uterus 6.2 Colon ca. SW480 1.3 Placenta 45.4 Colon ca.* SW620
(SW480 met) 2.9 Prostate 12.3 Colon ca. HT29 0.9 Prostate ca.*
(bone met) PC-3 0.0 Colon ca. HCT-116 0.0 Testis 8.9 Colon ca.
CaCo-2 5.9 Melanoma Hs688(A).T 1.0 Colon ca. HCT-15 4.9 Melanoma*
(met) Hs688(B).T 1.2 Colon ca. HCC-2998 1.5 Melanoma UACC-62 1.6
Gastric ca.* (liver met) NCI-N87 6.3 Melanoma M14 9.7 Bladder 5.4
Melanoma LOX IMVI 9.8 Trachea 8.5 Melanoma* (met) SK-MEL-5 5.8
Kidney 4.5 Melanoma SK-MEL-28 9.2 Kidney (fetal) 11.8 Column A -
Rel. Exp.(%) Ag37, Run 87355114
[0576]
55TABLE AL Panel 1.2 Tissue Name A B Tissue Name A B Endothelial
cells 21.9 17.3 Renal ca. 786-0 6.1 6.7 Heart (Fetal) 100.0 40.9
Renal ca. A498 13.0 9.3 Pancreas 14.7 0.4 Renal ca. RXF 393 8.4 6.0
Pancreatic ca. CAPAN 2 3.4 0.7 Renal ca. ACHN 10.7 4.9 Adrenal
Gland 26.4 10.4 Renal ca. UO-31 8.8 3.6 Thyroid 18.8 0.3 Renal ca.
TK-10 15.4 6.3 Salivary gland 21.5 5.8 Liver 15.2 3.2 Pituitary
gland 23.8 1.4 Liver (fetal) 13.3 5.9 Brain (fetal) 33.9 1.4 Liver
ca. (hepatoblast) HepG2 21.8 12.1 Brain (whole) 69.3 3.8 Lung 19.3
0.7 Brain (amygdala) 13.9 3.6 Lung (fetal) 28.1 4.5 Brain
(cerebellum) 67.4 3.0 Lung ca. (small cell) LX-1 24.5 24.0 Brain
(hippocampus) 45.1 12.6 Lung ca. (small cell) NCI-H69 8.1 12.7
Brain (thalamus) 19.1 26.4 Lung ca. (s. cell var.) SHP-77 3.8 1.8
Cerebral Cortex 53.2 30.1 Lung ca. (large cell)NCI-H460 40.3 19.5
Spinal cord 45.4 7.2 Lung ca. (non-sm. cell) A549 13.1 13.9
glio/astro U87-MG 7.8 21.3 Lung ca. (non-s. cell) NCI-H23 28.1 24.5
glio/astro U-118-MG 6.8 13.5 Lung ca. (non-s. cell) HOP-62 42.3 9.7
astrocytoma SW1783 2.0 1.8 Lung ca. (non-s. cl) NCI-H522 90.1 44.1
neuro*; met SK-N-AS 50.3 27.5 Lung ca. (squam.) SW 900 37.4 57.8
astrocytoma SF-539 20.3 21.2 Lung ca. (squam.) NCI-H596 9.8 4.2
astrocytoma SNB-75 13.7 2.8 Mammary gland 42.6 9.0 glioma SNB-19
19.5 28.9 Breast ca.* (pl. ef) MCF-7 85.3 56.6 glioma U251 15.2 0.1
Breast ca.* (pl. ef) MDA-MB-231 5.3 1.4 glioma SF-295 15.0 5.1
Breast ca.* (pl. ef) T47D 5.6 16.6 Heart 63.3 12.2 Breast ca.
BT-549 5.0 2.6 Skeletal Muscle 27.7 0.8 Breast ca. MDA-N 16.7 3.7
Bone marrow 2.9 0.9 Ovary 49.0 23.0 Thymus 4.3 2.8 Ovarian ca.
OVCAR-3 46.3 50.7 Spleen 33.4 15.6 Ovarian ca. OVCAR-4 11.3 10.6
Lymph node 23.7 5.4 Ovarian ca. OVCAR-5 28.5 66.9 Colorectal Tissue
8.4 2.4 Ovarian ca. OVCAR-8 19.8 100.0 Stomach 36.3 9.6 Ovarian ca.
IGROV-1 18.2 13.3 Small intestine 27.9 11.7 Ovarian ca. (ascites)
SK-OV-3 25.7 10.3 Colon ca. SW480 6.1 1.8 Uterus 24.5 3.5 Colon
ca.* SW620 (SW480 met) 10.4 40.3 Placenta 90.1 82.9 Colon ca. HT29
3.0 2.5 Prostate 28.5 15.0 Colon ca. HCT-116 6.9 11.0 Prostate ca.*
(bone met) PC-3 39.0 15.4 Colon ca. CaCo-2 13.1 28.9 Testis 8.4 0.6
Colon ca. Tissue (ODO3866) 3.1 1.8 Melanoma Hs688(A).T 3.8 1.2
Colon ca. HCC-2998 12.6 9.5 Melanoma* (met) Hs688(B).T 2.9 0.8
Gastric ca.* (liver met) NCI-N87 18.4 17.6 Melanoma UACC-62 23.2
11.4 Bladder 40.1 15.5 Melanoma M14 13.7 6.7 Trachea 13.8 6.0
Melanoma LOX IMVI 9.6 1.8 Kidney 19.5 42.9 Melanoma* (met) SK-MEL-5
27.4 8.9 Kidney (fetal) 30.4 61.6 Coumn A - Rel. Exp.(%) Ag1215,
Run 129140825 Column B - Rel. Exp.(%) Ag1382, Run 134498635
[0577]
56TABLE AM Panel 2.2 Tissue Name A Tissue Name A Normal Colon 31.4
Kidney Margin (OD04348) 39.8 Colon cancer (OD06064) 34.4 Kidney
malignant cancer (OD06204B) 15.4 Colon Margin (OD06064) 18.7 Kidney
normal adjacent tissue 10.4 (OD06204E) Colon cancer (OD06159) 2.7
Kidney Cancer (OD04450-01) 15.3 Colon Margin (OD06159) 22.5 Kidney
Margin (OD04450-03) 20.3 Colon cancer (OD06297-04) 3.2 Kidney
Cancer 8120613 1.4 Colon Margin (OD06297-05) 30.8 Kidney Margin
8120614 20.4 CC Gr.2 ascend colon (ODO3921) 11.7 Kidney Cancer
9010320 10.5 CC Margin (ODO3921) 8.4 Kidney Margin 9010321 9.0
Colon cancer metastasis (OD06104) 5.1 Kidney Cancer 8120607 32.3
Lung Margin (OD06104) 8.3 Kidney Margin 8120608 12.2 Colon mets to
lung (OD04451-01) 15.3 Normal Uterus 31.9 Lung Margin (OD04451-02)
4.3 Uterine Cancer 064011 31.4 Normal Prostate 11.2 Normal Thyroid
3.0 Prostate Cancer (OD04410) 10.0 Thyroid Cancer 064010 14.9
Prostate Margin (OD04410) 14.3 Thyroid Cancer A302152 37.9 Normal
Ovary 74.7 Thyroid Margin A302153 7.0 Ovarian cancer (OD06283-03)
27.5 Normal Breast 35.4 Ovarian Margin (OD06283-07) 6.4 Breast
Cancer (OD04566) 15.7 Ovarian Cancer 064008 16.3 Breast Cancer 1024
51.4 Ovarian cancer (OD06145) 8.8 Breast Cancer (OD04590-01) 36.9
Ovarian Margin (OD06145) 24.7 Breast Cancer Mets (OD04590-03) 21.0
Ovarian cancer (OD06455-03) 9.9 Breast Cancer Metastasis
(OD04655-05) 66.9 Ovarian Margin (OD06455-07) 12.9 Breast Cancer
064006 18.9 Normal Lung 18.0 Breast Cancer 9100266 27.7 Invasive
poor diff. lung adeno 11.8 Breast Margin 9100265 21.6 (ODO4945-01
Lung Margin (ODO4945-03) 13.8 Breast Cancer A209073 15.1 Lung
Malignant Cancer (OD03126) 31.6 Breast Margin A2090734 28.5 Lung
Margin (OD03126) 5.6 Breast cancer (OD06083) 100.0 Lung Cancer
(OD05014A) 17.8 Breast cancer node metastasis (OD06083) 65.1 Lung
Margin (OD05014B) 13.8 Normal Liver 17.2 Lung cancer (OD06081) 10.2
Liver Cancer 1026 15.1 Lung Margin (OD06081) 8.2 Liver Cancer 1025
36.3 Lung Cancer (OD04237-01) 15.1 Liver Cancer 6004-T 18.0 Lung
Margin (OD04237-02) 24.1 Liver Tissue 6004-N 6.5 Ocular Melanoma
Metastasis 25.5 Liver Cancer 6005-T 33.4 Ocular Melanoma Margin
(Liver) 18.0 Liver Tissue 6005-N 31.6 Melanoma Metastasis 41.5
Liver Cancer 064003 9.0 Melanoma Margin (Lung) 9.1 Normal Bladder
14.7 Normal Kidney 7.5 Bladder Cancer 1023 6.0 Kidney Ca, Nuclear
grade 2 (OD04338) 34.6 Bladder Cancer A302173 28.9 Kidney Margin
(OD04338) 7.6 Normal Stomach 33.7 Kidney Ca Nuclear grade 1/2
(OD04339) 38.4 Gastric Cancer 9060397 3.6 Kidney Margin (OD04339)
6.8 Stomach Margin 9060396 12.2 Kidney Ca, Clear cell type
(OD04340) 19.2 Gastric Cancer 9060395 15.1 Kidney Margin (OD04340)
18.8 Stomach Margin 9060394 21.2 Kidney Ca, Nuclear grade 3
(OD04348) 10.4 Gastric Cancer 064005 17.4 Colmn A - Rel. Exp. (%)
Ag1215, Run 175165684
[0578]
57TABLE AN Panel 4.1D Tissue Name A Tissue Name A Secondary Th1 act
10.0 HUVEC IL-1beta 19.8 Secondary Th2 act 27.4 HUVEC IFN gamma 5.4
Secondary Tr1 act 15.8 HUVEC TNF alpha + IFN gamma 0.0 Secondary
Th1 rest 8.8 HUVEC TNF alpha + IL4 5.6 Secondary Th2 rest 9.9 HUVEC
IL-11 0.0 Secondary Tr1 rest 0.0 Lung Microvascular EC none 8.1
Primary Th1 act 1.5 Lung Microvascular EC TNF alpha + IL-1beta 13.7
Primary Th2 act 18.7 Microvascular Dermal EC none 0.0 Primary Tr1
act 5.1 Microsvasular Dermal EC TNF alpha + IL-1beta 18.2 Primary
Th1 rest 9.3 Bronchial epithelium TNF alpha + IL1beta 13.2 Primary
Th2 rest 13.9 Small airway epithelium none 0.0 Primary Tr1 rest 0.0
Small airway epithelium TNF alpha + IL-1beta 47.3 CD45RA CD4
lymphocyte act 4.5 Coronery artery SMC rest 1.7 CD45RO CD4
lymphocyte act 6.9 Coronery artery SMC TNF alpha + IL-1beta 6.4 CD8
lymphocyte act 14.4 Astrocytes rest 0.0 Secondary CD8 lymphocyte
rest 0.0 Astrocytes TNF alpha + IL-1beta 9.3 Secondary CD8
lymphocyte act 0.0 KU-812 (Basophil) rest 33.7 CD4 lymphocyte none
22.5 KU-812 (Basophil) PMA/ionomycin 3.2 2ry Th1/Th2/Tr1_anti-CD95
CH11 31.4 CCD1106 (Keratinocytes) none 3.8 LAK cells rest 6.6
CCD1106 (Keratinocytes) TNF alpha + IL-1beta 2.1 LAK cells IL-2 7.6
Liver cirrhosis 17.9 LAK cells IL-2 + IL-12 0.0 NCI-H292 29.9 LAK
cells IL-2 + IFN gamma 7.7 NCI-H292 IL-4 43.8 LAK cells IL-2 +
IL-18 6.4 NCI-H292 IL-9 40.6 LAK cells PMA/ionomycin 0.0 NCI-H292
IL-13 84.1 NK Cells IL-2 rest 39.2 NCI-H292 IFN gamma 29.7 Two Way
MLR 3 day 3.5 HPAEC none 6.4 Two Way MLR 5 day 0.0 HPAEC TNF alpha
+ IL-1beta 0.0 Two Way MLR 7 day 0.0 Lung fibroblast none 13.0 PBMC
rest 0.0 Lung fibroblast TNF alpha + IL-1beta 26.1 PBMC PWM 4.9
Lung fibroblast IL-4 0.0 PBMC PHA-L 0.0 Lung fibroblast IL-9 10.9
Ramos (B cell) none 0.0 Lung fibroblast IL-13 0.0 Ramos (B cell)
ionomycin 0.0 Lung fibroblast IFN gamma 12.5 B lymphocytes PWM 10.6
Dermal fibroblast CCD1070 rest 14.6 B lymphocytes CD40L and IL-4
14.3 Dermal fibroblast CCD1070 TNF alpha 30.8 EOL-1 dbcAMP 100.0
Dermal fibroblast CCD1070 IL-1beta 4.8 EOL-1 dbcAMP PMA/ionomycin
39.2 Dermal fibroblast IFN gamma 0.0 Dendritic cells none 0.0
Dermal fibroblast IL-4 15.9 Dendritic cells LPS 0.0 Dermal
Fibroblasts rest 6.7 Dendritic cells anti-CD40 4.6 Neutrophils TNFa
+ LPS 0.0 Monocytes rest 0.0 Neutrophils rest 0.0 Monocytes LPS 2.9
Colon 3.6 Macrophages rest 3.6 Lung 6.1 Macrophages LPS 0.0 Thymus
0.0 HUVEC none 0.0 Kidney 6.7 HUVEC starved 3.7 Colun A - Rel.
Exp.(%) Ag7452, Run 306288903
[0579]
58TABLE AO Panel 4D Tissue Name A B Tissue Name A B Secondary Th1
act 27.9 19.6 HUVEC IL-1beta 3.9 2.6 Secondary Th2 act 35.4 25.5
HUVEC IFN gamma 19.2 17.1 Secondary Tr1 act 42.0 37.6 HUVEC TNF
alpha + IFN gamma 2.1 2.4 Secondary Th1 rest 29.5 18.8 HUVEC TNF
alpha + IL4 15.3 12.2 Secondary Th2 rest 27.5 21.9 HUVEC IL-11 13.6
15.3 Secondary Tr1 rest 33.7 23.2 Lung Microvascular EC none 19.9
20.4 Primary Th1 act 35.1 28.1 Lung Microvascular EC TNF alpha +
IL-1beta 18.0 14.9 Primary Th2 act 31.4 25.7 Microvascular Dermal
EC none 29.7 26.8 Primary Tr1 act 55.9 42.6 Microsvasular Dermal EC
TNF alpha + IL-1beta 36.9 33.4 Primary Th1 rest 91.4 100.0
Bronchial epithelium TNF alpha + IL1beta 41.5 25.7 Primary Th2 rest
68.8 64.6 Small airway epithelium none 13.3 8.8 Primary Tr1 rest
55.5 52.1 Small airway epithelium TNF alpha + IL-1beta 56.6 45.4
CD45RA CD4 lymphocyte act 21.6 17.8 Coronery artery SMC rest 22.2
19.8 CD45RO CD4 lymphocyte act 24.5 17.2 Coronery artery SMC TNF
alpha + IL-1beta 28.1 19.9 CD8 lymphocyte act 22.1 15.5 Astrocytes
rest 13.0 20.2 Secondary CD8 lymphocyte rest 17.1 12.4 Astrocytes
TNF alpha + IL-1beta 23.7 18.6 Secondary CD8 lymphocyte act 29.7
18.2 KU-812 (Basophil) rest 97.3 75.3 CD4 lymphocyte none 17.9 15.4
KU-812 (Basophil) PMA/ionomycin 100.0 90.1 2ry
Th1/Th2/Trl_anti-CD95 71.7 67.4 CCD1106 (Keratinocytes) none 16.6
13.8 CH11 LAK cells rest 17.1 10.3 CCD1106 (Keratinocytes) TNF
alpha + IL-1beta 67.8 11.3 LAK cells IL-2 21.5 15.7 Liver cirrhosis
14.3 12.3 LAK cells IL-2 + IL-12 18.4 17.1 Lupus kidney 28.7 18.8
LAK cells IL-2 + IFN gamma 29.5 22.2 NCI-H292 none 27.4 28.3 LAK
cells IL-2 + IL-18 18.0 22.1 NCI-H292 IL-4 61.1 55.1 LAK cells
PMA/ionomycin 10.7 7.3 NCI-H292 IL-9 37.6 31.6 NK Cells IL-2 rest
31.9 17.2 NCI-H292 IL-13 44.4 42.9 Two Way MLR 3 day 9.5 7.6
NCI-H292 IFN gamma 21.0 20.3 Two Way MLR 5 day 6.3 6.8 HPAEC none
21.5 17.2 Two Way MLR 7 day 10.5 8.5 HPAEC TNF alpha + IL-1beta
11.0 9.5 PBMC rest 10.7 10.3 Lung fibroblast none 31.0 20.6 PBMC
PWM 50.7 38.4 Lung fibroblast TNF alpha + IL-1beta 26.1 23.5 PBMC
PHA-L 32.5 31.2 Lung fibroblast IL-4 40.6 37.1 Ramos (B cell) none
0.0 0.0 Lung fibroblast IL-9 21.3 17.7 Ramos (B cell) ionomycin 0.0
0.0 Lung fibroblast IL-13 56.3 53.2 B lymphocytes PWM 36.1 34.2
Lung fibroblast IFN gamma 59.5 45.4 B lymphocytes CD40L and IL-4
18.3 17.2 Dermal fibroblast CCD1070 rest 47.0 33.4 EOL-1 dbcAMP
43.8 30.1 Dermal fibroblast CCD1070 TNF alpha 73.7 55.1 EOL-1
dbcAMP PMA/ionomycin 73.2 63.7 Dermal fibroblast CCD1070 IL-1beta
36.6 37.4 Dendritic cells none 1.6 0.6 Dermal fibroblast IFN gamma
8.8 9.7 Dendritic cells LPS 1.2 5.0 Dermal fibroblast IL-4 20.2
19.6 Dendritic cells anti-CD40 0.9 0.9 IBD Colitis 2 4.1 2.0
Monocytes rest 2.9 1.8 IBD Crohn's 3.1 3.1 Monocytes LPS 29.7 19.2
Colon 29.9 24.3 Macrophages rest 4.9 2.3 Lung 46.0 38.4 Macrophages
LPS 7.2 4.4 Thymus 60.3 54.0 HUVEC none 9.2 6.5 Kidney 32.1 26.4
HUVEC starved 18.6 14.7 Column A - Rel. Exp.(%) Ag1215, Run
140416188 Column B - Rel. Exp.(%) Ag1382, Run 145608863
[0580]
59TABLE AP Panel 5 Islet Tissue Name A Tissue Name A
97457_Patient-02go_adipose 20.7 94709_Donor 2 AM - A_adipose 28.5
97476_Patient-07sk_skeletal muscle 16.5 94710_Donor 2 AM -
B_adipose 15.3 97477_Patient-07ut_uterus 28.7 94711_Donor 2 AM -
C_adipose 10.9 97478_Patient-07pl_placenta 47.3 94712_Donor 2 AD -
A_adipose 14.0 99167_Bayer Patient 1 82.4 94713_Donor 2 AD -
B_adipose 17.9 97482_Patient-08ut_uterus 26.8 94714_Donor 2 AD -
C_adipose 28.3 97483_Patient-08pl_placenta 25.3 94742_Donor 3 U -
A_Mesenchymal Stem 12.0 Cells 97486_Patient-09sk_skeletal muscle
3.3 94743_Donor 3 U - B_Mesenchymal Stem 17.2 Cells
97487_Patient-09ut_uterus 15.7 94730_Donor 3 AM - A_adipose 21.2
97488_Patient-09pl_placenta 21.5 94731_Donor 3 AM - B_adipose 16.2
97492_Patient-10ut_uterus 26.8 94732_Donor 3 AM - C_adipose 21.0
97493_Patient-10pl_placenta 100.0 94733_Donor 3 AD - A_adipose 18.2
97495_Patient-11go_adipose 19.8 94734_Donor 3 AD - B_adipose 12.1
97496_Patient-11sk_skeletal muscle 12.9 94735_Donor 3 AD -
C_adipose 19.9 97497_Patient-11ut_uterus 38.7
77138_Liver_HepG2untreated 68.8 97498_Patient-11pl_placenta 31.9
73556_Heart_Cardiac stromal cells 9.4 (primary)
97500_Patient-12go_adipose 39.2 81735_Small Intestine 27.4
97501_Patient-12sk_skeletal muscle 43.2 72409_Kidney Proximal
Convoluted 1.6 Tubule 97502_Patient-12ut_uterus 52.1 82685_Small
intestine_Duodenum 8.3 97503_Patient-12pl_placenta 31.2
90650_Adrenal_Adrenocortical adenoma 5.7 94721_Donor 2 U -
A_Mesenchymal 17.6 72410_Kidney_HRCE 24.3 Stem Cells 94722_Donor 2
U - B_Mesenchymal Stem 16.3 72411_Kidney_HRE 21.0 Cells 94723_Donor
2 U - C_Mesenchymal Stem 22.1 73139_Uterus_Uterine smooth muscle
cells 7.5 Cells Colum A - Rel. Exp.(%) Ag1215, Run 258292593
[0581]
60TABLE AQ Panel CNS 1 Tissue Name A Tissue Name A BA4 Control 20.3
BA17 PSP 31.2 BA4 Control2 34.6 BA17 PSP2 9.9 BA4 Alzheimer's2 9.0
Sub Nigra Control 54.7 BA4 Parkinson's 33.0 Sub Nigra Control2 31.9
BA4 Parkinson's2 47.0 Sub Nigra Alzheimer's2 24.3 BA4 Huntington's
36.3 Sub Nigra Parkinson's2 92.0 BA4 Huntingson's2 11.1 Sub Nigra
Huntington's 80.7 BA4 PSP 15.8 Sub Nigra Huntington's2 43.2 BA4
PSP2 46.0 Sub Nigra PSP2 24.8 BA4 Depression 19.8 Sub Nigra
Depression 18.6 BA4 Depression2 7.8 Sub Nigra Depression2 12.1 BA7
Control 26.2 Glob Palladus Control 20.3 BA7 Control2 33.0 Glob
Palladus Control2 10.7 BA7 Alzheimer's2 4.5 Glob Palladus
Alzheimer's 21.6 BA7 Parkinson's 23.0 Glob Palladus Alzheimer's2
5.0 BA7 Parkinson's2 35.6 Glob Palladus Parkinson's 100.0 BA7
Huntington's 39.2 Glob Palladus Parkinson's2 23.0 BA7 Huntington's2
40.6 Glob Palladus PSP 10.2 BA7 PSP 31.2 Glob Palladus PSP2 11.8
BA7 PSP2 32.3 Glob Palladus Depression 23.5 BA7 Depression 6.3 Temp
Pole Control 7.7 BA9 Control 12.5 Temp Pole Control2 29.3 BA9
Control2 48.6 Temp Pole Alzheimer's 4.0 BA9 Alzheimer's 6.9 Temp
Pole Alzheimer's2 3.6 BA9 Alzheimer's2 5.4 Temp Pole Parkinson's
22.5 BA9 Parkinson's 25.3 Temp Pole Parkinson's2 22.2 BA9
Parkinson's2 33.4 Temp Pole Huntington's 30.6 BA9 Huntington's 45.1
Temp Pole PSP 3.8 BA9 Huntington's2 13.8 Temp Pole PSP2 2.3 BA9 PSP
23.5 Temp Pole Depression2 7.1 BA9 PSP2 5.0 Cing Gyr Control 59.0
BA9 Depression 10.8 Cing Gyr Control2 40.3 BA9 Depression2 11.8
Cing Gyr Alzheimer's 19.3 BA17 Control 39.8 Cing Gyr Alzheimer's2
11.8 BA17 Control2 40.1 Cing Gyr Parkinson's 42.3 BA17Alzheimer's2
7.6 Cing Gyr Parkinson's2 35.8 BA17 Parkinson's 31.2 Cing Gyr
Huntington's 62.4 BA17 Parkinson's2 35.4 Cing Gyr Huntington's2
28.7 BA17 Huntington's 47.3 Cing Gyr PSP 66.9 BA17 Huntington's2
25.0 Cing Gyr PSP2 5.2 BA17 Depression 22.2 Cing Gyr Depression
14.8 BA17 Depression2 41.8 Cing Gyr Depression2 32.1 Colum A - Rel.
Exp.(%) Ag1215, Run 171629509
[0582]
61TABLE AR general oncology screening panel_v_2.4 Tissue Name A
Tissue Name A Colon cancer 1 18.0 Bladder cancer NAT2 0.4 Colon
cancer NAT 1 21.0 Bladder cancer NAT 3 0.9 Colon cancer 2 33.4
Bladder cancer NAT 4 9.0 Colon cancer NAT 2 3.6 Prostate
adenocarcinoma 1 46.3 Colon cancer 3 26.8 Prostate adenocarcinoma 2
3.5 Colon cancer NAT 3 19.2 Prostate adenocarcinoma 3 11.9 Colon
malignant cancer 4 27.5 Prostate adenocarcinoma 4 16.8 Colon normal
adjacent tissue 4.2 Prostate cancer NAT 5 4.9 4 Lung cancer 1 11.4
Prostate adenocarcinoma 6 6.4 Lung NAT 1 1.3 Prostate
adenocarcinoma 7 6.6 Lung cancer 2 100.0 Prostate adenocarcinoma 8
3.3 Lung NAT 2 2.2 Prostate adenocarcinoma 9 33.9 Squamous cell
carcinoma 3 30.4 Prostate cancer NAT 10 2.5 Lung NAT 3 0.3 Kidney
cancer 1 44.1 metastatic melanoma 1 24.5 KidneyNAT 1 15.2 Melanoma
2 3.2 Kidney cancer 2 68.8 Melanoma 3 2.8 Kidney NAT 2 18.4
metastatic melanoma 4 62.9 Kidney cancer 3 29.9 metastatic melanoma
5 42.9 Kidney NAT 3 6.1 Bladder cancer 1 2.3 Kidney cancer 4 18.9
Bladder cancer NAT 1 0.0 Kidney NAT 4 7.4 Bladder cancer 2 5.8
Column A - Rel. Exp.(%) Ag1215, Run 267211472
[0583] Ardais Panel v.1.0 Summary: Ag1215 Expression of this gene
is higher in lung cancer samples compared to adjacent normal
tissue. Therefore, therapeutic modulation of this gene or its
protein product through the use of small molecule or antibodies may
be useful in the treatment of lung cancer.
[0584] CNS_neurodegeneration_v1.0 Summary: Ag1215/Ag7452 This panel
confirms the expression of this gene at low levels in the brains of
an independent group of individuals.
[0585] General_screening_panel_v1.4 Summary: Ag1215/Ag37 Two
experiments with two different primer-probe sets are in agreement.
Highest expression of this gene is seen in a breast cancer MCF-7
cell line (CTs=24-24.8). High expression of this gene is also seen
in cluster of cancer cell lines derived from pancreatic, gastric,
colon, lung, liver, renal, breast, ovarian, prostate, squamous cell
carcinoma, melanoma and brain cancers. Thus, expression of this
gene could be used as a marker to detect the presence of these
cancers. Furthermore, therapeutic modulation of the expression or
function of this gene may be effective in the treatment of
pancreatic, gastric, colon, lung, liver, renal, breast, ovarian,
prostate, squamous cell carcinoma, melanoma type and brain
cancers.
[0586] Among tissues with metabolic or endocrine function, this
gene is expressed at moderate to high levels in pancreas, adipose,
adrenal gland, thyroid, pituitary gland, skeletal muscle, heart,
liver and the gastrointestinal tract. Therefore, therapeutic
modulation of the activity of this gene may prove useful in the
treatment of endocrine/metabolically related diseases, such as
obesity and diabetes.
[0587] In addition, this gene is expressed at high levels in all
regions of the central nervous system examined, including amygdala,
hippocampus, substantia nigra, thalamus, cerebellum, cerebral
cortex, and spinal cord. Therefore, therapeutic modulation of this
gene product may be useful in the treatment of central nervous
system In addition, this gene is expressed at high levels in all
regions of the central nervous system examined, including amygdala,
hippocampus, substantia nigra, thalamus, cerebellum, cerebral
cortex, and spinal cord. Therefore, therapeutic modulation of this
gene product may be useful in the treatment of central nervous
system disorders such as Alzheimer's disease, Parkinson's disease,
epilepsy, multiple sclerosis, schizophrenia and depression.
[0588] This gene is expressed at much higher levels in fetal
(CTs=25-28) when compared to adult lung and liver (CTs=29-32). This
suggests that expression of this gene can be used to distinguish
fetal from adult lung and liver. In addition, the relative
overexpression of this gene in fetal tissue suggests that the
protein product may enhance lung and liver growth or development in
the fetus and may also act in a regenerative capacity in the adult.
Therefore, therapeutic modulation of the protein encoded by this
gene could be useful in treatment of liver related diseases.
[0589] General_screening_panel_v1.7 Summary: Ag7452 Highest
expression of this gene is seen in a breast cancer MCF7 cell line
(CT=27.4). This gene shows wide spread expression in this panel and
this pattern of expression is in agreement with panel 1.4 discussed
above.
[0590] HASS Panel v1.0 Summary: Ag1215 This gene shows widespread
expression in all the samples in this panel with high expression in
MCF7 cell lines (CTs=24-25) and thus could be potentially used as a
diagnostic and/therapeutic for breast cancer.
[0591] Oncology_cell_line_screening_panel_v3.2 Summary: Ag1215
Highest expression of this gene is seen in a lung cancer DMS-79
cell line (CT=27.9). Moderate to low expression of this gene is
seen in number of cancer cell lines derived from tongue, breast,
bone, bladder, fibrosarcoma, pancreatic, renal, T cell and
erythroleukemia, colon, gastric, lung and brain cancers. Therefore,
therapeutic modulation of this gene or its protein product may be
used for the treatment of these cancers.
[0592] Panel 1 Summary: Ag37 This gene is expressed at high to
moderate levels in the majority of the samples on this panel.
Expression of this gene is highest in a breast cancer cell line
(CT=22). In addition there is substantial expression in placental
tissue and in a number of brain regions. Moreover, therapeutic
modulation of this gene or its protein product, through the use of
small molecule drugs, antibodies or protein therapeutics, might be
of benefit in the treatment of breast cancer. In general, however,
expression appears to be higher in normal cells than in the cancer
cell lines.
[0593] Among tissues with endocrine or metabolic activity, this
gene is expressed at high levels in pancreas, adrenal gland,
thyroid, pituitary gland, hypothalamus, heart, skeletal muscle, and
liver. This observation indicates an importance for this gene in
endocrine and metaboic physiology. Therefore, therapeutic
modulation of this gene and/or gene product may prove useful in the
treatment of diseases associated with these physiological systems,
including diabetes and obesity.
[0594] Expression of the gene is very high throughout the central
nervous system including in the spinal cord, amygdala, cerebellum,
hippocampus, thalamus, and substantia nigra. This gene encodes a
protein with homology to sempaphorins. Semaphorins can act as axon
guidance proteins, specifically as chemorepellents which inhibit
CNS regenerative capacity. Therefore, therapeutic modulation of the
activity and or amount of this protein may be of use in inducing a
compensatory synaptogenic response to neuronal death in Alzheimer's
disease, Parkinson's disease, Huntington's disease, spinocerebellar
ataxia, progressive supranuclear palsy, multiple sclerosis, ALS,
head trauma, stroke, or any other disease/condition associated with
neuronal loss.
[0595] Panel 1.2 Summary: Ag1215/Ag1382 This gene is expressed at
high levels across most of the tissues on this panel with highest
expression in ovarian cancer cell line (CTs=22-23). Furthermore,
the expression of this gene seems to be associated with
reproductive tissues and cancer cell lines which is in agreement
with expression seen in panels 1 and 1.4.
[0596] Panel 2.2 Summary: Ag1215 This gene shows a widespread
expression. Specifically, highest expression is seen in breast
cancer cells. This is reasonably consistent with the results
obtained from Panel 1.2. In addition, there is also some
correlation with expression in normal kidney tissue when compared
to kidney cancers, also consistent with the observations in Panel
1.2. Thus, therapeutic modulation of this gene or gene product may
be useful in the treatment of breast cancer, ovarian cancer or
kidney cancer.
[0597] Panel 4.1D Summary: Ag7452 Highest expression of this gene
is seen in eosinophils (CT=33.3). Low but significant expression of
this gene is also seen in resting IL-2 treated NK cells, activated
eosinophils, activated NCI-H292 cells, activated small airway
epithelium and activated lung fibroblasts. Therefore, therapeutic
modulation of this gene through the use of small molecule drug or
antibodies may be useful in the treatment of inflammation in lung
especially chronic obstructive pulmonary disease, asthma, allergy,
and emphysema. In addition, regulating the expression of this gene
or the encoded protein may be useful in the treatment of
hematopoietic disorders involving eosinphils, parasitic infections
and asthma.
[0598] Panel 4D Summary: Ag1215/Ag1382 Results from two replicate
experiments performed using probe and primer sets of identical
sequences are in reasonable agreement. This gene is widely
expressed in cell lines from this panel (CTs=25-30), including
thymus, lung, muco-epidermoid cell lines, fibroblasts from diverse
origin, and activated T cells. In addition, this gene is expressed
in normal colon but not in colons from patients with Crohn's
disease or colitis. Thus, protein therapeutics designed with the
putative semaphorin encoded by this protein could reduce or
eliminate inflammation and tissue destruction due to IBD. High
expression of this gene was found on primary resting Th1 T cells,
and also primary resting Th2 and Tr1 T cells. The high expression
in secondary T cells treated with CD95 suggests that protein
encoded by this gene is involved in activation of cell death.
Furthermore, high expression of this gene is also found in
activated basophils and eosinophils, suggesting a role for this
protein in allergic disorder such as asthma, contact
hypersensitivity, and hypersensitive immediate reactions. Antibody
or protein therapeutics designed against the protein encoded for by
this gene could therefore reduce or inhibit inflammation in
allergy, asthma, emphysema, psoriasis and/or autoimmunity.
[0599] Panel 5 Islet Summary: Ag1215 Highest expression of this
gene is detected in placenta of a diabetic patient (CT=29.8). This
gene shows a widespread expression in this panel. This pattern is
in agreement with the expression profile in panel 1.4.
[0600] Panel CNS.sub.--1 Summary: Ag1215 This gene shows wide
spread expression in this panel. This gene codes for semaphorin 4C.
Semaphorins can act as axon guidance proteins, specifically through
their ability to act as chemorepellents that inhibit CNS
regenerative capacity. Therefore therapeutic modulation of levels
of this gene or its protein product may therefore be of use in
inducing a compensatory synaptogenic response to neuronal death in
Alzheimer's disease, Parkinson's disease, Huntington's disease,
spinocerebellar ataxia, progressive supranuclear palsy, multiple
sclerosis, ALS, head trauma, stroke, or any other disease/condition
associated with neuronal loss.
[0601] General oncology screening panel_v.sub.--2.4 Summary: Ag1215
Highest expression of this gene is seen in a lung cancer sample
(CT=26.6). Interestingly, expression of this gene is upregulated in
lung, colon, bladder, metastatic melanoma, prostate and kidney
cancers. Therefore, expression of this gene may be used as marker
to detect the presence of these cancers and also, therapeutic
modulation of this gene or its protein product through the use of
small molecule drug or antibodies may be useful in the treatment of
these cancers.
[0602] B. CG51896-04: Semaphorin 6A Precursor.
[0603] Expression of gene CG51896-04 was assessed using the
primer-probe sets Ag2772, Ag88 and Ag6309, described in Tables BA,
BB and BC. Results of the RTQ-PCR runs are shown in Tables BD, BE,
BF, BG, BH, BI, BJ, BK and BL.
62TABLE BA Probe Name Ag2772 Start SEQ ID Primers Sequences Length
Position No Forward 5'-actggaagcatctgcttgact-3' 21 2117 141 Probe
TET-5'-cacctgacagcacagaccctttgg-3'-TAMRA 24 2093 142 Reverse
5'-atcactcccttcttgtcttggt-3' 22 2050 143
[0604]
63TABLE BB Probe Name Ag88 Start SEQ ID Primers Sequences Length
Position No Forward 5'-catcttcaacaggccatggtt-3' 21 2770 144 Probe
TET-5'-tgagaacaatggtcagataccgccttaccaa-3'-TAMRA 31 2737 145 Reverse
5'-agcagctgtgtccactgcaa-3' 20 2715 146
[0605]
64TABLE BC Probe Name Ag6309 Start SEQ ID Primers Sequences Length
Position No Forward 5'-atacactgtgttgtaagacatttcattatc-3' 30 2223
147 Probe TET-5'-tggcactgaatgacatttcaactcctc-3'-TAMRA 27 2258 148
Reverse 5'-gggactgtcacaattcctttg-3' 21 2285 149
[0606]
65TABLE BD CNS_neurodegeneration_v1.0 Tissue Name A B Tissue Name A
B AD 1 Hippo 3.1 8.0 Control (Path) 3 Temporal Ctx 6.0 5.3 AD 2
Hippo 41.8 31.6 Control (Path) 4 Temporal Ctx 4.7 19.6 AD 3 Hippo
12.8 2.3 AD 1 Occipital Ctx 37.6 7.5 AD 4 Hippo 12.3 7.9 AD 2
Occipital Ctx (Missing) 0.0 0.0 AD 5 Hippo 60.7 19.6 AD 3 Occipital
Ctx 5.6 4.7 AD 6 Hippo 76.8 72.2 AD 4 Occipital Ctx 43.8 37.6
Control 2 Hippo 67.8 52.9 AD 5 Occipital Ctx 38.2 33.4 Control 4
Hippo 21.9 26.6 AD 6 Occipital Ctx 0.0 14.7 Control (Path) 3 Hippo
0.9 9.7 Control 1 Occipital Ctx 4.5 4.1 AD 1 Temporal Ctx 48.3 11.1
Control 2 Occipital Ctx 77.4 61.6 AD 2 Temporal Ctx 32.1 32.1
Control 3 Occipital Ctx 29.1 24.0 AD 3 Temporal Ctx 8.5 3.4 Control
4 Occipital Ctx 22.8 17.3 AD 4 Temporal Ctx 17.0 35.1 Control
(Path) 1 Occipital Ctx 100.0 100.0 AD 5 Inf Temporal Ctx 22.5 48.0
Control (Path) 2 Occipital Ctx 4.1 16.3 AD 5 Sup Temporal Ctx 48.6
41.5 Control (Path) 3 Occipital Ctx 10.4 4.2 AD 6 Inf Temporal Ctx
66.4 45.4 Control (Path) 4 Occipital Ctx 13.4 11.0 AD 6 Sup
Temporal Ctx 61.1 18.4 Control 1 Parietal Ctx 22.4 13.1 Control 1
Temporal Ctx 10.3 13.2 Control 2 Parietal Ctx 52.9 33.4 Control 2
Temporal Ctx 39.0 44.4 Control 3 Parietal Ctx 3.1 19.5 Control 3
Temporal Ctx 33.0 27.4 Control (Path) 1 Parietal Ctx 41.2 38.7
Control 3 Temporal Ctx 3.4 12.9 Control (Path) 2 Parietal Ctx 10.2
28.5 Control (Path) 1 Temporal Ctx 47.0 38.2 Control (Path) 3
Parietal Ctx 6.8 5.1 Control (Path) 2 Temporal Ctx 28.9 31.0
Control (Path) 4 Parietal Ctx 42.0 29.3 Column A - Rel. Exp.(%
Ag2772, Run 208699007 Column B - Rel. Exp.(%) Ag6309, Run
259476984
[0607]
66TABLE BE General_screening_panel_v1.5 Tissue Name A Tissue Name A
Adipose 2.4 Renal ca. TK-10 1.1 Melanoma* Hs688(A).T 0.0 Bladder
0.5 Melanoma* Hs688(B).T 0.0 Gastric ca. (liver met.) NCI-N87 0.0
Melanoma* M14 1.0 Gastric ca. KATO III 0.0 Melanoma* LOXIMVI 0.0
Colon ca. SW-948 0.0 Melanoma* SK-MEL-5 7.1 Colon ca. SW480 0.0
Squamous Cell carcinoma SCC-4 0.0 Colon ca.* (SW480 met) SW620 0.1
Testis Pool 0.8 Colon ca. HT29 0.0 Prostate ca.* (bone met) PC-3
0.0 Colon ca. HCT-116 0.0 Prostate Pool 0.6 Colon ca. CaCo-2 1.4
Placenta 1.8 Colon cancer tissue 1.0 Uterus Pool 0.5 Colon ca.
SW1116 0.0 Ovarian ca. OVCAR-3 0.0 Colon ca. Colo-205 0.3 Ovarian
ca. SK-OV-3 0.2 Colon ca. SW-48 0.0 Ovarian ca. OVCAR-4 0.0 Colon
Pool 0.3 Ovarian ca. OVCAR-5 0.7 Small Intestine Pool 0.8 Ovarian
ca. IGROV-1 5.8 Stomach Pool 0.6 Ovarian ca. OVCAR-8 0.0 Bone
Marrow Pool 0.3 Ovary 0.0 Fetal Heart 0.5 Breast ca. MCF-7 0.0
Heart Pool 0.4 Breast ca. MDA-MB-231 0.0 Lymph Node Pool 0.0 Breast
ca. BT 549 0.0 Fetal Skeletal Muscle 0.6 Breast ca. T47D 0.8
Skeletal Muscle Pool 0.6 Breast ca. MDA-N 0.0 Spleen Pool 0.2
Breast Pool 0.0 Thymus Pool 0.0 Trachea 0.2 CNS cancer (glio/astro)
U87-MG 0.0 Lung 0.4 CNS cancer (glio/astro) U-118-MG 0.0 Fetal Lung
10.4 CNS cancer (neuro; met) SK-N-AS 0.4 Lung ca. NCI-N417 0.6 CNS
cancer (astro) SF-539 0.2 Lung ca. LX-1 0.0 CNS cancer (astro)
SNB-75 0.0 Lung ca. NCI-H146 0.0 CNS cancer (glio) SNB-19 4.1 Lung
ca. SHP-77 0.0 CNS cancer (glio) SF-295 0.0 Lung ca. A549 0.0 Brain
(Amygdala) Pool 51.1 Lung ca. NCI-H526 0.0 Brain (cerebellum) 86.5
Lung ca. NCI-H23 0.7 Brain (fetal) 80.7 Lung ca. NCI-H460 0.4 Brain
(Hippocampus) Pool 53.6 Lung ca. HOP-62 0.0 Cerebral Cortex Pool
54.3 Lung ca. NCI-H522 0.0 Brain (Substantia nigra) Pool 32.5 Liver
0.0 Brain (Thalamus) Pool 72.2 Fetal Liver 0.0 Brain (whole) 61.1
Liver ca. HepG2 0.0 Spinal Cord Pool 100.0 Kidney Pool 0.5 Adrenal
Gland 2.4 Fetal Kidney 2.1 Pituitary gland Pool 0.4 Renal ca. 786-0
4.2 Salivary Gland 0.5 Renal ca. A498 0.0 Thyroid (female) 0.0
Renal ca. ACHN 0.2 Pancreatic ca. CAPAN2 0.0 Renal ca. UO-31 0.0
Pancreas Pool 0.1 Column A - Rel. Exp.(% Ag6309, Run 259428262
[0608]
67TABLE BF HASS Panel v1.0 Tissue Name A Tissue Name A MCF-7 C1 3.1
U87-MG F1 (B) 0.1 MCF-7 C2 6.0 U87-MG F2 0.0 MCF-7 C3 3.0 U87-MG F3
0.3 MCF-7 C4 6.2 U87-MG F4 0.1 MCF-7 C5 3.8 U87-MG F5 0.2 MCF-7 C6
2.5 U87-MG F6 1.1 MCF-7 C7 5.5 U87-MG F7 0.2 MCF-7 C9 3.2 U87-MG F8
0.6 MCF-7 C10 6.6 U87-MG F9 0.1 MCF-7 C11 1.1 U87-MG F10 0.5 MCF-7
C12 1.7 U87-MG F11 0.7 MCF-7 C13 3.6 U87-MG F12 0.2 MCF-7 C15 2.1
U87-MG F13 0.2 MCF-7 C16 2.7 U87-MG F14 0.7 MCF-7 C17 2.3 U87-MG
F15 0.2 T24 D1 0.7 U87-MG F16 0.2 T24 D2 0.0 U87-MG F17 0.3 T24 D3
0.1 LnCAP A1 29.9 T24 D4 0.1 LnCAP A2 26.1 T24 D5 0.0 LnCAP A3 46.7
T24 D6 0.0 LnCAP A4 26.6 T24 D7 0.0 LnCAP A5 39.8 T24 D9 0.0 LnCAP
A6 32.8 T24 D10 0.0 LnCAP A7 16.4 T24 D11 0.0 LnCAP A8 42.9 T24 D12
0.0 LnCAP A9 18.9 T24 D13 0.0 LnCAP A10 20.0 T24 D15 0.1 LnCAP A11
45.1 T24 D16 0.0 LnCAP A12 7.9 T24 D17 0.0 LnCAP A13 3.5 CAPaN B1
1.6 LnCAP A14 2.9 CAPaN B2 0.5 LnCAP A15 5.3 CAPaN B3 0.5 LnCAP A16
54.7 CAPaN B4 0.7 LnCAP A17 48.0 CAPaN B5 0.7 Primary Astrocytes
2.3 CAPaN B6 0.8 Primary Renal Proximal Tubule 13.1 Epithelial cell
A2 CAPaN B7 0.4 Primary melanocytes A5 31.0 CAPaN B8 0.1 126443 -
341 medullo 1.5 CAPaN B9 0.4 126444 - 487 medullo 100.0 CAPaN B10
0.8 126445 - 425 medullo 1.3 CAPaN B11 1.4 126446 - 690 medullo
46.7 CAPaN B12 1.0 126447 - 54 adult glioma 0.1 CAPaN B13 1.0
126448 - 245 adult glioma 15.1 CAPaN B14 0.1 126449 - 317 adult
glioma 20.6 CAPaN B15 0.2 126450 - 212 glioma 48.6 CAPaN B16 1.1
126451 - 456 glioma 84.7 CAPaN B17 1.2 Column A - Rel. Exp.(%)
Ag2772, Run 264977485
[0609]
68TABLE BG Panel 1 Tissue Name A Tissue Name A Endothelial cells
0.4 Renal ca. 786-0 66.9 Endothelical cells (treated) 1.6 Renal ca.
A498 0.8 Pancreas 18.2 Renal ca. RXF 393 19.8 Pancreatic ca. CAPAN
2 0.2 Renal ca. ACHN 8.1 Adrenal gland 51.8 Renal ca. UO-31 0.2
Thyroid 4.6 Renal ca. TK-10 18.3 Salivary gland 8.5 Liver 3.7
Pituitary gland 1.8 Liver (fetal) 2.1 Brain (fetal) 8.5 Liver ca.
(hepatoblast) HepG2 3.3 Brain (whole) 46.3 Lung 5.7 Brain
(amygdala) 9.4 Lung (fetal) 8.0 Brain (cerebellum) 100.0 Lung ca.
(small cell) LX-1 0.0 Brain (hippocampus) 34.4 Lung ca. (small
cell) NCI-H69 15.7 Brain (substantia nigra) 50.7 Lung ca. (s.cell
var.) SHP-77 0.0 Brain (thalamus) 15.4 Lung ca. (large
cell)NCI-H460 0.0 Brain (hypothalamus) 2.9 Lung ca. (non-sm. cell)
A549 0.0 Spinal cord 22.5 Lung ca. (non-s.cell) NCI-H23 0.5
glio/astro U87-MG 0.1 Lung ca. (non-s.cell) HOP-62 0.4 glio/astro
U-118-MG 0.0 Lung ca. (non-s.cl) NCI-H522 0.1 astrocytoma SW1783
0.0 Lung ca. (squam.) SW 900 0.7 neuro*; met SK-N-AS 4.6 Lung ca.
(squam.) NCI-H596 30.8 astrocytoma SF-539 0.2 Mammary gland 18.8
astrocytoma SNB-75 1.1 Breast ca.* (pl.ef) MCF-7 1.5 glioma SNB-19
7.1 Breast ca.* (pl.ef) MDA-MB-231 0.0 glioma U251 0.6 Breast ca.*
(pl.ef) T47D 30.6 glioma SF-295 0.1 Breast ca. BT-549 0.0 Heart 3.3
Breast ca. MDA-N 0.0 Skeletal muscle 1.1 Ovary 7.0 Bone marrow 0.9
Ovarian ca. OVCAR-3 1.2 Thymus 20.6 Ovarian ca. OVCAR-4 0.0 Spleen
2.5 Ovarian ca. OVCAR-5 11.8 Lymph node 3.2 Ovarian ca. OVCAR-8 0.4
Colon (ascending) 11.5 Ovarian ca. IGROV-1 12.0 Stomach 11.4
Ovarian ca. (ascites) SK-OV-3 0.9 Small intestine 5.5 Uterus 6.4
Colon ca. SW480 0.1 Placenta 43.8 Colon ca.* SW620 (SW480 met) 0.4
Prostate 3.1 Colon ca. HT29 0.0 Prostate ca.* (bone met) PC-3 0.0
Colon ca. HCT-116 0.0 Testis 35.6 Colon ca. CaCo-2 19.6 Melanoma
Hs688(A).T 0.0 Colon ca. HCT-15 0.0 Melanoma* (met) Hs688(B).T 0.0
Colon ca. HCC-2998 1.1 Melanoma UACC-62 1.4 Gastric ca.* (liver
met) NCI-N87 0.3 Melanoma M14 11.4 Bladder 1.6 Melanoma LOX IMVI
0.8 Trachea 5.0 Melanoma* (met) SK-MEL-5 18.9 Kidney 4.7 Melanoma
SK-MEL-28 30.6 Kidney (fetal) 13.7 Column A - Rel. Exp.(%) Ag88,
Run 87586103
[0610]
69TABLE BH Panel 1.3D Tissue Name A Tissue Name A Liver
adenocarcinoma 2.4 Kidney (fetal) 13.6 Pancreas 5.3 Renal ca. 786-0
36.6 Pancreatic ca. CAPAN 2 0.2 Renal ca. A498 2.1 Adrenal gland
22.2 Renal ca. RXF 393 27.0 Thyroid 4.4 Renal ca. ACHN 10.0
Salivary gland 5.1 Renal ca. UO-31 0.2 Pituitary gland 2.4 Renal
ca. TK-10 6.9 Brain (fetal) 3.3 Liver 1.8 Brain (whole) 15.9 Liver
(fetal) 3.1 Brain (amygdala) 14.9 Liver ca. (hepatoblast) HepG2 3.7
Brain (cerebellum) 8.8 Lung 14.9 Brain (hippocampus) 27.0 Lung
(fetal) 10.7 Brain (substantia nigra) 11.1 Lung ca. (small cell)
LX-1 0.4 Brain (thalamus) 18.2 Lung ca. (small cell) NCI-H69 9.3
Cerebral Cortex 52.1 Lung ca. (s.cell var.) SHP-77 0.4 Spinal cord
66.0 Lung ca. (large cell)NCI-H460 3.0 glio/astro U87-MG 0.5 Lung
ca. (non-sm. cell) A549 0.2 glio/astro U-118-MG 0.3 Lung ca.
(non-s.cell) NCI-H23 1.6 astrocytoma SW1783 0.0 Lung ca.
(non-s.cell) HOP-62 0.8 neuro*; met SK-N-AS 3.2 Lung ca. (non-s.cl)
NCI-H522 0.0 astrocytoma SF-539 0.4 Lung ca. (squam.) SW 900 0.8
astrocytoma SNB-75 1.0 Lung ca. (squam.) NCI-H596 19.3 glioma
SNB-19 15.1 Mammary gland 6.5 glioma U251 1.0 Breast ca.* (pl.ef)
MCF-7 2.5 glioma SF-295 0.2 Breast ca.* (pl.ef) MDA-MB-231 0.1
Heart (fetal) 5.8 Breast ca.* (pl.ef) T47D 16.4 Heart 4.5 Breast
ca. BT-549 0.4 Skeletal muscle (fetal) 100.0 Breast ca. MDA-N 0.0
Skeletal muscle 5.5 Ovary 48.0 Bone marrow 0.7 Ovarian ca. OVCAR-3
1.8 Thymus 14.2 Ovarian ca. OVCAR-4 0.0 Spleen 5.9 Ovarian ca.
OVCAR-5 7.0 Lymph node 1.2 Ovarian ca. OVCAR-8 0.7 Colorectal 31.9
Ovarian ca. IGROV-1 11.7 Stomach 1.3 Ovarian ca.* (ascites) SK-OV-3
1.9 Small intestine 9.3 Uterus 5.2 Colon ca. SW480 0.4 Placenta
30.6 Colon ca.* SW620(SW480 met) 0.7 Prostate 2.8 Colon ca. HT29
0.2 Prostate ca.* (bone met)PC-3 0.1 Colon ca. HCT-116 1.5 Testis
5.6 Colon ca. CaCo-2 32.1 Melanoma Hs688(A).T 0.2 Colon ca.
tissue(ODO3866) 5.7 Melanoma* (met) Hs688(B).T 0.1 Colon ca.
HCC-2998 1.8 Melanoma UACC-62 1.7 Gastric ca.* (liver met) NCI-N87
0.1 Melanoma M14 3.4 Bladder 14.1 Melanoma LOX IMVI 0.2 Trachea 5.7
Melanoma* (met) SK-MEL-5 15.4 Kidney 6.1 Adipose 6.6 Colun A - Rel.
Exp.(%) Ag2772, Run 164024167
[0611]
70TABLE BI Panel 2D Tissue Name A B Tissue Name A B Normal Colon
46.7 28.9 Kidney Margin 8120608 2.1 3.1 CC Well to Mod Diff
(ODO3866) 2.2 0.8 Kidney Cancer 8120613 0.8 2.0 CC Margin (ODO3866)
9.7 9.7 Kidney Margin 8120614 2.8 1.3 CC Gr.2 rectosigmoid
(ODO3868) 2.2 3.0 Kidney Cancer 9010320 8.9 10.3 CC Margin
(ODO3868) 2.7 2.1 Kidney Margin 9010321 11.0 10.0 CC Mod Diff
(ODO3920) 2.2 1.3 Normal Uterus 1.7 2.0 CC Margin (ODO3920) 13.1
11.5 Uterus Cancer 064011 5.9 5.2 CC Gr.2 ascend colon (ODO3921)
16.5 7.8 Normal Thyroid 3.4 6.2 CC Margin (ODO3921) 10.2 5.8
Thyroid Cancer 064010 1.1 2.3 CC from Partial Hepatectomy 6.7 23.7
Thyroid Cancer A302152 1.1 1.3 (ODO4309) Mets Liver Margin
(ODO4309) 9.5 2.7 Thyroid Margin A302153 5.0 4.4 Colon mets to lung
(OD04451-01) 4.5 2.6 Normal Breast 12.7 19.2 Lung Margin
(OD04451-02) 4.8 4.7 Breast Cancer (OD04566) 1.3 0.8 Normal
Prostate 6546-1 27.5 6.8 Breast Cancer (OD04590-01) 3.8 2.3
Prostate Cancer (OD04410) 17.4 14.2 Breast Cancer Mets 5.1 4.8
(OD04590-03) Prostate Margin (OD04410) 10.2 6.7 Breast Cancer
Metastasis 22.8 24.1 (OD04655-05) Prostate Cancer (OD04720-01) 6.7
2.8 Breast Cancer 064006 3.0 2.3 Prostate Margin (OD04720-02) 12.8
8.4 Breast Cancer 1024 8.2 4.4 Normal Lung 061010 22.8 15.7 Breast
Cancer 9100266 6.1 5.5 Lung Met to Muscle (ODO4286) 0.6 0.8 Breast
Margin 9100265 6.9 5.4 Muscle Margin (ODO4286) 1.5 1.6 Breast
Cancer A209073 9.0 3.9 Lung Malignant Cancer (OD03126) 3.5 4.8
Breast Margin A209073 9.7 11.4 Lung Margin (OD03126) 36.3 17.7
Normal Liver 3.3 2.6 Lung Cancer (OD04404) 3.3 3.1 Liver Cancer
064003 1.4 0.6 Lung Margin (OD04404) 6.8 8.7 Liver Cancer 1025 3.2
3.4 Lung Cancer (OD04565) 1.2 1.6 Liver Cancer 1026 1.7 1.7 Lung
Margin (OD04565) 6.8 6.3 Liver Cancer 6004-T 3.9 5.0 Lung Cancer
(OD04237-01) 4.2 4.2 Liver Tissue 6004-N 1.5 0.7 Lung Margin
(OD04237-02) 10.8 9.9 Liver Cancer 6005-T 1.6 1.7 Ocular Mel Met to
Liver (ODO4310) 100.0 100.0 Liver Tissue 6005-N 1.6 1.3 Liver
Margin (ODO4310) 5.4 5.8 Normal Bladder 8.2 6.6 Melanoma Mets to
Lung (OD04321) 65.5 55.5 Bladder Cancer 1023 0.5 0.6 Lung Margin
(OD04321) 28.9 26.6 Bladder Cancer A302173 6.7 4.4 Normal Kidney
18.4 18.4 Bladder Cancer (OD04718-01) 0.5 0.2 Kidney Ca, Nuclear
grade 2 27.7 17.3 Bladder Normal Adjacent 5.0 3.7 (OD04338)
(OD04718-03) Kidney Margin (OD04338) 5.9 6.1 Normal Ovary 5.6 3.4
Kidney Ca Nuclear grade 1/2 12.4 9.3 Ovarian Cancer 064008 9.3 6.7
(OD04339) Kidney Margin (OD04339) 10.7 12.6 Ovarian Cancer
(OD04768-07) 1.7 0.6 Kidney Ca, Clear cell type (OD04340) 58.2 44.4
Ovary Margin (OD04768-08) 2.8 3.4 Kidney Margin (OD04340) 10.7 15.4
Normal Stomach 12.9 7.0 Kidney Ca, Nuclear grade 3 1.5 1.6 Gastric
Cancer 9060358 1.7 2.2 (OD04348) Kidney Margin (OD04348) 6.7 9.5
Stomach Margin 9060359 2.7 1.2 Kidney Cancer (OD04622-01) 12.5 21.3
Gastric Cancer 9060395 3.0 2.8 Kidney Margin (OD04622-03) 2.0 1.9
Stomach Margin 9060394 4.3 2.3 Kidney Cancer (OD04450-01) 34.2 35.4
Gastric Cancer 9060397 27.9 11.0 Kidney Margin (OD04450-03) 5.8 7.2
Stomach Margin 9060396 1.3 0.4 Kidney Cancer 8120607 6.8 5.1
Gastric Cancer 064005 6.7 2.0 Clumn A - Rel. Exp.(%) Ag2772, Run
162440317 Column B - Rel. Exp.(%) Ag88, Run 144771649
[0612]
71TABLE BJ Panel 3D Tissue Name A Tissue Name A
Daoy-Medulloblastoma 1.0 Ca Ski-Cervical epidermoid carcinoma 0.0
(metastasis) TE671-Medulloblastoma 37.4 ES-2-Ovarian clear cell
carcinoma 0.0 D283 Med-Medulloblastoma 1.3 Ramos-Stimulated with
PMA/ionomycin 6 h 0.0 PFSK-1-Primitive Neuroectodermal 17.8
Ramos-Stimulated with PMA/ionomycin 14 h 0.0 XF-498-CNS 4.0
MEG-01-Chronic myelogenous leukemia 4.9 (megokaryoblast)
SNB-78-Glioma 0.0 Raji-Burkitt's lymphoma 0.0 SF-268-Glioblastoma
0.0 Daudi-Burkitt's lymphoma 0.3 T98G-Glioblastoma 1.0 U266-B-cell
plasmacytoma 0.0 SK-N-SH-Neuroblastoma (metastasis) 7.2
CA46-Burkitt's lymphoma 0.0 SF-295-Glioblastoma 1.0
RL-non-Hodgkin's B-cell lymphoma 0.0 Cerebellum 58.2 JM1-pre-B-cell
lymphoma 0.0 Cerebellum 44.8 Jurkat-T cell leukemia 0.0
NCI-H292-Mucoepidermoid lung 0.0 TF-1-Erythroleukemia 2.2 carcinoma
DMS-114-Small cell lung cancer 0.3 HUT 78-T-cell lymphoma 0.1
DMS-79-Small cell lung cancer 49.0 U937-Histiocytic lymphoma 0.7
NCI-H146-Small cell lung cancer 18.0 KU-812-Myelogenous leukemia
0.6 NCI-H526-Small cell lung cancer 0.2 769-P-Clear cell renal
carcinoma 100.0 NCI-N417-Small cell lung cancer 50.0 Caki-2-Clear
cell renal carcinoma 5.9 NCI-H82-Small cell lung cancer 9.3 SW
839-Clear cell renal carcinoma 79.6 NCI-H157-Squamous cell lung
cancer 0.0 Rhabdoid kidney tumor 0.0 (metastasis) NCI-H1155-Large
cell lung cancer 0.4 Hs766T-Pancreatic carcinoma (LN 0.0
metastasis) NCI-H1299-Large cell lung cancer 5.7 CAPAN-1-Pancreatic
adenocarcinoma (liver 0.0 metastasis) NCI-H727-Lung carcinoid 7.4
SU86.86-Pancreatic carcinoma (liver 1.3 metastasis) NCI-UMC-11-Lung
carcinoid 28.1 BxPC-3-Pancreatic adenocarcinoma 0.0 LX-1-Small cell
lung cancer 0.3 HPAC-Pancreatic adenocarcinoma 0.1 Colo-205-Colon
cancer 6.4 MIA PaCa-2-Pancreatic carcinoma 0.0 KM12-Colon cancer
2.9 CFPAC-1-Pancreatic ductal adenocarcinoma 0.7 KM20L2-Colon
cancer 0.0 PANC-1-Pancreatic epithelioid ductal 0.2 carcinoma
NCI-H716-Colon cancer 33.9 T24-Bladder carcinma (transitional cell)
0.0 SW-48-Colon adenocarcinoma 2.8 5637-Bladder carcinoma 1.3
SW1116-Colon adenocarcinoma 0.0 HT-1197-Bladder carcinoma 3.0 LS
174T-Colon adenocarcinoma 0.6 UM-UC-3-Bladder carcinma
(transitional 0.0 cell) SW-948-Colon adenocarcinoma 0.3
A204-Rhabdomyosarcoma 0.0 SW-480-Colon adenocarcinoma 0.0
HT-1080-Fibrosarcoma 0.7 NCI-SNU-5-Gastric carcinoma 0.3
MG-63-Osteosarcoma 0.0 KATO III-Gastric carcinoma 0.0
SK-LMS-1-Leiomyosarcoma (vulva) 0.0 NCI-SNU-16-Gastric carcinoma
1.0 SJRH30-Rhabdomyosarcoma (met to bone 50.7 marrow)
NCI-SNU-1-Gastric carcinoma 14.7 A431-Epidermoid carcinoma 0.0
RF-1-Gastric adenocarcinoma 2.8 WM266-4-Melanoma 2.5 RF-48-Gastric
adenocarcinoma 2.4 DU 145-Prostate carcinoma (brain metastasis) 0.0
MKN-45-Gastric carcinoma 0.3 MDA-MB-468-Breast adenocarcinoma 0.0
NCI-N87-Gastric carcinoma 0.0 SCC-4-Squamous cell carcinoma of
tongue 0.0 OVCAR-5-Ovarian carcinoma 0.0 SCC-9-Squamous cell
carcinoma of tongue 0.0 RL95-2-Uterine carcinoma 0.0
SCC-15-Squamous cell carcinoma of tongue 0.0 HelaS3-Cervical
adenocarcinoma 0.0 CAL 27-Squamous cell carcinoma of tongue 0.0
Clumn A - Rel. Exp.(%) Ag88, Run 153109696
[0613]
72TABLE BK Panel 4D Tissue Name A B Tissue Name A B Secondary Th1
act 0.8 0.0 HUVEC IL-1beta 0.5 1.1 Secondary Th2 act 1.5 0.4 HUVEC
IFN gamma 2.5 4.1 Secondary Tr1 act 2.8 0.0 HUVEC TNF alpha + IFN
gamma 1.1 0.0 Secondary Th1 rest 0.7 0.0 HUVEC TNF alpha + IL4 2.0
2.4 Secondary Th2 rest 1.7 0.0 HUVEC IL-11 3.0 3.0 Secondary Tr1
rest 1.0 0.0 Lung Microvascular EC none 1.8 0.9 Primary Th1 act 3.2
0.0 Lung Microvascular EC TNF alpha + IL-1beta 1.5 0.3 Primary Th2
act 2.6 0.0 Microvascular Dermal EC none 3.9 3.3 Primary Tr1 act
5.0 0.0 Microsvasular Dermal EC TNF alpha + IL-1beta 2.5 2.4
Primary Th1 rest 7.1 0.0 Bronchial epithelium TNF alpha + IL1beta
18.3 22.5 Primary Th2 rest 2.8 0.0 Small airway epithelium none 1.2
5.1 Primary Tr1 rest 2.5 0.0 Small airway epithelium TNF alpha +
IL-1beta 24.0 23.3 CD45RA CD4 lymphocyte act 1.4 0.6 Coronery
artery SMC rest 1.1 1.8 CD45RO CD4 lymphocyte act 1.6 0.3 Coronery
artery SMC TNF alpha + IL-1beta 0.6 0.9 CD8 lymphocyte act 2.0 0.0
Astrocytes rest 19.8 14.1 Secondary CD8 lymphocyte rest 2.7 0.0
Astrocytes TNF alpha + IL-1beta 3.4 4.4 Secondary CD8 lymphocyte
act 0.3 0.2 KU-812 (Basophil) rest 2.4 1.2 CD4 lymphocyte none 0.7
0.0 KU-812 (Basophil) PMA/ionomycin 11.3 11.3 2ry
Th1/Th2/Tr1_anti-CD95 1.5 0.0 CCD1106 (Keratinocytes) none 0.1 0.2
CH11 LAK cells rest 2.6 0.4 CCD1106 (Keratinocytes) TNF alpha +
IL-1beta 0.0 1.3 LAK cells IL-2 2.6 0.0 Liver cirrhosis 11.9 15.0
LAK cells IL-2 + IL-12 2.5 1.3 Lupus kidney 7.3 23.8 LAK cells IL-2
+ IFN gamma 5.6 0.8 NCI-H292 none 1.0 0.0 LAK cells IL-2 + IL-18
4.2 0.9 NCI-H292 IL-4 2.0 0.0 LAK cells PMA/ionomycin 1.8 0.0
NCI-H292 IL-9 1.9 0.0 NK Cells IL-2 rest 2.4 0.0 NCI-H292 IL-13 1.1
0.2 Two Way MLR 3 day 2.7 0.0 NCI-H292 IFN gamma 1.1 0.0 Two Way
MLR 5 day 1.7 0.0 HPAEC none 1.2 1.7 Two Way MLR 7 day 0.7 0.4
HPAEC TNF alpha + IL-1beta 2.3 1.3 PBMC rest 1.0 0.0 Lung
fibroblast none 0.4 0.4 PBMC PWM 12.9 10.3 Lung fibroblast TNF
alpha + IL-1beta 1.9 1.9 PBMC PHA-L 2.1 1.4 Lung fibroblast IL-4
2.4 0.4 Ramos (B cell) none 1.6 0.0 Lung fibroblast IL-9 0.9 1.9
Ramos (B cell) ionomycin 12.7 0.0 Lung fibroblast IL-13 2.0 1.3 B
lymphocytes PWM 5.8 3.8 Lung fibroblast IFN gamma 0.9 1.7 B
lymphocytes CD40L and IL-4 7.2 0.4 Dermal fibroblast CCD1070 rest
3.8 2.6 EOL-1 dbcAMP 1.3 0.0 Dermal fibroblast CCD1070 TNF alpha
3.1 0.5 EOL-1 dbcAMP PMA/ionomycin 1.9 0.0 Dermal fibroblast
CCD1070 IL-1beta 0.2 0.3 Dendritic cells none 0.9 0.4 Dermal
fibroblast IFN gamma 2.3 0.9 Dendritic cells LPS 1.5 1.4 Dermal
fibroblast IL-4 1.6 1.0 Dendritic cells anti-CD40 2.3 0.3 IBD
Colitis 2 1.0 4.1 Monocytes rest 1.4 0.0 IBD Crohn's 4.9 6.9
Monocytes LPS 1.7 0.7 Colon 100.0 87.1 Macrophages rest 2.9 0.5
Lung 18.4 24.3 Macrophages LPS 0.4 0.0 Thymus 34.4 100.0 HUVEC none
2.0 1.0 Kidney 20.9 34.4 HUVEC starved 4.8 0.8 Coumn A - Rel.
Exp.(%) Ag2772, Run 161924079 Column B - Rel. Exp.(%) Ag88, Run
139410561
[0614]
73TABLE BL general oncology screening panel_v_2.4 Tissue Name A B
Tissue Name A B Colon cancer 1 5.0 14.4 Bladder NAT 2 0.0 0.3 Colon
NAT 1 7.0 9.2 Bladder NAT 3 0.0 0.1 Colon cancer 2 0.0 1.2 Bladder
NAT 4 6.5 1.6 Colon NAT 2 18.3 12.3 Prostate adenocarcinoma 1 0.0
7.7 Colon cancer 3 15.9 11.3 Prostate adenocarcinoma 2 0.0 1.0
Colon NAT 3 33.9 25.0 Prostate adenocarcinoma 3 0.0 4.6 Colon
malignant cancer 4 0.0 7.7 Prostate adenocarcinoma 4 0.0 2.1 Colon
NAT 4 13.9 9.3 Prostate NAT 5 7.5 0.5 Lung cancer 1 8.0 0.9
Prostate adenocarcinoma 6 0.0 2.7 Lung NAT 1 12.5 1.2 Prostate
adenocarcinoma 7 0.0 2.8 Lung cancer 2 100.0 100.0 Prostate
adenocarcinoma 8 0.0 0.6 Lung NAT 2 31.2 4.3 Prostate
adenocarcinoma 9 0.0 6.4 Squamous cell carcinoma 3 20.0 2.5
Prostate NAT 10 0.0 0.3 Lung NAT 3 5.0 0.2 Kidney cancer 1 33.4
12.9 Metastatic melanoma 1 0.0 14.9 Kidney NAT 1 0.0 5.8 Melanoma 2
0.0 1.1 Kidney cancer 2 40.6 80.7 Melanoma 3 0.0 1.9 Kidney NAT 2
0.0 4.6 Metastatic melanoma 4 0.0 18.8 Kidney cancer 3 6.3 49.3
Metastatic melanoma 5 7.5 32.1 Kidney NAT 3 0.0 2.4 Bladder cancer
1 0.0 0.6 Kidney cancer 4 6.8 19.1 Bladder NAT 1 0.0 0.0 Kidney NAT
4 0.0 4.3 Bladder cancer 2 0.0 1.0 Column A - Rel. Exp.(%) Ag6309
Run 259804334 Column B - Rel. Exp.(%) Ag88, Run 262228151
[0615] CNS_neurodegeneration_v1.0 Summary: Ag2772/Ag6309 This panel
confirms the expression of this gene at low levels in the brains of
an independent group of individuals.
[0616] General_screening_panel_v1.5 Summary: Ag6309 Highest
expression of this gene is detected in spinal cord (CT=29.4).
Moderate expression of this gene is mainly seen in all the region
of central nervous system examined, including amygdala,
hippocampus, substantia nigra, thalamus, cerebellum, cerebral
cortex, and spinal cord. This gene codes for semaphorin 6A protein
(Sema6A). Sema6A is shown to be expressed in thalamocortical
neurons and required for their axons to project properly (Leighton
Pa., Mitchell K J, Goodrich L V, Lu X, Pinson K, Scherz P, Skarnes
W C, Tessier-Lavigne M. 2001, Nature 410(6825):174-9). Therefore,
therapeutic modulation of this gene product may be useful in the
treatment of central nervous system disorders such as Alzheimer's
disease, Parkinson's disease, epilepsy, multiple sclerosis,
schizophrenia and depression.
[0617] Low expression of this gene is also seen in number of cancer
cell lines derived from brain, ovarian, melanoma and a renal
cancer. Therefore, therapeutic modulation of the expression of this
gene or Sema6A protien encoded by this gene through the use of
small molecules or antibodies may be useful in the treatment of
these cancer, especially in inhibiting migration of these cancer
cell lines.
[0618] HASS Panel v1.0 Summary: Ag2772 Highest expression of this
gene is seen in a brain cancer (487 medullo) sample (CT=27.3). High
to moderate expression of this gene is seen in medulloblastoma and
glioma brain cancer samples and prostate cancer (LnCAP) cell line.
Expression of this gene is downregulated in LnCAP cells under
acidic plus hypoxic environment. In addition, low expression of
this gene is also seen in MCF7 cells. Therefore, therapeutic
modulation of this gene or its protein product may be useful in the
treatment of brain, prostate and breast cancers.
[0619] Panel 1 Summary: Ag88 Highest expression of this gene is
seen in cerebellum (CT=24.5). High expression of this gene is
mainly seen in all the regions of central nervous system examined.
Please see panel 1.5 for further discussion of this gene.
[0620] High to moderate expression of this gene is also seen in
tissues with metabolic/endocrine functions including, pancreas,
thyroid, adrenal gland, pituitary gland, skeletal muscle, heart,
liver and the gastrointestinal tract. Therefore, therapeutic
modulation of the activity of this gene may prove useful in the
treatment of endocrine/metabolically related diseases, such as
obesity and diabetes.
[0621] High to moderate expression of this gene is also seen in
number of cancer cell lines derived from melanoma, ovarian, renal,
colon, liver and brain cancers. Therefore therapeutic modulation of
this gene or its protein product may be useful in the treatment of
these cancers.
[0622] Panel 1.3D Summary: Ag2772 Highest expression of this gene
is seen in fetal skeletal muscle (CT=27.4). Interestingly, this
gene is expressed at much higher levels in fetal (CT=27.4) when
compared to adult skeletal muscle (CT=31.5). This observation
suggests that expression of this gene can be used to distinguish
fetal from adult skeletal muscle. In addition, the relative
overexpression of this gene in fetal tissue suggests that the
protein product may enhance muscle growth or development in the
fetus and thus may also act in a regenerative capacity in the
adult. Therefore, therapeutic modulation of the protein encoded by
this gene could be useful in treatment of muscle related
diseases.
[0623] Some expression pattern correlates with (ex: cancer cell
lines) that seen in panel 1.
[0624] Panel 2D Summary: Ag2772/Ag88 Two experiments with different
probe primer sets are in excellent agreement, with highest
expression of this gene seen in a liver cancer (ODO4310) sample
(CTs=25-28). This gene shows a widespread expression in this panel,
with high to moderate expression in normal and cancer samples from
stomach, ovary, bladder, colon, liver, lung, metastatic melanoma,
kidney, uterus, thyroid and breast. Interestingly, expression of
this gene is upregulated in metastatic melanoma, gastric, liver and
kidney cancers. Therefore, expression of this gene may be used as
marker to detect the presence of metastatic melanoma, gastric,
liver and kidney cancers, furthermore, therapeutic modulation of
this gene or its protein product may be useful in the treatment of
these cancers.
[0625] Panel 3D Summary: Ag88 Highest expression of this gene is
detected in a renal cancer cell line (CT=30). Moderate expression
of this gene is also seen number of cancer cell lines derived from
brain, lung, colon, gastric, renal and bone cancers. Therefore,
therapeutic modulation of this gene or its protein product may be
useful in the treatment of these cancers.
[0626] Panel 4D Summary: Ag2772/Ag88 Two experiments with different
probe-primer sets are in good agreement with highest expression of
this gene seen in colon and thymus (CTs 27-30). This gene shows
moderate to low expression in most of samples in this panel.
Expression of this gene is upregulated in activated bronchial and
small airway epithelium, basophils, liver cirrhosis and lupus
kidney. Therefore therapeutic modulation of this gene or its
protein product may be useful in the treatment of asthma,
allergies, chronic obstructive pulmonary disease, Crohn's disease,
ulcerative colitis, liver cirrhosis and lupus erythematosus.
[0627] General oncology screening panel_v.sub.--2.4 Summary:
Ag6309/Ag88 Highest expression of this gene is seen in lung cancer
sample (CTs=27-34.7). Moderate to low expression of this gene is
seen in normal and cancer samples from lung, colon, metastatic
melanoms, prostate, and kidney. Expression of this gene is
upregulated in kidney, metastatic melanoma and lung cancers, which
is in agreement with expression seen in panel 2D. Please see panel
2D for further discussion of this gene.
[0628] C. CG52324-01: Phosphatidylethanolamine-Binding Protein.
[0629] Expression of gene CG52324-01 was assessed using the
primer-probe sets Ag2678 and Ag36, described in Tables CA and CB.
Results of the RTQ-PCR runs are shown in Tables CC, CD, CE, CF, CG,
CH and CI.
74TABLE CA Probe Name Ag2678 Start SEQ ID Primers Sequences Length
Position No Forward 5'-cgcaacctatatcctggtgat-3' 21 373 150 Probe
TET-5'-atccagatgcccctagcagagcagaa-3'-TAMRA 26 399 151 Reverse
5'-agccaatgtctccagaatctct-3' 22 432 152
[0630]
75TABLE CB Probe Name Ag36 Start SEQ ID Primers Sequences Length
Position No Forward 5'-caggtggaaacggttcagaaa-3' 21 650 153 Probe
TET-5'-ctgtccattttccaagagcctcgagttttgt-3'-TAMRA 31 618 154 Reverse
5'-catctctctccttcccaaggaa-3' 22 595 155
[0631]
76TABLE CC CNS_neurodegeneration_v1.0 Tissue Name A B C Tissue Name
A B C AD 1 Hippo 25.0 20.4 21.9 Control (Path) 3 Temporal Ctx 9.3
5.4 3.9 AD 2 Hippo 45.7 37.4 25.9 Control (Path) 4 Temporal Ctx
27.4 20.4 33.9 AD 3 Hippo 7.4 3.1 6.4 AD 1 Occipital Ctx 12.3 14.0
12.1 AD 4 Hippo 12.9 15.3 19.5 AD 2 Occipital Ctx (Missing) 0.0 0.0
0.0 AD 5 hippo 68.3 66.9 73.7 AD 3 Occipital Ctx 10.2 4.2 2.9 AD 6
Hippo 94.6 100.0 100.0 AD 4 Occipital Ctx 28.3 35.8 21.2 Control 2
Hippo 68.3 51.1 62.0 AD 5 Occipital Ctx 22.4 49.7 55.5 Control 4
Hippo 50.7 40.6 40.1 AD 6 Occipital Ctx 58.6 39.5 39.2 Control
(Path) 3 Hippo 12.8 4.2 8.3 Control 1 Occipital Ctx 2.6 3.7 6.1 AD
1 Temporal Ctx 23.8 23.3 13.2 Control 2 Occipital Ctx 64.6 73.7
45.1 AD 2 Temporal Ctx 42.6 23.8 29.5 Control 3 Occipital Ctx 29.3
17.3 0.0 AD 3 Temporal Ctx 5.6 3.7 4.3 Control 4 Occipital Ctx 22.4
14.0 15.9 AD 4 Temporal Ctx 23.3 21.9 28.1 Control (Path) 1
Occipital Ctx 84.7 64.2 97.9 AD 5 Inf Temporal Ctx 87.1 81.8 57.8
Control (Path) 2 Occipital Ctx 15.1 14.8 20.2 AD 5 Sup Temporal Ctx
100.0 80.7 90.8 Control (Path) 3 Occipital Ctx 0.7 2.4 2.8 AD 6 Inf
Temporal Ctx 57.0 52.5 55.5 Control (Path) 4 Occipital Ctx 8.2 8.3
16.8 AD 6 Sup Temporal Ctx 55.1 43.8 58.2 Control 1 Parietal Ctx
13.4 6.8 6.4 Control 1 Temporal Ctx 4.2 5.0 7.2 Control 2 Parietal
Ctx 60.7 48.0 32.8 Control 2 Temporal Ctx 44.1 47.6 34.4 Control 3
Parietal Ctx 27.9 19.8 36.1 Control 3 Temporal Ctx 32.1 33.7 43.2
Control (Path) 1 Parietal Ctx 99.3 71.2 65.1 Control 4 Temporal Ctx
16.3 20.7 12.9 Control (Path) 2 Parietal Ctx 37.1 36.1 32.3 Control
(Path) 1 Temporal Ctx 49.0 45.4 51.8 Control (Path) 3 Parietal Ctx
6.6 6.5 2.8 Control (Path) 2 Temporal Ctx 35.1 34.9 52.9 Control
(Path) 4 Parietal Ctx 28.1 26.4 42.3 Column A - Rel. Exp.(% Ag2678,
Run 206231405 Column B - Rel. Exp.(%) Ag2678, Run 206271178 Column
C - Rel. Exp.(%) Ag36, Run 206943639
[0632]
77TABLE CD Panel 1 Tissue Name A Tissue Name A Endothelial cells
0.0 Renal ca. 786-0 0.0 Endothelial cells (treated) 0.0 Renal ca.
A498 0.0 Pancreas 2.0 Renal ca. RXF 393 0.0 Pancreatic ca. CAPAN 2
0.0 Renal ca. ACHN 0.0 Adrenal gland 0.9 Renal ca. UO-31 0.0
Thyroid 21.2 Renal ca. TK-10 0.0 Salivary gland 7.6 Liver 0.1
Pituitary gland 0.1 Liver (fetal) 0.0 Brain (fetal) 0.2 Liver ca.
(hepatoblast) HepG2 0.0 Brain (whole) 2.0 Lung 6.2 Brain (amygdala)
1.0 Lung (fetal) 7.1 Brain (cerebellum) 4.6 Lung ca. (small cell)
LX-1 0.0 Brain (hippocampus) 3.0 Lung ca. (small cell) NCI-H69 0.0
Brain (substantia nigra) 6.3 Lung ca. (s.cell var.) SHP-77 0.0
Brain (thalamus) 4.8 Lung ca. (large cell) NCI-H460 0.0 Brain
(hypothalamus) 10.5 Lung ca. (non-sm. cell) A549 0.0 Spinal cord
1.8 Lung ca. (non-s.cell) NCI-H23 0.0 glio/astro U87-MG 0.0 Lung
ca. (non-s.cell) HOP-62 0.0 glio/astro U-118-MG 0.0 Lung ca.
(non-s.cl) NCI-H522 0.0 astrocytoma SW1783 0.0 Lung ca. (squam.) SW
900 0.0 neuro*; met SK-N-AS 0.0 Lung ca. (squam.) NCI-H596 0.1
astrocytoma SF-539 0.1 Mammary gland 0.5 astrocytoma SNB-75 0.0
Breast ca.* (pl.ef) MCF-7 0.0 glioma SNB-19 0.0 Breast ca.* (pl.ef)
MDA-MB-231 0.0 glioma U251 0.0 Breast ca.* (pl.ef) T47D 0.0 glioma
SF-295 0.0 Breast ca. BT-549 0.0 Heart 33.9 Breast ca. MDA-N 0.0
Skeletal muscle 100.0 Ovary 0.1 Bone marrow 1.0 Ovarian ca. OVCAR-3
0.0 Thymus 0.3 Ovarian ca. OVCAR-4 0.0 Spleen 0.1 Ovarian ca.
OVCAR-5 0.0 Lymph node 0.3 Ovarian ca. OVCAR-8 0.0 Colon
(ascending) 0.0 Ovarian ca. IGROV-1 0.0 Stomach 0.6 Ovarian ca.
(ascites) SK-OV-3 0.0 Small intestine 0.1 Uterus 0.5 Colon ca.
SW480 0.0 Placenta 0.2 Colon ca.* SW620 (SW480 met) 0.0 Prostate
15.0 Colon ca. HT29 0.0 Prostate ca.* (bone met) PC-3 0.0 Colon ca.
HCT-116 0.0 Testis 8.7 Colon ca. CaCo-2 0.0 Melanoma Hs688(A).T 0.0
Colon ca. HCT-15 0.0 Melanoma* (met) Hs688(B).T 0.0 Colon ca.
HCC-2998 0.0 Melanoma UACC-62 0.0 Gastric ca. * (liver met) NCI-N87
0.0 Melanoma M14 0.0 Bladder 0.2 Melanoma LOX IMVI 0.0 Trachea 1.2
Melanoma* (met) SK-MEL-5 0.0 Kidney 0.4 Melanoma SK-MEL-28 0.0
Kidney (fetal) 0.3 Column A - Rel. Exp.(%) Ag36, Run 87587974
[0633]
78TABLE CE Panel 1.3D Tissue Name A B Tissue Name A B Liver
adenocarcinoma 0.0 0.0 Kidney (fetal) 0.1 0.0 Pancreas 0.8 0.2
Renal ca. 786-0 0.0 0.0 Pancreatic ca. CAPAN 2 0.0 0.0 Renal ca.
A498 0.0 0.0 Adrenal gland 0.2 0.2 Renal ca. RXF 393 0.0 0.0
Thyroid 22.7 6.8 Renal ca. ACHN 0.0 0.0 Salivary gland 3.9 1.0
Renal ca. UO-31 0.0 0.0 Pituitary gland 3.6 0.5 Renal ca. TK-10 0.0
0.0 Brain (fetal) 0.1 0.1 Liver 1.0 0.2 Brain (whole) 1.4 0.5 Liver
(fetal) 0.0 0.0 Brain (amygdala) 1.0 0.5 Liver ca. (hepatoblast)
HepG2 0.0 0.0 Brain (cerebellum) 0.9 0.6 Lung 59.9 12.8 Brain
(hippocampus) 7.9 0.9 Lung (fetal) 11.0 5.8 Brain (substantia
nigra) 3.8 1.4 Lung ca. (small cell) LX-1 0.1 0.0 Brain (thalamus)
6.0 2.2 Lung ca. (small cell) NCI-H69 0.0 0.0 Cerebral Cortex 7.2
6.2 Lung ca. (s. cell var.) SHP-77 0.0 0.0 Spinal cord 1.1 1.7 Lung
ca. (large cell)NCI-H460 0.0 0.0 glio/astro U87-MG 0.0 0.0 Lung ca.
(non-sm. cell) A549 0.0 0.0 glio/astro U-118-MG 0.1 0.0 Lung ca.
(non-s. cell) NCI-H23 0.0 0.0 astrocytoma SW1783 0.0 0.0 Lung ca.
(non-s. cell) HOP-62 0.0 0.0 neuro*; met SK-N-AS 0.0 0.0 Lung ca.
(non-s. cl) NCI-H522 0.0 0.0 astrocytoma SF-539 0.2 0.1 Lung ca.
(squam.) SW 900 0.0 0.0 astrocytoma SNB-75 0.1 0.0 Lung ca.
(squam.) NCI-H596 0.0 0.0 glioma SNB-19 0.0 0.0 Mammary gland 1.0
0.3 glioma U251 0.0 0.0 Breast ca.* (pl. ef) MCF-7 0.0 0.0 glioma
SF-295 0.0 0.0 Breast ca.* (pl. ef) MDA-MB-231 0.0 0.0 Heart
(fetal) 8.1 7.4 Breast ca.* (pl. ef) T47D 0.0 0.0 Heart 21.2 27.7
Breast ca. BT-549 0.1 0.0 Skeletal muscle (fetal) 100.0 85.3 Breast
ca. MDA-N 0.0 0.0 Skeletal muscle 51.4 100.0 Ovary 0.8 0.6 Bone
marrow 0.5 0.1 Ovarian ca. OVCAR-3 0.0 0.0 Thymus 0.2 0.3 Ovarian
ca. OVCAR-4 0.0 0.0 Spleen 0.2 0.0 Ovarian ca. OVCAR-5 0.1 0.0
Lymph node 0.3 0.0 Ovarian ca. OVCAR-8 0.0 0.1 Colorectal 0.2 0.1
Ovarian ca. IGROV-1 0.0 0.0 Stomach 1.5 0.3 Ovarian ca.* (ascites)
SK-OV-3 0.0 0.0 Small intestine 0.1 0.0 Uterus 0.0 0.0 Colon ca.
SW480 0.0 0.0 Placenta 0.4 0.0 Colon ca.* SW620(SW480 met) 0.0 0.0
Prostate 8.2 4.6 Colon ca. HT29 0.0 0.0 Prostate ca.* (bone
met)PC-3 0.0 0.0 Colon ca. HCT-116 0.0 0.0 Testis 6.1 2.0 Colon ca.
CaCo-2 0.0 0.0 Melanoma Hs688(A).T 0.0 0.0 Colon ca.
tissue(ODO3866) 0.1 0.0 Melanoma* (met) Hs688(B).T 0.0 0.0 Colon
ca. HCC-2998 0.0 0.0 Melanoma UACC-62 0.0 0.0 Gastric ca.* (liver
met) NCI-N87 0.0 0.0 Melanoma M14 0.0 0.0 Bladder 0.0 0.0 Melanoma
LOX IMVI 0.0 0.0 Trachea 4.8 1.7 Melanoma* (met) SK-MEL-5 0.0 0.0
Kidney 0.2 0.2 Adipose 1.2 0.6 Colun A - Rel. Exp.(%) Ag2678, Run
158535212 Column B - Rel. Exp.(%) Ag36, Run 161682677
[0634]
79TABLE CF Panel 2D Tissue Name A B Tissue Name A B Normal Colon
6.5 5.3 Kidney Margin 8120608 0.3 0.4 CC Well to Mod Diff (ODO3866)
0.1 0.0 Kidney Cancer 8120613 0.1 0.0 CC Margin (ODO3866) 0.1 0.1
Kidney Margin 8120614 1.5 1.4 CC Gr.2 rectosigmoid (ODO3868) 0.0
0.0 Kidney Cancer 9010320 0.1 0.1 CC Margin (ODO3868) 0.1 0.1
Kidney Margin 9010321 1.1 0.8 CC Mod Diff (ODO3920) 0.0 0.0 Normal
Uterus 0.2 0.1 CC Margin (ODO3920) 0.0 0.1 Uterus Cancer 064011 0.3
0.4 CC Gr.2 ascend colon (ODO3921) 0.0 0.0 Normal Thyroid 22.1 23.5
CC Margin (ODO3921) 0.0 0.0 Thyroid Cancer 064010 6.6 10.3 CC from
Partial Hepatectomy 0.1 0.2 Thyroid Cancer A302152 14.1 20.9
(ODO4309) Mets Liver Margin (ODO4309) 0.5 0.3 Thyroid Margin
A302153 24.3 18.6 Colon mets to lung (OD04451-01) 10.4 14.0 Normal
Breast 1.2 0.3 Lung Margin (OD04451-02) 36.6 50.0 Breast Cancer
(OD04566) 0.0 0.1 Normal Prostate 6546-1 13.1 13.6 Breast Cancer
(OD04590-01) 3.1 2.5 Prostate Cancer (OD04410) 4.9 6.5 Breast
Cancer Mets 0.3 0.3 (OD04590-03) Prostate Margin (OD04410) 13.6
26.8 Breast Cancer Metastasis 0.3 0.2 (OD04655-05) Prostate Cancer
(OD04720-01) 11.4 18.9 Breast Cancer 064006 0.2 0.3 Prostate Margin
(OD04720-02) 30.4 38.7 Breast Cancer 1024 1.4 0.8 Normal Lung
061010 35.8 46.0 Breast Cancer 9100266 0.2 0.1 Lung Met to Muscle
(ODO4286) 0.0 0.0 Breast Margin 9100265 0.7 0.4 Muscle Margin
(ODO4286) 54.3 55.5 Breast Cancer A209073 0.3 0.4 Lung Malignant
Cancer (OD03126) 11.7 14.9 Breast Margin A209073 0.7 0.9 Lung
Margin (OD03126) 92.7 100.0 Normal Liver 0.0 0.0 Lung Cancer
(OD04404) 7.7 12.5 Liver Cancer 064003 0.0 0.0 Lung Margin
(OD04404) 22.5 29.5 Liver Cancer 1025 0.0 0.2 Lung Cancer (OD04565)
0.1 0.1 Liver Cancer 1026 0.1 0.0 Lung Margin (OD04565) 18.9 29.1
Liver Cancer 6004-T 0.1 0.1 Lung Cancer (OD04237-01) 1.1 1.1 Liver
Tissue 6004-N 0.0 0.0 Lung Margin (OD04237-02) 29.9 27.0 Liver
Cancer 6005-T 0.1 0.1 Ocular Mel Met to Liver (ODO4310) 0.0 0.1
Liver Tissue 6005-N 0.1 0.0 Liver Margin (ODO4310) 0.2 0.1 Normal
Bladder 0.1 0.1 Melanoma Mets to Lung (OD04321) 0.5 0.4 Bladder
Cancer 1023 0.1 0.2 Lung Margin (OD04321) 100.0 92.0 Bladder Cancer
A302173 0.0 0.1 Normal Kidney 0.7 0.7 Bladder Cancer (OD04718-01)
0.1 0.1 Kidney Ca, Nuclear grade 2 0.7 0.3 Bladder Normal Adjacent
0.0 0.0 (OD04338) (OD04718-03) Kidney Margin (OD04338) 1.4 0.8
Normal Ovary 0.3 0.1 Kidney Ca Nuclear grade 1/2 0.1 0.0 Ovarian
Cancer 064008 0.4 0.1 (OD04339) Kidney Margin (OD04339) 0.6 1.4
Ovarian Cancer (OD04768-07) 0.0 0.0 Kidney Ca, Clear cell type
(OD04340) 0.3 0.3 Ovary Margin (OD04768-08) 0.1 0.0 Kidney Margin
(OD04340) 1.2 0.7 Normal Stomach 0.5 0.7 Kidney Ca, Nuclear grade 3
0.0 0.2 Gastric Cancer 9060358 0.2 0.3 (OD04348) Kidney Margin
(OD04348) 0.3 0.4 Stomach Margin 9060359 1.2 1.1 Kidney Cancer
(OD04622-01) 0.3 0.1 Gastric Cancer 9060395 0.2 0.2 Kidney Margin
(OD04622-03) 0.0 0.2 Stomach Margin 9060394 0.8 0.8 Kidney Cancer
(OD04450-01) 0.0 0.0 Gastric Cancer 9060397 0.2 0.1 Kidney Margin
(OD04450-03) 1.0 0.6 Stomach Margin 9060396 0.3 0.5 Kidney Cancer
8120607 0.2 0.0 Gastric Cancer 064005 0.0 0.1 Coumn A - Rel.
Exp.(%) Ag2678, Run 158536099 Column B - Rel. Exp.(%) Ag36, Run
161680686
[0635]
80TABLE CG Panel 3D Tissue Name A Tissue Name A
Daoy-Medulloblastoma 4.1 Ca Ski-Cervical epidermoid carcinoma 0.0
(metastasis) TE671-Medulloblastoma 0.0 ES-2-Ovarian clear cell
carcinoma 0.0 D283 Med-Medulloblastoma 0.0 Ramos-Stimulated with
PMA/ionomycin 6 h 0.0 PFSK-1-Primitive Neuroectodermal 2.7
Ramos-Stimulated with PMA/ionomycin 14 h 0.0 XF-498-CNS 0.0
MEG-01-Chronic myelogenous leukemia 0.0 (megokaryoblast)
SNB-78-Glioma 0.0 Raji-Burkitt's lymphoma 0.0 SF-268-Glioblastoma
0.0 Daudi-Burkitt's lymphoma 3.4 T98G-Glioblastoma 0.7 U266-B-cell
plasmacytoma 0.8 SK-N-SH-Neuroblastoma (metastasis) 0.0
CA46-Burkitt's lymphoma 0.7 SF-295-Glioblastoma 1.4
RL-non-Hodgkin's B-cell lymphoma 0.0 Cerebellum 100.0
JM1-pre-B-cell lymphoma 0.0 Cerebellum 98.6 Jurkat-T cell leukemia
0.0 NCI-H292-Mucoepidermoid lung 1.2 TF-1-Erythroleukemia 0.0
carcinoma DMS-114-Small cell lung cancer 0.6 HUT 78-T-cell lymphoma
0.0 DMS-79-Small cell lung cancer 1.4 U937-Histiocytic lymphoma 1.8
NCI-H146-Small cell lung cancer 0.0 KU-812-Myelogenous leukemia 0.0
NCI-H526-Small cell lung cancer 0.0 769-P-Clear cell renal
carcinoma 0.0 NCI-N417-Small cell lung cancer 0.0 Caki-2-Clear cell
renal carcinoma 0.0 NCI-H82-Small cell lung cancer 0.0 SW 839-Clear
cell renal carcinoma 0.0 NCI-H157-Squamous cell lung cancer 0.0
Rhabdoid kidney tumor 0.0 (metastasis) NCI-H1155-Large cell lung
cancer 0.0 Hs766T-Pancreatic carcinoma (LN metastasis) 1.3
NCI-H1299-Large cell lung cancer 0.0 CAPAN-1-Pancreatic
adenocarcinoma (liver 0.0 metastasis) NCI-H727-Lung carcinoid 0.6
SU86.86-Pancreatic carcinoma (liver 0.0 metastasis) NCI-UMC-11-Lung
carcinoid 0.0 BxPC-3-Pancreatic adenocarcinoma 0.3 LX-1-Small cell
lung cancer 0.0 HPAC-Pancreatic adenocarcinoma 0.0 Colo-205-Colon
cancer 0.0 MIA PaCa-2-Pancreatic carcinoma 0.0 KM12-Colon cancer
0.8 CFPAC-1-Pancreatic ductal adenocarcinoma 0.8 KM20L2-Colon
cancer 0.0 PANC-1-Pancreatic epithelioid ductal 0.0 carcinoma
NCI-H716-Colon cancer 0.0 T24-Bladder carcinma (transitional cell)
0.0 SW-48-Colon adenocarcinoma 0.0 5637-Bladder carcinoma 0.0
SW1116-Colon adenocarcinoma 0.0 HT-1197-Bladder carcinoma 2.6 LS
174T-Colon adenocarcinoma 0.0 UM-UC-3-Bladder carcinma
(transitional cell) 0.0 SW-948-Colon adenocarcinoma 0.0
A204-Rhabdomyosarcoma 0.0 SW-480-Colon adenocarcinoma 0.0
HT-1080-Fibrosarcoma 0.0 NCI-SNU-5-Gastric carcinoma 0.0
MG-63-Osteosarcoma 1.5 KATO III-Gastric carcinoma 0.0
SK-LMS-1-Leiomyosarcoma (vulva) 0.0 NCI-SNU-16-Gastric carcinoma
0.0 SJRH30-Rhabdomyosarcoma (met to bone 0.0 marrow)
NCI-SNU-1-Gastric carcinoma 0.0 A431-Epidermoid carcinoma 0.0
RF-1-Gastric adenocarcinoma 0.6 WM266-4-Melanoma 0.0 RF-48-Gastric
adenocarcinoma 0.0 DU 145-Prostate carcinoma (brain metastasis) 0.0
MKN-45-Gastric carcinoma 0.0 MDA-MB-468-Breast adenocarcinoma 0.0
NCI-N87-Gastric carcinoma 0.0 SCC-4-Squamous cell carcinoma of
tongue 0.0 OVCAR-5-Ovarian carcinoma 0.0 SCC-9-Squamous cell
carcinoma of tongue 0.0 RL95-2-Uterine carcinoma 0.0
SCC-15-Squamous cell carcinoma of tongue 0.0 HelaS3-Cervical
adenocarcinoma 0.0 CAL 27-Squamous cell carcinoma of tongue 0.0
Clumn A - Rel. Exp.(%) Ag36, Run 161682678
[0636]
81TABLE CH Panel 4D Tissue Name A B Tissue Name A B Secondary Th1
act 0.0 0.0 HUVEC IL-1beta 0.0 0.0 Secondary Th2 act 0.0 0.0 HUVEC
IFN gamma 0.0 0.0 Secondary Tr1 act 0.0 0.0 HUVEC TNF alpha + IFN
gamma 0.0 0.0 Secondary Th1 rest 0.0 0.0 HUVEC TNF alpha + IL4 0.0
0.0 Secondary Th2 rest 0.0 0.0 HUVEC IL-11 0.0 0.0 Secondary Tr1
rest 0.2 0.2 Lung Microvascular EC none 0.0 0.1 Primary Th1 act 0.0
0.0 Lung Microvascular EC TNF alpha + IL-1beta 0.0 0.1 Primary Th2
act 0.0 0.0 Microvascular Dermal EC none 0.0 0.0 Primary Tr1 act
0.0 0.0 Microsvasular Dermal EC TNF alpha + IL-1beta 0.0 0.0
Primary Th1 rest 0.0 0.0 Bronchial epithelium TNF alpha + IL1beta
0.2 0.0 Primary Th2 rest 0.0 0.1 Small airway epithelium none 0.0
0.0 Primary Tr1 rest 0.3 0.1 Small airway epithelium TNF alpha +
IL-1beta 0.0 0.1 CD45RA CD4 lymphocyte act 0.2 0.0 Coronery artery
SMC rest 0.0 0.0 CD45RO CD4 lymphocyte act 0.0 0.0 Coronery artery
SMC TNF alpha + IL-1beta 0.0 0.0 CD8 lymphocyte act 0.0 0.0
Astrocytes rest 0.0 0.0 Secondary CD8 lymphocyte rest 0.0 0.0
Astrocytes TNF alpha + IL-1beta 0.3 0.0 Secondary CD8 lymphocyte
act 0.0 0.0 KU-812 (Basophil) rest 0.0 0.0 CD4 lymphocyte none 0.0
0.0 KU-812 (Basophil) PMA/ionomycin 0.0 0.0 2ry
Th1/Th2/Tr1_anti-CD95 CH11 0.0 0.0 CCD1106 (Keratinocytes) none 0.0
0.0 LAK cells rest 0.0 0.0 CCD1106 (Keratinocytes) TNF alpha +
IL-1beta 0.0 0.0 LAK cells IL-2 0.2 0.0 Liver cirrhosis 1.4 0.0 LAK
cells IL-2 + IL-12 0.0 0.0 Lupus kidney 0.0 0.1 LAK cells IL-2 +
IFN gamma 0.0 0.0 NCI-H292 none 2.4 1.8 LAK cells IL-2 + IL-18 0.4
0.0 NCI-H292 IL-4 2.2 1.8 LAK cells PMA/ionomycin 0.1 0.0 NCI-H292
IL-9 3.2 0.7 NK Cells IL-2 rest 0.0 0.0 NCI-H292 IL-13 2.4 0.3 Two
Way MLR 3 day 0.2 0.0 NCI-H292 IFN gamma 0.8 0.1 Two Way MLR 5 day
0.0 0.0 HPAEC none 0.0 0.0 Two Way MLR 7 day 0.0 0.0 HPAEC TNF
alpha + IL-1beta 0.0 0.0 PBMC rest 0.0 0.0 Lung fibroblast none 0.2
0.0 PBMC PWM 0.0 0.0 Lung fibroblast TNF alpha + IL-1beta 0.7 0.0
PBMC PHA-L 0.0 0.0 Lung fibroblast IL-4 1.1 0.3 Ramos (B cell) none
0.0 0.0 Lung fibroblast IL-9 0.4 0.4 Ramos (B cell) ionomycin 0.7
0.1 Lung fibroblast IL-13 0.2 0.3 B lymphocytes PWM 0.0 0.0 Lung
fibroblast IFN gamma 0.4 0.1 B lymphocytes CD40L and IL-4 0.9 0.5
Dermal fibroblast CCD1070 rest 0.2 0.2 EOL-1 dbcAMP 0.0 0.0 Dermal
fibroblast CCD1070 TNF alpha 0.0 0.0 EOL-1 dbcAMP PMA/ionomycin 0.1
0.0 Dermal fibroblast CCD1070 IL-1beta 0.0 0.6 Dendritic cells none
0.0 0.0 Dermal fibroblast IFN gamma 0.0 0.3 Dendritic cells LPS 0.4
0.3 Dermal fibroblast IL-4 0.2 0.0 Dendritic cells anti-CD40 0.2
0.0 IBD Colitis 2 0.0 0.0 Monocytes rest 0.0 0.0 IBD Crohn's 0.1
0.1 Monocytes LPS 0.0 0.0 Colon 1.5 0.2 Macrophages rest 0.6 0.9
Lung 100.0 100.0 Macrophages LPS 0.0 0.1 Thymus 1.9 3.2 HUVEC none
0.0 0.0 Kidney 2.5 2.1 HUVEC starved 0.0 0.0 Colmn A - Rel. Exp.(%)
Ag2678, Run 158536138 Column B - Rel. Exp.(%) Ag36, Run
161681141
[0637]
82TABLE CI Panel 5D Tissue Name A Tissue Name A
97457_Patient-02go_adipose 0.3 94709_Donor 2 AM - A_adipose 0.3
97476_Patient-07sk_skeletal muscle 5.0 94710_Donor 2 AM - B_adipose
0.4 97477_Patient-07ut_uterus 0.2 94711_Donor 2 AM - C_adipose 0.1
97478_Patient-07pl_placenta 0.4 94712_Donor 2 AD - A_adipose 0.1
97481_Patient-08sk_skeletal muscle 11.1 94713_Donor 2 AD -
B_adipose 0.2 97482_Patient-08ut_uterus 0.0 94714_Donor 2 AD -
C_adipose 0.2 97483_Patient-08pl_placenta 0.1 94742_Donor 3 U -
A_Mesenchymal Stem 0.0 Cells 97486_Patient-09sk_skeleta- l muscle
14.5 94743_Donor 3 U - B_Mesenchymal Stem 0.0 Cells
97487_Patient-09ut_uterus 0.1 94730_Donor 3 AM - A_adipose 0.1
97488_Patient-09pl_placenta 0.3 94731_Donor 3 AM - B_adipose 0.0
97492_Patient-10ut_uterus 0.3 94732_Donor 3 AM - C_adipose 0.0
97493_Patient-10pl_placenta 0.2 94733_Donor 3 AD - A_adipose 26.8
97495_Patient-11go_adipose 0.1 94734_Donor 3 AD - B_adipose 0.0
97496_Patient-11sk_skeletal muscle 36.9 94735_Donor 3 AD -
C_adipose 0.0 97497_Patient-11ut_uterus 0.1
77138_Liver_HepG2untreated 0.0 97498_Patient-11pl_placenta 0.2
73556_Heart_Cardiac stromal cells 0.1 (primary)
97500_Patient-12go_adipose 0.1 81735_Small Intestine 1.1
97501_Patient-12sk_skeletal muscle 100.0 72409_Kidney_Proximal
Convoluted 0.1 Tubule 97502_Patient-12ut_uterus 0.1 82685_Small
intestine_Duodenum 0.0 97503_Patient-12pl_placenta 0.0
90650_Adrenal_Adrenocortical adenoma 0.0 94721_Donor 2 U -
A_Mesenchymal 0.0 72410_Kidney_HRCE 0.1 Stem Cells 94722_Donor 2 U
- B_Mesenchymal Stem 0.1 72411_Kidney_HRE 0.0 Cells 94723_Donor 2 U
- C_Mesenchymal Stem 0.0 73139_Uterus_Uterine smooth muscle cells
0.0 Cells Column A - Rel. Exp.(%) Ag36, Run 169315041
[0638] CNS_neurodegeneration_v1.0 Summary: Ag2678/Ag36 This panel
confirms the expression of this gene at low levels in the brains of
an independent group of individuals.
[0639] Panel 1 Summary: Ag36 Highest expression of this gene is
seen in skeletal muscle (CT=23.8). The pattern of expression in
this panel is in agreement with that seen in panel 1.3D.
[0640] Panel 1.3D Summary: Ag2678/Ag36 Two experiments with
different probe-primer sets are in good agreement. Highest
expression of this gene is seen in fetal and adult skeletal muscle
(CTs=25). Therefore, therapeutic modulation of this gene expression
or its protein product may be useful in the treatment of muscle
related diseases. In addition, significant expression of this gene
is also seen in other tissues with metabolic/endocrine functions
including adipose, pancreas, thyroid, adrenal gland, pituitary
gland, heart, liver and gastrointestinal tract. Therefore,
therapeutic modulation of the activity of this gene may prove
useful in the treatment of endocrine/metabolically related
diseases, such as obesity and diabetes.
[0641] Moderate expression of this gene is also seen in all the
regions of central nervous system examined including amygdala,
hippocampus, substantia nigra, thalamus, cerebellum, cerebral
cortex, and spinal cord. Therefore, therapeutic modulation of this
gene product may be useful in the treatment of central nervous
system disorders such as Alzheimer's disease, Parkinson's disease,
epilepsy, multiple sclerosis, schizophrenia and depression.
[0642] Panel 2D Summary: Ag2678/Ag36 Two experiments with different
probe-primer sets are in good agreement. Highest expression of this
gene is seen in normal lung margin samples (CTs=24.8-26). Lung
margins show consistently higher expression than the corresponsing
lung cancer tissues. Interestingly, expression of this gene is
higher in normal tissues compared to the cancer sample. Therefore,
therapeutic modulation of the activity of this gene or its protein
product, through the use of small molecule drugs, protein
therapeutics or antibodies, might be beneficial in the treatment of
lung, stomach, breast, prostate, thyroid, kidney, and colon
cancers.
[0643] Panel 3D Summary: Ag36 Highest expression of this gene is
mainly seen in cerebellum sample (CTs=30). Therefore, therapeutic
modulation of this gene or its protein product may be useful in the
treatment of cerebellum related diseases such as ataxia and
autism.
[0644] Panel 4D Summary: Ag2678/Ag36 Two experiments with different
probe-primer sets are in good agreement. Highest expression of this
gene is seen in lung (CTs=27). Moderate to low expression of this
gene is also seen in resting and activated mucoepidermoid cell line
NCI-H292, colon, thymus and kidney. Therefore, therapeutic
modulation of this gene may be used for the treatment of
inflammatory/autoimmune diseases that affect colon, lung and kidney
including asthma, allergies, inflammatory bowel disease, lupus
erythematosus, psoriasis, and emphysema.
[0645] Panel 5D Summary: Ag36 Highest expression of this gene is
seen in a diabetic patient on insulin (patient 12)(CT=26.6).
Significant expression of this gene is also seen in skeletal muscle
from non-diabetic but obese patients and also in adipose tissue.
Therefore, therapeutic modulation of the activity of this gene or
its protein product may be useful in the treatment of metabolic
disease such as diabetes and obesity.
[0646] D. CG53054-02: Wnt-14 Protein Precursor.
[0647] Expression of gene CG53054-02 was assessed using the
primer-probe sets Ag2261 and Ag3035, described in Tables DA and DB.
Results of the RTQ-PCR runs are shown in Tables DC, DD, DE, DF, DG,
DH, DI, DJ and DK.
83TABLE DA Probe Name Ag2261 Start SEQ ID Primers Sequences Length
Position No Forward 5'-ggatgactcgcctagcttct-3' 20 882 156 Probe
TET-5'-gccgtaggtgccaccgtgagaag-3'-TAMRA 23 935 157 Reverse
5'-agcagatgctctcgcagtt-3' 19 958 158
[0648]
84TABLE DB Probe Name Ag3035 Start SEQ ID Primers Sequences Length
Position No Forward 5'-acagcagcaagttcgtcaag-3' 20 527 159 Probe
TET-5'-agacggtcaagcaaggatctgcgag-3'-TAMRA 25 559 160 Reverse
5'-cagaggttgttgtggaagt-3' 20 593 161
[0649]
85TABLE DC AI comprehensive panel v1.0 Tissue Name A Tissue Name A
110967 COPD-F 13.1 112427 Match Control Psoriasis-F 84.1 110980
COPD-F 44.1 112418 Psoriasis-M 11.5 110968 COPD-M 32.3 112723 Match
Control Psoriasis-M 2.6 110977 COPD-M 100.0 112419 Psoriasis-M 17.3
110989 Emphysema-F 48.0 112424 Match Control Psoriasis-M 9.9 110992
Emphysema-F 28.7 112420 Psoriasis-M 45.1 110993 Emphysema-F 17.9
112425 Match Control Psoriasis-M 36.1 110994 Emphysema-F 7.4 104689
(MF) OA Bone-Backus 16.6 110995 Emphysema-F 50.3 104690 (MF) Adj
"Normal" Bone-Backus 10.2 110996 Emphysema-F 17.3 104691 (MF) OA
Synovium-Backus 27.9 110997 Asthma-M 1.6 104692 (BA) OA
Cartilage-Backus 0.0 111001 Asthma-F 13.1 104694 (BA) OA
Bone-Backus 21.3 111002 Asthma-F 46.7 104695 (BA) Adj "Normal"
Bone-Backus 8.1 111003 Atopic Asthma-F 25.3 104696 (BA) OA
Synovium-Backus 22.1 111004 Atopic Asthma-F 44.4 104700 (SS) OA
Bone-Backus 6.0 111005 Atopic Asthma-F 22.4 104701 (SS) Adj
"Normal" Bone-Backus 12.4 111006 Atopic Asthma-F 6.9 104702 (SS) OA
Synovium-Backus 39.5 111417 Allergy-M 22.1 117093 OA Cartilage Rep7
21.3 112347 Allergy-M 2.2 112672 OA Bone5 17.9 112349 Normal Lung-F
0.9 112673 OA Synovium5 10.1 112357 Normal Lung-F 76.8 112674 OA
Synovial Fluid cells5 7.6 112354 Normal Lung-M 11.3 117100 OA
Cartilage Rep14 12.8 112374 Crohns-F 30.1 112756 OA Bone9 24.0
112389 Match Control Crohns-F 20.6 112757 OA Synovium9 49.3 112375
Crohns-F 29.9 112758 OA Synovial Fluid Cells9 5.7 112732 Match
Control Crohns-F 14.3 117125 RA Cartilage Rep2 17.3 112725 Crohns-M
6.4 113492 Bone2 RA 25.5 112387 Match Control Crohns-M 18.8 113493
Synovium2 RA 6.3 112378 Crohns-M 1.6 113494 Syn Fluid Cells RA 16.8
112390 Match Control Crohns-M 25.9 113499 Cartilage4 RA 13.1 112726
Crohns-M 14.1 113500 Bone4 RA 21.8 112731 Match Control Crohns-M
25.9 113501 Synovium4 RA 13.9 112380 Ulcer Col-F 33.0 113502 Syn
Fluid Cells4 RA 6.8 112734 Match Control Ulcer Col-F 19.2 113495
Cartilage3 RA 14.4 112384 Ulcer Col-F 21.8 113496 Bone3 RA 13.9
112737 Match Control Ulcer Col-F 3.6 113497 Synovium3 RA 10.0
112386 Ulcer Col-F 10.8 113498 Syn Fluid Cells3 RA 23.0 112738
Match Control Ulcer Col-F 25.5 117106 Normal Cartilage Rep20 15.5
112381 Ulcer Col-M 0.2 113663 Bone3 Normal 0.0 112735 Match Control
Ulcer Col-M 1.9 113664 Synovium3 Normal 0.0 112382 Ulcer Col-M 14.4
113665 Syn Fluid Cells3 Normal 0.1 112394 Match Control Ulcer Col-M
3.1 117107 Normal Cartilage Rep22 3.6 112383 Ulcer Col-M 31.6
113667 Bone4 Normal 18.3 112736 Match Control Ulcer Col-M 11.3
113668 Synovium4 Normal 16.4 112423 Psoriasis-F 7.1 113669 Syn
Fluid Cells4 Normal 43.8 Column A - Rel. Exp.(% Ag3035, Run
311087483
[0650]
86TABLE DD HASS Panel v1.0 Tissue Name A Tissue Name A MCF-7 C1 1.7
U87-MG F1 (B) 0.0 MCF-7 C2 2.6 U87-MG F2 0.0 MCF-7 C3 2.8 U87-MG F3
0.1 MCF-7 C4 2.9 U87-MG F4 0.2 MCF-7 C5 5.6 U87-MG F5 0.5 MCF-7 C6
2.2 U87-MG F6 0.3 MCF-7 C7 6.3 U87-MG F7 0.0 MCF-7 C9 4.4 U87-MG F8
0.0 MCF-7 C10 2.4 U87-MG F9 0.0 MCF-7 C11 0.9 U87-MG F10 0.4 MCF-7
C12 1.6 U87-MG F11 0.2 MCF-7 C13 4.5 U87-MG F12 0.3 MCF-7 C15 3.4
U87-MG F13 0.0 MCF-7 C16 5.7 U87-MG F14 0.0 MCF-7 C17 4.8 U87-MG
F15 0.1 T24 D1 1.4 U87-MG F16 0.5 T24 D2 0.2 U87-MG F17 0.7 T24 D3
4.8 LnCAP A1 2.4 T24 D4 0.5 LnCAP A2 1.2 T24 D5 3.1 LnCAP A3 2.6
T24 D6 0.3 LnCAP A4 2.3 T24 D7 2.0 LnCAP A5 6.0 T24 D9 4.5 LnCAP A6
3.8 T24 D10 0.7 LnCAP A7 7.2 T24 D11 1.8 LnCAP A8 10.7 T24 D12 0.3
LnCAP A9 14.6 T24 D13 1.5 LnCAP A10 3.1 T24 D15 2.6 LnCAP A11 7.9
T24 D16 0.5 LnCAP A12 2.9 T24 D17 1.2 LnCAP A13 1.7 CAPaN B1 68.8
LnCAP A14 3.0 CAPaN B2 36.9 LnCAP A15 3.2 CAPaN B3 16.5 LnCAP A16
9.3 CAPaN B4 28.3 LnCAP A17 7.3 CAPaN B5 44.1 Primary Astrocytes
2.3 CAPaN B6 44.4 Primary Renal Proximal Tubule Epithelial 1.9 cell
A2 CAPaN B7 34.9 Primary melanocytes A5 0.5 CAPaN B8 34.2 126443 -
341 medullo 0.5 CAPaN B9 45.1 126444 - 487 medullo 9.6 CAPaN B10
47.3 126445 - 425 medullo 0.0 CAPaN B11 51.1 126446 - 690 medullo
2.8 CAPaN B12 64.2 126447 - 54 adult glioma 1.1 CAPaN B13 29.7
126448 - 245 adult glioma 0.1 CAPaN B14 26.4 126449 - 317 adult
glioma 0.4 CAPaN B15 16.4 126450 - 212 glioma 0.2 CAPaN B16 77.4
126451 - 456 glioma 0.6 CAPaN B17 100.0 Column A Rel.Exp. (%)
Ag3035, Run 259153969
[0651]
87TABLE DE Oncology_cell_line_screening_panel_v3.2 Tissue Name A
Tissue Name A 94905_Daoy_Medulloblast- oma/Cerebellum_ssc 0.2
94954_Ca Ski_Cervical epidermoid 8.4 DNA carcinoma
(metastasis)_sscDNA 94906_TE671_Medulloblastoma/Cerebell- um_ssc
0.5 94955_ES-2_Ovarian clear cell 0.0 DNA carcinoma_sscDNA
94907_D283 0.2 94957_Ramos/6 h stim_Stimulated with 0.0
Med_Medulloblastoma/Cerebellum_sscDNA PMA/ionomycin 6 h_sscDNA
94908_PFSK-1_Primitive 0.0 94958_Ramos/14 h stim_Stimulated 0.0
Neuroectodermal/Cerebellum_sscDNA with PMA/ionomycin 14 h_sscDNA
94909_XF-498_CNS_sscDNA 0.2 94962_MEG-01_Chronic myelogenous 0.0
leukemia (megokaryoblast)_sscDNA 94910_SNB-78_CNS/glioma_sscDNA 0.5
94963_Raji_Burkitt's 0.0 lymphoma_sscDNA
94911_SF-268_CNS/glioblastoma_sscDNA 0.3 94964_Daudi_Burkitt's 0.0
lymphoma_sscDNA 94912_T98G_Glioblastoma_sscDNA 0.5
94965_U266_B-cell 0.0 plasmacytoma/myeloma_sscDNA
96776_SK-N-SH_Neuroblastoma 0.0 94968_CA46_Burkitt's 0.0
(metastasis)_sscDNA lymphoma_sscDNA 94913_SF-295_CNS/glioblastoma-
_sscDNA 0.6 94970_RL_non-Hodgkin's B-cell 0.0 lymphoma_sscDNA
132565_NT2pool_sscDNA 3.9 94972_JM1_pre-B-cell 1.1
lymphoma/leukemia_sscDNA 94914_Cerebellum_sscDNA 0.7 94973_Jurkat_T
cell leukemia_sscDNA 0.0 96777_Cerebellum_sscDNA 0.8
94974_TF-1_Erythroleukemia_sscDNA 0.0 94916_NCI-H292_Mucoepide-
rmoid lung 11.7 94975_HUT 78_T-cell 0.1 carcinoma_sscDNA
lymphoma_sscDNA 94917_DMS-114_Small cell lung 0.3
94977_U937_Histocytic 0.0 cancer_sscDNA lymphoma_sscDNA
94918_DMS-79_Small cell lung 100.0 94980_KU-812_Myelogenous 1.3
cancer/neuroendocrine_sscDNA leukemia_sscDNA 94919_NCI-H146_Small
cell lung 16.4 94981_769-P_Clear cell renal 1.9
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94920_NCI-H526_Small
cell lung 0.0 94983_Caki-2_Clear cell renal 1.8
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94921_NCI-N417_Small
cell lung 0.0 94984_SW 839_Clear cell renal 0.8
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94923_NCI-H82_Small
cell lung 0.4 94986_G401_Wilms' tumor_sscDNA 0.4
cancer/neuroendocrine_sscDNA 94924_NCI-H157_Squamous cell lung
cancer 2.4 126768_293 cells_sscDNA 1.9 (metastasis)_sscDNA
94925_NCI-H1155_Large cell lung 15.5 94987_Hs766T_Pancreatic
carcinoma 2.1 cancer/neuroendocrine_sscDNA (LN metastasis)_sscDNA
94926_NCI-H1299_Large cell lung 3.4 94988_CAPAN-1_Pancreatic 3.2
cancer/neuroendocrine_sscDNA adenocarcinoma (liver
metastasis)_sscDNA 94927_NCI-H727_Lung carcinoid_sscDNA 9.5
94989_SU86.86_Pancreatic carcinoma 1.6 (liver metastasis)_sscDNA
94928_NCI-UMC-11_Lung carcinoid_sscDNA 20.2 94990_BxPC-3_Pancreatic
3.0 adenocarcinoma_sscDNA 94929_LX-1_Small cell lung cancer_sscDNA
0.0 94991_HPAC_Pancreatic 0.9 adenocarcinoma_sscDNA
94930_Colo-205_Colon cancer_sscDNA 0.0 94992_MIA PaCa-2_Pancreatic
1.1 carcinoma_sscDNA 94931_KM12_Colon cancer_sscDNA 0.0
94993_CFPAC-1_Pancreatic ductal 0.2 adenocarcinoma_sscDNA
94932_KM20L2_Colon cancer_sscDNA 0.0 94994_PANC-1_Pancreatic
epithelioid 22.8 ductal carcinoma_sscDNA 94933_NCI-H716_Colon
cancer_sscDNA 4.3 94996_T24_Bladder carcinma 0.2 (transitional
cell)_sscDNA 94935_SW-48_Colon adenocarcinoma_sscDNA 0.0
94997_5637_Bladder 0.5 carcinoma_sscDNA 94936_SW1116_Colon 1.2
94998_HT-1197_Bladder 12.5 adenocarcinoma_sscDNA carcinoma_sscDNA
94937_LS 174T_Colon 0.6 94999_UM-UC-3_Bladder carcinma 0.0
adenocarcinoma_sscDNA (transitional cell)_sscDNA 94938_SW-948_Colon
0.0 95000_A204_Rhabdomyosarcoma_ssc 0.0 adenocarcinoma_sscDNA DNA
94939_SW-480_Colon 0.0 95001_HT-1080_Fibrosarcoma_sscDNA 1.6
adenocarcinoma_sscDNA 94940_NCI-SNU-5_Gastric carcinoma_sscDNA 6.9
95002_MG-63_Osteosarcoma 14.7 (bone)_sscDNA 112197_KATO
III_Stomach_sscDNA 0.3 95003_SK-LMS-1_Leiomyosarcoma 0.0
(vulva)_sscDNA 94943_NCI-SNU-16_Gastric 0.6
95004_SJRH30_Rhabdomyosarcoma 1.3 carcinoma_sscDNA (met to bone
marrow)_sscDNA 94944_NCI-SNU-1_Gastric carcinoma_sscDNA 2.9
95005_A431_Epidermoid 5.9 carcinoma_sscDNA 94946_RF-1_Gastric
adenocarcinoma_sscDNA 0.0 95007_WM266-4_Melanoma_sscDN- A 0.0
94947_RF-48_Gastric 0.0 112195_DU 145_Prostate_sscDNA 1.1
adenocarcinoma_sscDNA 96778_MKN-45_Gastric carcinoma_sscDNA 0.6
95012_MDA-MB-468_Breast 2.2 adenocarcinoma_sscDNA
94949_NCI-N87_Gastric carcinoma_sscDNA 3.9
112196_SSC-4_Tongue_sscDNA 2.6 94951_OVCAR-5_Ovarian
carcinoma_sscDNA 1.3 112194_SSC-9_Tongue_ssc- DNA 3.3
94952_RL95-2_Uterine carcinoma_sscDNA 3.0
112191_SSC-15_Tongue_sscDNA 1.8 94953_HelaS3_Cervical 1.7 95017_CAL
27_Squamous cell 1.2 adenocarcinoma_sscDNA carcinoma of
tongue_sscDNA Column A - Rel. Exp.(%) Ag3035, Run 259180377
[0652]
88TABLE DF Panel 1.3D Tissue Name A B C Tissue Name A B C Liver
adenocarcinoma 22.4 19.6 71.2 Kidney (fetal) 2.1 0.0 2.7 Pancreas
3.9 2.5 2.8 Renal ca. 786-0 0.0 0.0 0.0 Pancreatic ca. CAPAN 2 5.3
3.5 9.5 Renal ca. A498 10.2 5.3 9.2 Adrenal gland 2.1 0.6 2.0 Renal
ca. RXF 393 0.0 0.0 0.0 Thyroid 7.0 9.8 3.9 Renal ca. ACHN 0.0 2.2
0.0 Salivary gland 1.9 2.1 4.2 Renal ca. UO-31 0.0 0.0 0.0
Pituitary gland 1.0 2.2 6.7 Renal ca. TK-10 0.0 0.0 0.0 Brain
(fetal) 6.8 4.9 10.8 Liver 0.0 0.0 0.0 Brain (whole) 4.8 3.0 1.4
Liver (fetal) 7.6 0.0 0.0 Brain (amygdala) 4.6 5.3 1.5 Liver ca.
(hepatoblast) HepG2 0.0 0.0 0.0 Brain (cerebellum) 1.6 1.6 2.0 Lung
14.3 15.8 9.2 Brain (hippocampus) 7.5 11.3 0.6 Lung (fetal) 15.1
15.4 7.4 Brain (substantia nigra) 1.2 2.6 1.3 Lung ca. (small cell)
LX-1 1.6 0.0 0.0 Brain (thalamus) 2.5 1.7 2.6 Lung ca. (small cell)
NCI-H69 29.5 19.1 31.2 Cerebral Cortex 0.0 0.0 5.0 Lung ca. (s.
cell var.) SHP-77 11.0 5.1 37.4 Spinal cord 1.7 2.1 2.7 Lung ca.
(large cell)NCI-H460 0.0 0.0 0.0 glio/astro U87-MG 0.0 0.0 0.0 Lung
ca. (non-sm. cell) A549 0.0 1.2 1.6 glio/astro U-118-MG 55.1 50.3
42.9 Lung ca. (non-s. cell) NCI-H23 0.0 1.3 0.8 astrocytoma SW1783
0.0 7.5 0.0 Lung ca. (non-s. cell) HOP-62 0.0 1.7 0.0 neuro*; met
SK-N-AS 0.0 0.0 0.7 Lung ca. (non-s. cl) NCI-H522 8.0 8.3 7.3
astrocytoma SF-539 1.9 4.7 9.9 Lung ca. (squam.) SW 900 4.0 0.0 1.8
astrocytoma SNB-75 2.0 4.9 6.9 Lung ca. (squam.) NCI-H596 15.8 10.2
58.2 glioma SNB-19 6.7 2.4 3.7 Mammary gland 7.2 4.1 4.4 glioma
U251 2.1 4.5 6.8 Breast ca.* (pl. ef) MCF-7 1.7 3.4 7.3 glioma
SF-295 10.0 0.6 4.6 Breast ca.* (pl. ef) 23.2 19.6 19.2 MDA-MB-231
Heart (fetal) 11.1 9.9 38.2 Breast ca.* (pl. ef) T47D 4.3 5.8 21.8
Heart 4.9 6.0 15.2 Breast ca. BT-549 0.0 4.2 2.2 Skeletal muscle
(fetal) 100.0 100.0 85.3 Breast ca. MDA-N 0.0 0.0 0.0 Skeletal
muscle 5.5 8.4 39.8 Ovary 3.6 3.1 8.1 Bone marrow 0.0 0.0 0.7
Ovarian ca. OVCAR-3 1.1 1.0 5.6 Thymus 10.0 3.9 6.4 Ovarian ca.
OVCAR-4 0.0 0.0 0.7 Spleen 3.8 4.2 1.6 Ovarian ca. OVCAR-5 0.0 0.0
11.5 Lymph node 5.0 1.1 1.4 Ovarian ca. OVCAR-8 1.3 4.3 4.1
Colorectal 3.4 5.4 6.8 Ovarian ca. IGROV-1 0.0 0.0 8.1 Stomach 6.0
15.4 3.1 Ovarian ca.* (ascites) 7.5 16.0 100.0 SK-OV-3 Small
intestine 15.9 18.7 2.3 Uterus 17.8 15.1 9.9 Colon ca. SW480 24.3
15.3 11.6 Placenta 4.6 8.2 2.1 Colon ca.* SW620(SW480 0.0 0.0 2.1
Prostate 3.6 5.3 0.6 met) Colon ca. HT29 0.0 0.0 0.0 Prostate ca.*
(bone met)PC-3 1.7 1.5 6.1 Colon ca. HCT-116 3.8 0.6 3.3 Testis
21.9 14.6 1.6 Colon ca. CaCo-2 0.0 0.8 0.3 Melanoma Hs688(A).T 3.1
4.7 1.4 Colon ca. tissue(ODO3866) 2.3 0.0 1.6 Melanoma* (met)
Hs688(B).T 0.4 1.3 0.0 Colon ca. HCC-2998 0.0 0.0 1.6 Melanoma
UACC-62 0.0 0.0 0.0 Gastric ca.* (liver met) 16.7 14.9 15.3
Melanoma M14 0.0 0.0 0.0 NCI-N87 Bladder 1.6 3.2 3.0 Melanoma LOX
IMVI 0.0 0.0 0.0 Trachea 24.3 33.7 5.7 Melanoma* (met) SK-MEL-5 0.0
2.0 0.7 Kidney 0.0 0.0 0.0 Adipose 6.7 7.2 21.2 Colun A - Rel.
Exp.(%) Ag2261, Run 150631675 Column B - Rel. Exp.(%) Ag2261, Run
152887692 Column C - Rel. Exp.(%) Ag3035, Run 167597764
[0653]
89TABLE DG Panel 2D Tissue Name A B Tissue Name A B Normal Colon
19.1 19.8 Kidney Margin 8120608 2.4 0.0 CC Well to Mod Diff
(ODO3866) 0.0 5.8 Kidney Cancer 8120613 14.6 7.3 CC Margin
(ODO3866) 19.5 12.5 Kidney Margin 8120614 4.8 1.5 CC Gr.2
rectosigmoid (ODO3868) 3.8 1.4 Kidney Cancer 9010320 0.0 0.0 CC
Margin (ODO3868) 2.6 5.1 Kidney Margin 9010321 0.0 0.0 CC Mod Diff
(ODO3920) 6.0 2.9 Normal Uterus 9.7 2.8 CC Margin (ODO3920) 23.8
6.4 Uterus Cancer 064011 85.9 41.5 CC Gr.2 ascend colon (ODO3921)
9.3 2.2 Normal Thyroid 15.2 7.3 CC Margin (ODO3921) 16.8 11.7
Thyroid Cancer 064010 0.0 3.0 CC from Partial Hepatectomy 2.4 0.0
Thyroid Cancer A302152 1.9 1.2 (ODO4309) Mets Liver Margin
(ODO4309) 2.6 0.0 Thyroid Margin A302153 2.6 2.8 Colon mets to lung
(OD04451-01) 7.9 4.5 Normal Breast 16.2 2.7 Lung Margin
(OD04451-02) 11.3 12.9 Breast Cancer (OD04566) 78.5 29.7 Normal
Prostate 6546-1 6.3 2.6 Breast Cancer (OD04590-01) 37.6 23.8
Prostate Cancer (OD04410) 17.8 7.3 Breast Cancer Mets 100.0 24.5
(OD04590-03) Prostate Margin (OD04410) 10.7 7.4 Breast Cancer
Metastasis 94.0 45.4 (OD04655-05) Prostate Cancer (OD04720-01) 4.7
4.4 Breast Cancer 064006 25.7 24.8 Prostate Margin (OD04720-02)
13.9 5.6 Breast Cancer 1024 23.2 7.1 Normal Lung 061010 36.6 14.3
Breast Cancer 9100266 33.0 7.5 Lung Met to Muscle (ODO4286) 1.0 0.0
Breast Margin 9100265 7.6 7.6 Muscle Margin (ODO4286) 31.0 38.2
Breast Cancer A209073 13.9 0.9 Lung Malignant Cancer (OD03126) 81.8
100.0 Breast Margin A209073 2.5 0.0 Lung Margin (OD03126) 35.8 18.2
Normal Liver 0.0 0.0 Lung Cancer (OD04404) 57.0 39.5 Liver Cancer
064003 0.0 0.0 Lung Margin (OD04404) 9.4 11.8 Liver Cancer 1025 4.8
1.7 Lung Cancer (OD04565) 37.1 42.0 Liver Cancer 1026 7.1 0.0 Lung
Margin (OD04565) 22.7 9.3 Liver Cancer 6004-T 4.8 0.0 Lung Cancer
(OD04237-01) 5.3 6.4 Liver Tissue 6004-N 4.4 1.8 Lung Margin
(OD04237-02) 78.5 32.8 Liver Cancer 6005-T 0.0 6.0 Ocular Mel Met
to Liver (ODO4310) 0.0 0.0 Liver Tissue 6005-N 0.0 1.8 Liver Margin
(ODO4310) 2.4 0.0 Normal Bladder 2.4 3.0 Melanoma Mets to Lung
(OD04321) 13.0 0.0 Bladder Cancer 1023 8.5 4.9 Lung Margin
(OD04321) 96.6 50.0 Bladder Cancer A302173 17.0 11.8 Normal Kidney
0.0 0.0 Bladder Cancer (OD04718-01) 10.0 5.7 Kidney Ca, Nuclear
grade 2 0.0 0.0 Bladder Normal Adjacent 19.3 27.5 (OD04338)
(OD04718-03) Kidney Margin (OD04338) 4.0 4.6 Normal Ovary 13.6 12.4
Kidney Ca Nuclear grade 1/2 0.0 3.3 Ovarian Cancer 064008 37.9 2.1
(OD04339) Kidney Margin (OD04339) 18.7 0.0 Ovarian Cancer
(OD04768-07) 18.4 3.7 Kidney Ca, Clear cell type (OD04340) 8.8 11.7
Ovary Margin (OD04768-08) 28.3 12.2 Kidney Margin (OD04340) 0.0 2.0
Normal Stomach 48.3 17.3 Kidney Ca, Nuclear grade 3 3.5 4.0 Gastric
Cancer 9060358 0.0 0.0 (OD04348) Kidney Margin (OD04348) 2.0 1.7
Stomach Margin 9060359 9.9 3.0 Kidney Cancer (OD04622-01) 9.3 0.0
Gastric Cancer 9060395 20.7 10.4 Kidney Margin (OD04622-03) 0.0 6.3
Stomach Margin 9060394 10.0 12.2 Kidney Cancer (OD04450-01) 0.0 0.0
Gastric Cancer 9060397 8.7 1.5 Kidney Margin (OD04450-03) 0.0 0.0
Stomach Margin 9060396 7.5 6.2 Kidney Cancer 8120607 0.0 0.7
Gastric Cancer 064005 10.7 4.8 Colmn A - Rel. Exp.(%) Ag2261, Run
150811744 Column B - Rel. Exp.(%) Ag2261, Run 152887693
[0654]
90TABLE DH Panel 4.1D Tissue Name A B Tissue Name A B Secondary Th1
act 0.0 0.0 HUVEC IL-1beta 1.7 0.0 Secondary Th2 act 0.0 0.0 HUVEC
IFN gamma 0.8 11.8 Secondary Tr1 act 0.0 0.0 HUVEC TNF alpha + IFN
gamma 0.2 0.0 Secondary Th1 rest 0.0 0.0 HUVEC TNF alpha + IL4 0.6
4.5 Secondary Th2 rest 0.0 2.0 HUVEC IL-11 1.1 17.2 Secondary Tr1
rest 0.0 0.0 Lung Microvascular EC none 2.7 7.2 Primary Th1 act 0.0
0.0 Lung Microvascular EC TNF alpha + IL-1beta 0.6 0.0 Primary Th2
act 0.0 0.0 Microvascular Dermal EC none 3.8 0.0 Primary Tr1 act
0.0 0.0 Microsvasular Dermal EC TNF alpha + IL-1beta 1.2 0.0
Primary Th1 rest 0.0 0.0 Bronchial epithelium TNF alpha + IL1beta
3.7 5.0 Primary Th2 rest 0.0 0.0 Small airway epithelium none 1.9
10.7 Primary Tr1 rest 0.0 0.0 Small airway epithelium TNF alpha +
IL-1beta 4.0 24.5 CD45RA CD4 lymphocyte act 0.0 2.3 Coronery artery
SMC rest 0.2 2.2 CD45RO CD4 lymphocyte act 0.0 0.0 Coronery artery
SMC TNF alpha + IL-1beta 0.0 0.0 CD8 lymphocyte act 0.0 0.0
Astrocytes rest 2.4 7.9 Secondary CD8 lymphocyte rest 0.0 0.0
Astrocytes TNF alpha + IL-1beta 1.3 0.0 Secondary CD8 lymphocyte
act 0.0 0.0 KU-812 (Basophil) rest 0.0 2.3 CD4 lymphocyte none 0.0
0.0 KU-812 (Basophil) PMA/ionomycin 2.1 4.5 2ry
Th1/Th2/Tr1_anti-CD95 0.0 0.0 CCD1106 (Keratinocytes) none 22.2
100.0 CH11 LAK cells rest 0.0 4.7 CCD1106 (Keratinocytes) TNF alpha
+ IL-1beta 18.8 52.5 LAK cells IL-2 0.0 0.0 Liver cirrhosis 0.7 4.2
LAK cells IL-2 + IL-12 0.0 0.0 NCI-H292 none 0.4 0.0 LAK cells IL-2
+ IFN gamma 0.0 0.0 NCI-H292 IL-4 1.5 0.0 LAK cells IL-2 + IL-18
0.0 3.0 NCI-H292 IL-9 2.0 8.8 LAK cells PMA/ionomycin 11.0 42.0
NCI-H292 IL-13 1.4 17.8 NK Cells IL-2 rest 0.0 5.2 NCI-H292 IFN
gamma 1.5 6.9 Two Way MLR 3 day 0.0 0.0 HPAEC none 3.1 18.7 Two Way
MLR 5 day 0.0 0.0 HPAEC TNF alpha + IL-1beta 0.5 0.0 Two Way MLR 7
day 0.0 0.0 Lung fibroblast none 6.2 29.9 PBMC rest 0.0 0.0 Lung
fibroblast TNF alpha + IL-1beta 2.1 0.0 PBMC PWM 0.5 0.0 Lung
fibroblast IL-4 4.2 9.8 PBMC PHA-L 0.4 0.0 Lung fibroblast IL-9 8.3
25.3 Ramos (B cell) none 0.0 0.0 Lung fibroblast IL-13 4.0 3.9
Ramos (B cell) ionomycin 0.0 0.0 Lung fibroblast IFN gamma 8.1 59.5
B lymphocytes PWM 0.0 0.0 Dermal fibroblast CCD1070 rest 0.4 0.0 B
lymphocytes CD40L and IL-4 0.0 0.0 Dermal fibroblast CCD1070 TNF
alpha 0.9 4.1 EOL-1 dbcAMP 0.0 2.7 Dermal fibroblast CCD1070
IL-1beta 2.9 0.0 EOL-1 dbcAMP PMA/ionomycin 1.0 1.7 Dermal
fibroblast IFN gamma 5.8 41.8 Dendritic cells none 0.0 0.0 Dermal
fibroblast IL-4 17.2 62.4 Dendritic cells LPS 0.0 0.0 Dermal
Fibroblast rest 4.8 12.9 Dendritic cells anti-CD40 0.0 0.0
Neutrophils TNFa + LPS 1.0 0.0 Monocytes rest 0.0 0.0 Neutrophils
rest 2.2 2.3 Monocytes LPS 0.6 0.0 Colon 2.6 0.0 Macrophages rest
0.0 0.0 Lung 8.8 0.0 Macrophages LPS 0.0 0.0 Thymus 17.1 1.8 HUVEC
none 2.4 7.6 Kidney 100.0 0.0 HUVEC starved 8.8 22.1 Colun A - Rel.
Exp.(%) Ag3035, Run 190944495 Column B - Rel. Exp.(%) Ag3035, Run
259180379
[0655]
91TABLE DI Panel 4D Tissue Name A B Tissue Name A B Secondary Th1
act 0.0 2.1 HUVEC IL-1beta 0.0 1.7 Secondary Th2 act 0.0 0.0 HUVEC
IFN gamma 3.7 11.5 Secondary Tr1 act 0.0 4.2 HUVEC TNF alpha + IFN
gamma 0.0 3.1 Secondary Th1 rest 0.0 0.0 HUVEC TNF alpha + IL4 4.3
5.1 Secondary Th2 rest 0.0 2.3 HUVEC IL-11 4.0 11.2 Secondary Tr1
rest 0.0 0.0 Lung Microvascular EC none 7.2 8.1 Primary Th1 act 0.0
0.0 Lung Microvascular EC TNF alpha + IL-1beta 0.0 0.0 Primary Th2
act 0.0 0.0 Microvascular Dermal EC none 8.4 14.5 Primary Tr1 act
0.0 0.0 Microvasular Dermal EC TNF alpha + IL-1beta 0.0 2.2 Primary
Th1 rest 0.0 0.0 Bronchial epithelium TNF alpha + IL1beta 0.0 16.3
Primary Th2 rest 0.0 0.0 Small airway epithelium none 5.9 18.8
Primary Tr1 rest 0.0 0.0 Small airway epithelium TNF alpha +
IL-1beta 24.3 58.6 CD45RA CD4 lymphocyte act 0.0 0.0 Coronery
artery SMC rest 0.0 2.0 CD45RO CD4 lymphocyte act 0.0 0.0 Coronery
artery SMC TNF alpha + IL-1beta 0.0 0.0 CD8 lymphocyte act 0.0 0.0
Astrocytes rest 3.3 13.5 Secondary CD8 lymphocyte rest 0.0 0.7
Astrocytes TNF alpha + IL-1beta 0.0 8.6 Secondary CD8 lymphocyte
act 1.6 0.0 KU-812 (Basophil) rest 0.0 0.0 CD4 lymphocyte none 0.0
0.0 KU-812 (Basophil) PMA/ionomycin 0.0 9.7 2ry
Th1/Th2/Tr1_anti-CD95 0.0 1.4 CCD1106 (Keratinocytes) none 47.3
100.0 CH11 LAK cells rest 3.5 0.0 CCD1106 (Keratinocytes) TNF alpha
+ IL-1beta 9.0 53.6 LAK cells IL-2 0.0 0.0 Liver cirrhosis 32.8 9.4
LAK cells IL-2 + IL-12 0.0 0.0 Lupus kidney 0.0 1.6 LAK cells IL-2
+ IFN gamma 0.0 4.0 NCI-H292 none 3.8 3.4 LAK cells IL-2 + IL-18
0.0 0.0 NCI-H292 IL-4 8.0 19.5 LAK cells PMA/ionomycin 26.1 50.7
NCI-H292 IL-9 0.0 4.2 NK Cells IL-2 rest 0.0 0.0 NCI-H292 IL-13
13.8 7.0 Two Way MLR 3 day 0.0 0.0 NCI-H292 IFN gamma 16.2 5.7 Two
Way MLR 5 day 0.0 0.0 HPAEC none 6.7 30.1 Two Way MLR 7 day 0.0 0.0
HPAEC TNF alpha + IL-1beta 0.0 0.0 PBMC rest 0.0 0.0 Lung
fibroblast none 7.6 42.0 PBMC PWM 0.0 0.0 Lung fibroblast TNF alpha
+ IL-1beta 3.1 6.3 PBMC PHA-L 0.0 0.0 Lung fibroblast IL-4 4.3 34.2
Ramos (B cell) none 0.0 0.0 Lung fibroblast IL-9 12.7 27.5 Ramos (B
cell) ionomycin 0.0 0.0 Lung fibroblast IL-13 6.8 19.9 B
lymphocytes PWM 0.0 0.0 Lung fibroblast IFN gamma 30.4 51.1 B
lymphocytes CD40L and IL-4 3.1 0.0 Dermal fibroblast CCD1070 rest
0.0 2.8 EOL-1 dbcAMP 0.0 0.0 Dermal fibroblast CCD1070 TNF alpha
5.2 19.6 EOL-1 dbcAMP PMA/ionomycin 3.5 2.7 Dermal fibroblast
CCD1070 IL-1beta 0.0 2.0 Dendritic cells none 0.0 0.0 Dermal
fibroblast IFN gamma 28.5 32.1 Dendritic cells LPS 0.0 0.0 Dermal
fibroblast IL-4 42.9 91.4 Dendritic cells anti-CD40 0.0 0.0 IBD
Colitis 2 2.2 5.5 Monocytes rest 0.0 0.0 IBD Crohn's 3.1 9.6
Monocytes LPS 0.0 0.0 Colon 100.0 58.6 Macrophages rest 0.0 0.0
Lung 36.3 26.1 Macrophages LPS 0.0 0.0 Thymus 0.0 0.0 HUVEC none
0.0 17.7 Kidney 4.0 33.0 HUVEC starved 17.4 51.1 Column A - Rel.
Exp. (%) Ag2261, Run 152887762 Column B - Rel. Exp. (%) Ag3035, Run
165242424
[0656]
92TABLE DJ Panel 5 Islet Tissue Name A Tissue Name A
97457_Patient-02go_adipose 19.3 94709_Donor 2 AM - A_adipose 0.8
97476_Patient-07sk_skeletal muscle 13.0 94710_Donor 2 AM -
B_adipose 0.0 97477_Patient-07ut_uterus 5.3 94711_Donor 2 AM -
C_adipose 1.8 97478_Patient-07pl_placenta 1.4 94712_Donor 2 AD -
A_adipose 5.3 99167_Bayer Patient 1 89.5 94713_Donor 2 AD -
B_adipose 3.2 97482_Patient-08ut_uterus 9.6 94714_Donor 2 AD -
C_adipose 0.0 97483_Patient-08pl_placenta 7.4 94742_Donor 3 U -
A_Mesenchymal Stem 2.6 Cells 97486_Patient-09sk_skeletal muscle
10.7 94743_Donor 3 U - B_Mesenchymal Stem 2.6 Cells
97487_Patient-09ut_uterus 4.7 94730_Donor 3 AM - A_adipose 0.8
97488_Patient-09pl_placenta 3.6 94731_Donor 3 AM - B_adipose 1.7
97492_Patient-10ut_uterus 6.1 94732_Donor 3 AM - C_adipose 1.5
97493_Patient-10pl_placenta 6.8 94733_Donor 3 AD - A_adipose 4.8
97495_Patient-11go_adipose 1.6 94734_Donor 3 AD - B_adipose 2.0
97496_Patient-11sk_skeletal muscle 20.7 94735_Donor 3 AD -
C_adipose 2.5 97497_Patient-11ut_uterus 3.2
77138_Liver_HepG2untreated 0.0 97498_Patient-11pl_placenta 1.8
73556_Heart_Cardiac stromal cells 0.0 (primary)
97500_Patient-12go_adipose 8.5 81735_Small Intestine 15.6
97501_Patient-12sk_skeletal muscle 100.0 72409_Kidney_Proximal
Convoluted 0.0 Tubule 97502_Patient-12ut_uterus 2.2 82685_Small
intestine_Duodenum 5.9 97503_Patient-12pl_placenta 0.0
90650_Adrenal_Adrenocortical adenoma 2.9 94721_Donor 2 U -
A_Mesenchymal 0.0 72410_Kidney_HRCE 7.5 Stem Cells 94722_Donor 2 U
- B_Mesenchymal Stem 1.5 72411_Kidney_HRE 3.3 Cells 94723_Donor 2 U
- C_Mesenchymal Stem 1.5 73139_Uterus_Uterine smooth muscle cells
0.0 Cells Colun A - Rel. Exp.(%) Ag3035, Run 259234350
[0657]
93TABLE DK general oncology screening panel_v_2.4 Tissue Name A
Tissue Name A Colon cancer 1 7.6 Bladder cancer NAT 2 0.0 Colon
cancer NAT 1 14.7 Bladder cancer NAT 3 0.0 Colon cancer 2 3.7
Bladder cancer NAT 4 25.7 Colon cancer NAT 2 10.0 Prostate
adenocarcinoma 1 8.3 Colon cancer 3 7.3 Prostate adenocarcinoma 2
1.6 Colon cancer NAT 3 25.9 Prostate adenocarcinoma 3 3.4 Colon
malignant cancer 4 1.7 Prostate adenocarcinoma 4 9.5 Colon normal
adjacent tissue 1.6 Prostate cancer NAT 5 2.4 4 Lung cancer 1 9.5
Prostate adenocarcinoma 6 0.0 Lung NAT 1 2.9 Prostate
adenocarcinoma 7 4.9 Lung cancer 2 42.3 Prostate adenocarcinoma 8
0.0 Lung NAT 2 23.3 Prostate adenocarcinoma 9 20.6 Squamous cell
carcinoma 3 73.7 Prostate cancer NAT 10 0.4 Lung NAT 3 1.3 Kidney
cancer 1 1.9 metastatic melanoma 1 37.1 KidneyNAT 1 8.1 Melanoma 2
9.4 Kidney cancer 2 34.4 Melanoma 3 1.4 Kidney NAT 2 7.6 metastatic
melanoma 4 100.0 Kidney cancer 3 12.9 metastatic melanoma 5 39.8
Kidney NAT 3 4.5 Bladder cancer 1 2.9 Kidney cancer 4 3.4 Bladder
cancer NAT 1 0.0 Kidney NAT 4 5.9 Bladder cancer 2 3.2 Column A -
Rel. Exp.(%) Ag3035, Run 259737910
[0658] AI_comprehensive panel_v1.0 Summary: Ag3035 Highest
expression of this gene is detected in a COPD sample (CT=30).
Moderate levels of expression of this gene are detected in samples
derived from normal and orthoarthitis/rheumatoid arthritis bone and
adjacent bone, cartilage, synovium and synovial fluid samples, from
normal lung, COPD lung, emphysema, atopic asthma, asthma, allergy,
Crohn's disease (normal matched control and diseased), ulcerative
colitis (normal matched control and diseased), and psoriasis
(normal matched control and diseased). Therefore, therapeutic
modulation of this gene product may ameliorate symptoms/conditions
associated with autoimmune and inflammatory disorders including
psoriasis, allergy, asthma, inflammatory bowel disease, rheumatoid
arthritis and osteoarthritis.
[0659] HASS Panel v1.0 Summary: Ag3035 Expression is seen at a low
level in LnCAP, T24, MCF-7 cell lines and at a moderate level in
CaPan cell line. The expression is slightly increased by a decrease
in oxygen tension in LnCAP cell line but not in any other cell line
suggesting a specific induction inthis prostate cancer cell line. A
low level of expression is also seen in 2 of 4 medulloblastomas and
primary astrocytes and primary renal proximal tubular epithelial
cells.
[0660] Oncology_cell_line_screening_panel_v3.2 Summary: Ag3035
Highest expression of this gene is seen in a lung cancer DMS-79
cell line (28.6). Moderate to low expression of this gene is seen
in number of cancer cell lines derived from tongue, bone, bladder,
pancreatic, cervical, uterine, gastric, colon and lung cancer.
Therefore, expression of this gene may be used as marker to detect
the presence of these cancer. Furthermore, therapeutic modulation
of the activity of this gene or its protein product may be useful
in the treatment of these cancers.
[0661] Panel 1.3D Summary: Ag2261 This gene is expressed at
moderate levels in a number of metabolic tissues, with highest
overall expression seen in fetal skeletal muscle (CTs=30.4-31.8).
The higher levels of expression in fetal skeletal muscle when
compared to adult skeletal muscle suggests that the protein product
encoded by this gene may be useful in treating muscular dystrophy,
Lesch-Nyhan syndrome, myasthenia gravis and other conditions that
result in weak or dystrophic muscle. This gene is also expressed in
adipose, thyroid and heart. Since biologic cross-talk between
adipose and thyroid is a component of some forms of obesity, this
gene product may be a protein therapeutic for the treatment of
metabolic disease, including obesity and Type 2 diabetes.
[0662] Ag3035 This probe/primer set recognizes a distinct portion
of this gene and shows a distinctive expression pattern when
compared to Ag2261. This observation may indicate that the
probe/primer sets can distinguish splice variants of this gene.
Expression of this gene is highest in an ovarian cancer cell line
(CT=30.6). As is the case for probe-primer set Ag2261, expression
of this gene using probe-primer Ag3035 also shows relatively high
levels in fetal skeletal muscle. However, in addition, this gene
shows increased levels of this gene in adult skeletal muscle as
well as in adult and fetal heart. Most other expression is similar
using both probe/primer sets.
[0663] Panel 2D Summary: Ag2261 The expression of this gene was
assessed in two independent runs on panel 2D. This gene was found
to be consistently expressed in samples of breast cancer, uterine
cancer and lung cancer when compared to their respective normal
adjacent tissue controls. Thus, the expression of this gene could
be used to distinguish breast cancer, lung cancer or uterine cancer
from their normal tissues. Moreover, therapeutic modulation of this
gene, through the use of small molecule drugs, antibodies or
protein therapeutics might be of use in the treatment of breast,
lung or uterine cancer.
[0664] Panel 4.1D Summary: Ag3035 This probe/primer set recognizes
a distinct portion of this gene and shows a distinctive expression
pattern when compared to Ag2261 in Panel 4D. This observation may
indicate that the probe/primer sets can distinguish splice variants
of this gene. Expression of this gene is highest in kidney
(CT=30.6). Expression in other samples in this panel is similar
using both probe/primer sets. This gene, a WNT-14 homolog is also
expressed at moderate to low levels in several unstimulated or
cytokine-activated keratinocyte and lung and dermal fibroblast
preparations (CT range 29-34). Thus, WNT-14 encoded by this gene
may be useful as a protein therapeutic that reduces or eliminates
the symptoms of chronic obstructive pulmonary disease, asthma,
emphysema, or psoriasis. In addition, due to its known effects on
development of vertebrate joints, the protein encoded by this gene
may also reduce or eliminate the symptoms of osetoarthritis (See
Hartmann and Tabin, 2001, Wnt-14 Plays a Pivotal Role in Inducing
Synovial Joint Formation in the Developing Appendicular Skeleton
Cell, Vol 104, 341-351).
[0665] Panel 4D Summary: Ag2261 This gene is expressed at low
levels in colon (CT=33.5). Low but significant levels of expression
are also found in the lung, keratinocytes and dermal fibroblast.
Thus, this transcript could be used as a marker for thymic, lung
and skin tissues. The putative Wnt-14 molecule encoded by this gene
may play an important role in the normal homeostasis of these
tissues. Therapeutics designed with the protein encoded by this
gene could be important for maintaining or restoring normal
function to these organs during inflammation.
[0666] Panel 5 Islet Summary: Ag3035 Highest expression of this
gene is seen in a diabetic patient (CT=31.8). Significant
expression of this gene is also seen in islet cells. Therefore,
therapeutic modulation of the activity of this gene or its protein
product may be useful in the treatment of metabolic related disease
such as obesity and diabetes, especially type II diabetes.
[0667] General oncology screening panel_v.sub.--2.4 Summary: Ag3035
Highest expression of this gene is detected in metastatic melanoma
(CT=31.3). Moderate to low expression of this gene is also seen in
cancer and normal adjacent samples from lung, kidney, colon,
prostate cancers. Expression of this gene is higher in metastatic
melanoma, prostate, lung and kidney cancers. Therefore, expression
of this gene may be used as marker to detect the presence of these
cancers and also, therapeutic modulation of the activity of this
gene or its protein product may be useful in the treatment of these
cancers.
[0668] E. CG54818-01: Semaphorin 3E.
[0669] Expression of gene CG54818-01 was assessed using the
primer-probe set Ag2060, described in Table EA. Results of the
RTQ-PCR runs are shown in Tables EB, EC, ED, EE, EF, EG, EH, EI,
EJ, EK, EL and EM.
94TABLE EA Probe Name Ag2060 Start SEQ ID Primers Sequences Length
Position No Forward 5'-atgtttcatgtcccaccattt-3' 21 5174 162 Probe
TET-5'-tttctccctctctctcccaacacaca-3'-TAMRA 26 5201 163 Reverse
5'-agtatgtgaggggtgtgtgtgt-3' 22 5228 164
[0670]
95TABLE EB AI comprehensive panel v1.0 Column A-Rel. Exp.(% Ag2060,
Run 276596902 Tissue Name A 110967 COPD-F 1.0 110980 COPD-F 3.4
110968 COPD-M 1.3 110977 COPD-M 11.3 110989 Emphysema-F 1.3 110992
Emphysema-F 2.9 110993 Emphysema-F 1.1 110994 Emphysema-F 0.3
110995 Emphysema-F 3.8 110996 Emphysema-F 1.2 110997 Asthma-M 1.2
111001 Asthma-F 1.7 111002 Asthma-F 4.3 111003 Atopic Asthma-F 4.0
111004 Atopic Asthma-F 15.6 111005 Atopic Asthma-F 7.5 111006
Atopic Asthma-F 2.8 111417 Allergy-M 4.6 l12347 Allergy-M 0.1
112349 Normal Lung-F 0.1 112357 Normal Lung-F 0.3 112354 Normal
Lung-M 1.3 112374 Crohns-F 0.6 112389 Match Control Crohns-F 1.8
112375 Crohns-F 0.2 112732 Match Control Crohns-F 0.3 112725
Crohns-M 0.9 112378 Match Control Crohns-M 0.2 112387 Crohns-M 0.2
112390 Match Control Crohns-M 7.8 112726 Crohns-M 1.8 112731 Match
Control Crohns-M 3.6 112380 Ulcer Col-F 2.9 112734 Match Control
Ulcer Col-F 2.2 112384 Ulcer Col-F 1.7 112737 Match Control Ulcer
Col-F 0.9 112386 Ulcer Col-F 0.4 112738 Match Control Ulcer Col-F
2.1 112381 Ulcer Col-M 0.1 112735 Match Control Ulcer Col-M 0.3
112382 Ulcer Col-M 6.6 112394 Match Control Ulcer Col-M 0.7 112383
Ulcer Col-M 1.3 112736 Match Control Ulcer Col-M 1.1 112423
Psoriasis-F 1.0 112427 Match Contol Psoriasis-F 10.4 112418
Psoriasis-M 0.9 112723 Match Control Psoriasis-M 0.0 112419
Psoriasis-M 2.4 112424 Match Control Psoriasis-M 2.3 112420
Psoriasis-M 1.6 112425 Match Control Psoriasis-M 18.9 104689 (MF)
OA Bone-Backus 10.7 104690 (MF) Adj "Normal" Bone-Backus 16.7
104691 (MF) OA Synovium-Backus 76.3 104692 (BA) OA Cartilage-Backus
100.0 104694 (BA) OA Bone-Backus 14.1 104695 (BA) Adj "Normal"
Bone-Backus 24.1 104696 (BA) OA Synovium-Backus 26.1 104700 (55) OA
Bone-Backus 6.7 104701 (55) Adj "Normal" Bone-Backus 15.0 104702
(55) OA Synovium-Backus 18.6 117093 OA Cartilage Rep7 0.4 112672 OA
Bone5 1.1 112673 OA Synovium5 0.5 112674 OA Synovial Fluid cells5
0.9 117100 OA Cartilage Rep14 0.1 112756 OA Bone9 1.4 112757 OA
Synovium9 0.4 112758 OA Synovial Fluid Cells9 0.7 117125 RA
Cartilage Rep2 0.6 113492 Bone2 RA 9.0 113493 Synovium2 RA 4.9
113494 Syn Fluid Cells RA 8.8 113499 Cartilage4 RA 9.9 113500 Bone4
RA 13.5 113501 Synovium4 RA 10.5 113502 Syn Fluid Cells4 RA 7.3
113495 Cartilage3 RA 6.0 113496 Bone3 RA 7.9 113497 Synovium3 RA
3.7 113498 Syn Fluid Cells3 RA 6.5 117106 Normal Cartilage Rep20
0.0 113663 Bone3 Normal 0.0 113664 Synovium3 Normal 0.0 113665 Syn
Fluid Cells3 Normal 0.1 117107 Normal Cartilage Rep22 0.1 113667
Bone4 Normal 1.1 113668 Synovium4 Normal 0.8 113669 Syn Fluid
Cells4 Normal 1.1
[0671]
96TABLE EC Ardais Panel v.1.0 Tissue Name A Tissue Name A
136799_Lung cancer(362) 2.6 136787_lung cancer(356) 15.4
136800_Lung NAT(363) 47.3 136788_lung NAT(357) 57.8 136813_Lung
cancer(372) 8.9 136804_Lung cancer(369) 23.3 136814_Lung NAT(373)
47.3 136805_Lung NAT(36A) 40.6 136815_Lung cancer(374) 20.6
136806_Lung cancer(36B) 6.3 136816_Lung NAT(375) 10.3 136807_Lung
NAT(36C) 29.9 136791_Lung cancer(35A) 45.4 136789_lung cancer(358)
4.4 136795_Lung cancer(35E) 97.9 136802_Lung cancer(365) 4.2
136797_Lung cancer(360) 2.0 136803_Lung cancer(368) 100.0
136794_lung NAT(35D) 33.9 136811_Lung cancer(370) 1.1 136818_Lung
NAT(377) 39.2 136810_Lung NAT(36F) 60.7 Column A - Rel. Exp.(%)
Ag2060, Run 267680190
[0672]
97TABLE ED CNS neurodegeneration v1.0 Tissue Name A Tissue Name A
AD 1 Hippo 9.7 Control (Path) 3 Temporal Ctx 0.7 AD 2 Hippo 32.8
Control (Path) 4 Temporal Ctx 11.6 AD 3 Hippo 3.1 AD 1 Occipital
Ctx 24.3 AD 4 Hippo 7.1 AD 2 Occipital Ctx (Missing) 0.0 AD 5 hippo
100.0 AD 3 Occipital Ctx 3.0 AD 6 Hippo 35.1 AD 4 Occipital Ctx
15.0 Control 2 Hippo 49.0 AD 5 Occipital Ctx 17.8 Control 4 Hippo
2.8 AD 6 Occipital Ctx 28.7 Control (Path) 3 Hippo 2.0 Control 1
Occipital Ctx 1.5 AD 1 Temporal Ctx 9.3 Control 2 Occipital Ctx
62.0 AD 2 Temporal Ctx 26.1 Control 3 Occipital Ctx 23.7 AD 3
Temporal Ctx 1.6 Control 4 Occipital Ctx 1.9 AD 4 Temporal Ctx 15.3
Control (Path) 1 Occipital Ctx 97.3 AD 5 Inf Temporal Ctx 57.0
Control (Path) 2 Occipital Ctx 12.7 AD 5 SupTemporal Ctx 19.6
Control (Path) 3 Occipital Ctx 1.4 AD 6 Inf Temporal Ctx 25.5
Control (Path) 4 Occipital Ctx 12.6 AD 6 Sup Temporal Ctx 21.0
Control 1 Parietal Ctx 3.0 Control 1 Temporal Ctx 1.5 Control 2
Parietal Ctx 28.3 Control 2 Temporal Ctx 24.5 Control 3 Parietal
Ctx 12.9 Control 3 Temporal Ctx 16.0 Control (Path) 1 Parietal Ctx
52.1 Control 4 Temporal Ctx 1.0 Control (Path) 2 Parietal Ctx 12.6
Control (Path) 1 33.0 Control (Path) 3 Parietal Ctx 1.5 Temporal
Ctx Control (Path) 2 29.3 Control (Path) 4 Parietal Ctx 15.5
Temporal Ctx Column A - Rel. Exp.(% Ag2060, Run 206231411
[0673]
98TABLE EE HASS Panel v1.0 Tissue Name A Tissue Name A MCF-7 C1 0.0
U87-MG F1 (B) 0.0 MCF-7 C2 0.0 U87-MG F2 0.0 MCF-7 C3 0.1 U87-MG F3
0.2 MCF-7 C4 0.1 U87-MG F4 0.3 MCF-7 C5 0.1 U87-MG F5 0.6 MCF-7 C6
0.0 U87-MG F6 0.8 MCF-7 C7 0.1 U87-MG F7 0.2 MCF-7 C9 0.2 U87-MG F8
0.6 MCF-7 C10 0.0 U87-MG F9 0.0 MCF-7 C11 0.0 U87-MG F10 0.2 MCF-7
C12 0.1 U87-MG F11 0.7 MCF-7 C13 0.1 U87-MG F12 0.6 MCF-7 C15 0.0
U87-MG F13 0.1 MCF-7 C16 0.1 U87-MG F14 0.2 MCF-7 C17 0.0 U87-MG
F15 0.3 T24 D1 0.0 U87-MG F16 0.7 T24 D2 0.0 U87-MG F17 1.0 T24 D3
0.0 LnCAP A1 0.0 T24 D4 0.2 LnCAP A2 0.0 T24 D5 0.1 LnCAP A3 0.0
T24 D6 0.2 LnCAP A4 0.0 T24 D7 0.0 LnCAP A5 0.0 T24 D9 0.0 LnCAP A6
0.0 T24 D10 0.0 LnCAP A7 0.0 T24 D11 0.0 LnCAP A8 0.0 T24 D12 0.2
LnCAP A9 0.0 T24 D13 0.0 LnCAP A10 0.0 T24 D15 0.0 LnCAP A11 0.0
T24 D16 0.0 LnCAP A12 0.0 T24 D17 0.0 LnCAP A13 0.0 CAPaN B1 0.1
LnCAP A14 0.0 CAPaN B2 0.0 LnCAP A15 0.0 CAPaN B3 0.0 LnCAP A16 0.0
CAPaN B4 0.1 LnCAP A17 0.0 CAPaN B5 0.0 Primary Astrocytes 1.4
CAPaN B6 0.0 Primary Renal Proximal Tubule Epithelial 0.3 cell A2
CAPaN B7 0.1 Primary melanocytes A5 2.8 CAPaN B8 0.1 126443 - 341
medullo 0.0 CAPaN B9 0.0 126444 - 487 medullo 0.0 CAPaN B10 0.1
126445 - 425 medullo 0.0 CAPaN B11 0.0 126446 - 690 medullo 0.0
CAPaN B12 0.0 126447 - 54 adult glioma 1.4 CAPaN B13 0.1 126448 -
245 adult glioma 100.0 CAPaN B14 0.0 126449 - 317 adult glioma 6.7
CAPaN B15 0.0 126450 - 212 glioma 0.0 CAPaN B16 0.0 126451 - 456
glioma 0.0 CAPaN B17 0.0 Column A - Rel. Exp.(%) Ag2060, Run
267674609
[0674]
99TABLE EF Oncology_cell_line_screening_panel_v3.2 Tissue Name A
Tissue Name A 94905_Daoy_Medulloblast- oma/Cerebellum_ssc 15.6
94954_Ca Ski_Cervical epidermoid 0.0 DNA carcinoma
(metastasis)_sscDNA 94906_TE671_Medulloblastom/Cerebellu- m_ssc 0.0
94955_ES-2_Ovarian clear cell 0.0 DNA carcinoma_sscDNA 94907_D283
0.2 94957_Ramos/6 h stim_Stimulated with 0.0
Med_Medulloblastoma/Cerebellum_sscDNA PMA/ionomycin 6 h_sscDNA
94908_PFSK-1_Primitive 100.0 94958_Ramos/14 h stim_Stimulated 0.0
Neuroectodermal/Cerebellum_sscDNA with PMA/ionomycin 14 h_sscDNA
94909_XF-498_CNS_sscDNA 51.1 94962_MEG-01_Chronic myelogenous 0.0
leukemia (megokaryoblast)_sscDNA 94910_SNB-78_CNS/glioma_sscDNA 0.0
94963_Raji_Burkitt's 0.0 lymphoma_sscDNA
94911_SF-268_CNS/glioblastoma_sscDNA 0.7 94964_Daudi_Burkitt's 0.0
lymphoma_sscDNA 94912_T98G_Glioblastoma_sscDNA 0.0
94965_U266_B-cell 0.0 plasmacytoma/myeloma_sscDNA
96776_SK-N-SH_Neuroblastoma 0.6 94968_CA46_Burkitt's 0.0
(metastasis)_sscDNA lymphoma_sscDNA 94913_SF-295_CNS/glioblastoma-
_sscDNA 0.0 94970_RL_non-Hodgkin's B-cell 0.0 lymphoma_sscDNA
132565_NT2 pool_sscDNA 0.0 94972_JM1_pre-B-cell 0.0
lymphoma/leukemia_sscDNA 94914_Cerebellum_sscDNA 20.6
94973_Jurkat_T cell leukemia_sscDNA 0.0 96777_Cerebellum_sscDNA 0.1
94974_TF-1_Erythroleukemia_sscDNA 0.0 94916_NCI-H292_Mucoepide-
rmoid lung 0.2 94975_HUT 78_T-cell 0.0 carcinoma_sscDNA
lymphoma_sscDNA 94917_DMS-114_Small cell lung 0.9
94977_U937_Histiocytic 0.0 cancer_sscDNA lymphoma_sscDNA
94918_DMS-79_Small cell lung 3.8 94980_KU-812_Myelogenous 40.9
cancer/neuroendocrine_sscDNA leukemia_sscDNA 94919_NCI-H146_Small
cell lung 3.0 94981_769-P_Clear cell renal 0.0
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94920_NCI-H526_Small
cell lung 2.4 94983_Caki-2_Clear cell renal 3.6
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94921_NCI-N417_Small
cell lung 6.4 94984_SW 839_Clear cell renal 0.3
cancer/neuroendocrine_sscDNA carcinoma_sscDNA 94923_NCI-H82_Small
cell lung 0.0 94986_G401_Wilms' tumor_sscDNA 0.9
cancer/neuroendocrine_sscDNA 94924_NCI-H157_Squamous cell lung
cancer 2.0 126768_293 cells_sscDNA 6.0 (metastasis)_sscDNA
94925_NCI-H1155_Large cell lung 1.4 94987_Hs766T_Pancreatic
carcinoma 0.0 cancer/neuroendocrine_sscDNA (LN metastasis)_sscDNA
94926_NCI-H1299_Large cell lung 4.2 94988_CAPAN-1_Pancreatic 4.3
cancer/neuroendocrine_sscDNA adenocarcinoma (liver
metastasis)_sscDNA 94927_NCI-H727_Lung carcinoid_sscDNA 0.9
94989_SU86.86_Pancreatic carcinoma 2.8 (liver metastasis)_sscDNA
94928_NCI-UMC-11_Lung carcinoid_sscDNA 0.0 94990_BxPC-3_Pancreatic
1.2 adenocarcinoma_sscDNA 94929_LX-1_Small cell lung cancer_sscDNA
0.3 94991_HPAC_Pancreatic 4.6 adenocarcinoma_sscDNA
94930_Colo-205_Colon cancer_sscDNA 0.0 94992_MIA PaCa-2_Pancreatic
0.3 carcinoma_sscDNA 94931_KM12_Colon cancer_sscDNA 0.0
94993_CFPAC-1_Pancreatic ductal 1.7 adenocarcinoma_sscDNA
94932_KM20L2_Colon cancer_sscDNA 0.9 94994_PANC-1_Pancreatic
epithelioid 6.8 ductal carcinoma_sscDNA 94933_NCI-H716_Colon
cancer_sscDNA 4.7 94996_T24_Bladder carcinma 0.0 (transitional
cell)_sscDNA 94935_SW-48_Colon adenocarcinoma_sscDNA 0.0
94997_5637_Bladder 0.0 carcinoma_sscDNA 94936_SW1116_Colon 0.0
94998_HT-1197_Bladder 0.0 adenocarcinoma_sscDNA carcinoma_sscDNA
94937_LS 174T_Colon 0.0 94999_UM-UC-3_Bladder carcinma 0.1
adenocarcinoma_sscDNA (transitional cell)_sscDNA 94938_SW-948_Colon
0.0 95000_A204_Rhabdomyosarcoma_ssc 0.0 adenocarcinoma_sscDNA DNA
94939_SW-480_Colon 0.0 95001_HT-1080_Fibrosarcoma_sscDNA 0.0
adenocarcinoma_sscDNA 94940_NCI-SNU-5_Gastric carcinoma_sscDNA 0.0
95002_MG-63_Osteosarcoma 0.3 (bone)_sscDNA 112197_KATO
III_Stomach_sscDNA 0.0 95003_SK-LMS-1_Leiomyosarcoma 52.9
(vulva)_sscDNA 94943_NCI-SNU-16_Gastric 1.9
95004_SJRH30_Rhabdomyosarcoma 0.2 carcinoma_sscDNA (met to bone
marrow)_sscDNA 94944_NCI-SNU-1_Gastric carcinoma_sscDNA 11.4
95005_A431_Epidermoid 0.0 carcinoma_sscDNA 94946_RF-1_Gastric
adenocarcinoma_sscDNA 0.0 95007_WM266-4_Melanoma_sscDN- A 21.8
94947_RF-48_Gastric 0.0 112195_DU 145_Prostate_sscDNA 0.0
adenocarcinoma_sscDNA 96778_MKN-45_Gastric carcinoma_sscDNA 0.0
95012_MDA-MB-468_Breast 14.3 adenocarcinoma_sscDNA
94949_NCI-N87_Gastric carcinoma_sscDNA 1.0
112196_SSC-4_Tongue_sscDNA 0.0 94951_OVCAR-5_Ovarian
carcinoma_sscDNA 0.9 112194_SSC-9_Tongue_ssc- DNA 0.3
94952_RL95-2_Uterine carcinoma_sscDNA 0.3
112191_SSC-15_Tongue_sscDNA 0.0 94953_HelaS3_Cervical 0.0 95017_CAL
27_Squamous cell 1.7 adenocarcinoma_sscDNA carcinoma of
tongue_sscDNA Column A - Rel. Exp.(%) Ag2060, Run 311303311
[0675]
100TABLE EG Panel 1.3D Tissue Name A Tissue Name A Liver
adenocarcinoma 0.2 Kidney (fetal) 0.7 Pancreas 0.8 Renal ca. 786-0
0.8 Pancreatic ca. CAPAN 2 0.5 Renal ca. A498 0.0 Adrenal gland 0.8
Renal ca. RXF 393 0.0 Thyroid 1.5 Renal ca. ACHN 2.9 Salivary gland
0.9 Renal ca. UO-31 41.8 Pituitary gland 2.7 Renal ca. TK-10 0.0
Brain (fetal) 17.3 Liver 0.0 Brain (whole) 42.9 Liver (fetal) 0.4
Brain (amygdala) 92.7 Liver ca. (hepatoblast) HepG2 0.0 Brain
(cerebellum) 4.8 Lung 28.3 Brain (hippocampus) 100.0 Lung (fetal)
21.3 Brain (substantia nigra) 13.6 Lung ca. (small cell) LX-1 0.5
Brain (thalamus) 47.0 Lung ca. (small cell) NCI-H69 0.0 Cerebral
Cortex 27.7 Lung ca. (s. cell var.) SHP-77 16.6 Spinal cord 11.0
Lung ca. (large cell) NCI-H460 6.1 glio/astro U87-MG 0.3 Lung ca.
(non-sm. cell) A549 0.4 glio/astro U-118-MG 11.9 Lung ca. (non-s.
cell) NCI-H23 13.6 astrocytoma SW1783 0.5 Lung ca. (non-s. cell)
HOP-62 2.6 neuro*; met SK-N-AS 1.5 Lung ca. (non-s. cl) NCI-H522
0.0 astrocytoma SF-539 0.3 Lung ca. (squam.) SW 900 7.1 astrocytoma
SNB-75 5.6 Lung ca. (squam.) NCI-H596 0.0 glioma SNB-19 0.2 Mammary
gland 12.2 glioma U251 6.8 Breast ca.* (pl.ef) MCF-7 0.0 glioma
SF-295 0.0 Breast ca.* (pl.ef) MDA-MB-231 1.0 Heart (fetal) 0.0
Breast ca.* (pl.ef) T47D 3.6 Heart 0.0 Breast ca. BT-549 0.2
Skeletal muscle (fetal) 1.2 Breast ca. MDA-N 3.1 Skeletal muscle
0.4 Ovary 0.9 Bone marrow 0.5 Ovarian ca. OVCAR-3 0.2 Thymus 3.7
Ovarian ca. OVCAR-4 0.2 Spleen 0.4 Ovarian ca. OVCAR-5 15.7 Lymph
node 0.2 Ovarian ca. OVCAR-8 13.5 Colorectal 6.9 Ovarian ca.
IGROV-1 0.0 Stomach 8.2 Ovarian ca.* (ascites) SK-OV-3 0.0 Small
intestine 32.1 Uterus 6.0 Colon ca. SW480 0.2 Placenta 0.4 Colon
ca.* SW620(SW480 met) 0.0 Prostate 6.5 Colon ca. HT29 1.4 Prostate
ca.* (bone met)PC-3 0.0 Colon ca. HCT-116 1.0 Testis 0.4 Colon ca.
CaCo-2 0.0 Melanoma Hs688(A).T 0.0 Colon ca. tissue(ODO3866) 1.0
Melanoma* (met) Hs688(B).T 0.1 Colon ca. HCC-2998 0.0 Melanoma
UACC-62 0.6 Gastric ca.* (liver met) NCI-N87 5.8 Melanoma M14 0.0
Bladder 1.2 Melanoma LOX IMVI 0.0 Trachea 10.5 Melanoma* (met)
SK-MEL-5 2.2 Kidney 0.0 Adipose 1.3 Colun A - Rel. Exp.(%) Ag2060,
Run 160163887
[0676]
101TABLE EH Panel 2.2 Tissue Name A Tissue Name A Normal Colon 21.8
Kidney Margin (OD04348) 11.6 Colon cancer (OD06064) 27.4 Kidney
malignant cancer (OD06204B) 1.5 Colon Margin (OD06064) 32.8 Kidney
normal adjacent tissue 3.2 (OD06204E) Colon cancer (OD06159) 0.1
Kidney Cancer (OD04450-01) 2.7 Colon Margin (OD06159) 31.0 Kidney
Margin (OD04450-03) 1.6 Colon cancer (OD06297-04) 0.0 Kidney Cancer
8120613 0.0 Colon Margin (OD06297-05) 31.0 Kidney Margin 8120614
0.0 CC Gr.2 ascend colon (ODO3921) 0.0 Kidney Cancer 9010320 0.0 CC
Margin (ODO3921) 2.8 Kidney Margin 9010321 0.0 Colon cancer
metastasis (OD06104) 0.0 Kidney Cancer 8120607 0.0 Lung Margin
(OD06104) 2.9 Kidney Margin 8120608 0.0 Colon mets to lung
(OD04451-01) 3.5 Normal Uterus 8.8 Lung Margin (OD04451-02) 72.2
Uterine Cancer 064011 1.7 Normal Prostate 14.9 Normal Thyroid 2.0
Prostate Cancer (OD04410) 0.3 Thyroid Cancer 064010 5.7 Prostate
Margin (OD04410) 27.0 Thyroid Cancer A302152 0.8 Normal Ovary 1.8
Thyroid Margin A302153 0.4 Ovarian cancer (OD06283-03) 0.0 Normal
Breast 55.1 Ovarian Margin (OD06283-07) 4.3 Breast Cancer (OD04566)
1.3 Ovarian Cancer 064008 2.0 Breast Cancer 1024 32.8 Ovarian
cancer (OD06145) 12.9 Breast Cancer (OD04590-01) 0.2 Ovarian Margin
(OD06145) 5.5 Breast Cancer Mets (OD04590-03) 1.5 Ovarian cancer
(OD06455-03) 39.5 Breast Cancer Metastasis (OD04655-05) 8.3 Ovarian
Margin (OD06455-07) 32.3 Breast Cancer 064006 19.6 Normal Lung 24.7
Breast Cancer 9100266 6.2 Invasive poor diff. lung adeno 18.9
Breast Margin 9100265 4.4 (ODO4945-01 Lung Margin (ODO4945-03) 40.1
Breast Cancer A209073 14.2 Lung Malignant Cancer (OD03126) 45.4
Breast Margin A2090734 5.9 Lung Margin (OD03126) 6.2 Breast cancer
(OD06083) 45.7 Lung Cancer (OD05014A) 3.1 Breast cancer node
metastasis (OD06083) 9.3 Lung Margin (OD05014B) 100.0 Normal Liver
5.2 Lung cancer (OD06081) 0.0 Liver Cancer 1026 0.0 Lung Margin
(OD06081) 18.7 Liver Cancer 1025 0.6 Lung Cancer (OD04237-01) 13.7
Liver Cancer 6004-T 2.8 Lung Margin (OD04237-02) 77.9 Liver Tissue
6004-N 1.4 Ocular Melanoma Metastasis 0.0 Liver Cancer 6005-T 0.0
Ocular Melanoma Margin (Liver) 0.0 Liver Tissue 6005-N 0.4 Melanoma
Metastasis 0.0 Liver Cancer 064003 0.0 Melanoma Margin (Lung) 37.4
Normal Bladder 0.0 Normal Kidney 1.0 Bladder Cancer 1023 0.0 Kidney
Ca, Nuclear grade 2 (OD04338) 3.2 Bladder Cancer A302173 3.6 Kidney
Margin (OD04338) 2.8 Normal Stomach 67.4 Kidney Ca Nuclear grade
1/2 (OD04339) 16.0 Gastric Cancer 9060397 0.9 Kidney Margin
(OD04339) 0.0 Stomach Margin 9060396 4.2 Kidney Ca, Clear cell type
(OD04340) 0.5 Gastric Cancer 9060395 10.1 Kidney Margin (OD04340)
3.8 Stomach Margin 9060394 8.4 Kidney Ca, Nuclear grade 3 (OD04348)
0.0 Gastric Cancer 064005 8.4 Coumn A - Rel. Exp.(%) Ag2060, Run
174166860
[0677]
102TABLE EI Panel 3D Tissue Name A Tissue Name A
Daoy-Medulloblastoma 42.6 Ca Ski-Cervical epidermoid carcinoma 0.0
(metastasis) TE671-Medulloblastoma 0.0 ES-2-Ovarian clear cell
carcinoma 0.0 D283 Med-Medulloblastoma 0.0 Ramos-Stimulated with
PMA/ionomycin 6 h 0.0 PFSK-1-Primitive Neuroectodermal 100.0
Ramos-Stimulated with PMA/ionomycin 14 h 0.0 XF-498-CNS 37.9
MEG-01-Chronic myelogenous leukemia 0.0 (megokaryoblast)
SNB-78-Glioma 0.0 Raji-Burkitt's lymphoma 0.0 SF-268-Glioblastoma
0.0 Daudi-Burkitt's lymphoma 0.0 T98G-Glioblastoma 0.5 U266-B-cell
plasmacytoma 0.0 SK-N-SH-Neuroblastoma (metastasis) 0.5
CA46-Burkitt's lymphoma 0.0 SF-295-Glioblastoma 0.0
RL-non-Hodgkin's B-cell lymphoma 0.0 Cerebellum 34.6 JM1-pre-B-cell
lymphoma 0.0 Cerebellum 1.2 Jurkat-T cell leukemia 0.0
NCI-H292-Mucoepidermoid lung 0.0 TF-1-Erythroleukemia 0.0 carcinoma
DMS-114-Small cell lung cancer 0.7 HUT 78-T-cell lymphoma 0.0
DMS-79-Small cell lung cancer 5.3 U937-Histiocytic lymphoma 0.0
NCI-H146-Small cell lung cancer 5.3 KU-812-Myelogenous leukemia
32.1 NCI-H526-Small cell lung cancer 3.8 769-P-Clear cell renal
carcinoma 0.0 NCI-N417-Small cell lung cancer 13.1 Caki-2-Clear
cell renal carcinoma 2.0 NCI-H82-Small cell lung cancer 5.3 SW
839-Clear cell renal carcinoma 2.0 NCI-H157-Squamous cell lung
cancer 0.0 Rhabdoid kidney tumor 0.3 (metastasis) NCI-H1155-Large
cell lung cancer 0.8 Hs766T-Pancreatic carcinoma (LN 0.0
metastasis) NCI-H1299-Large cell lung cancer 4.5 CAPAN-1-Pancreatic
adenocarcinoma (liver 2.8 metastasis) NCI-H727-Lung carcinoid 2.2
SU86.86-Pancreatic carcinoma (liver 3.6 metastasis) NCI-UMC-11-Lung
carcinoid 0.6 BxPC-3-Pancreatic adenocarcinoma 0.0 LX-1-Small cell
lung cancer 0.0 HPAC-Pancreatic adenocarcinoma 6.4 Colo-205-Colon
cancer 0.0 MIA PaCa-2-Pancreatic carcinoma 0.0 KM12-Colon cancer
0.0 CFPAC-1-Pancreatic ductal adenocarcinoma 0.0 KM20L2-Colon
cancer 2.2 PANC-1-Pancreatic epithelioid ductal 9.7 carcinoma
NCI-H716-Colon cancer 13.4 T24-Bladder carcinma (transitional cell)
1.2 SW-48-Colon adenocarcinoma 0.0 5637-Bladder carcinoma 0.0
SW1116-Colon adenocarcinoma 0.0 HT-1197-Bladder carcinoma 0.0 LS
174T-Colon adenocarcinoma 0.0 UM-UC-3-Bladder carcinma
(transitional 0.5 cell) SW-948-Colon adenocarcinoma 0.0
A204-Rhabdomyosarcoma 0.0 SW-480-Colon adenocarcinoma 0.0
HT-1080-Fibrosarcoma 0.0 NCI-SNU-5-Gastric carcinoma 0.0
MG-63-Osteosarcoma 0.8 KATO III-Gastric carcinoma 0.0
SK-LMS-1-Leiomyosarcoma (vulva) 80.7 NCI-SNU-16-Gastric carcinoma
1.8 SJRH30-Rhabdomyosarcoma (met to bone 0.0 marrow)
NCI-SNU-1-Gastric carcinoma 28.9 A431-Epidermoid carcinoma 0.0
RF-1-Gastric adenocarcinoma 0.0 WM266-4-Melanoma 30.8 RF-48-Gastric
adenocarcinoma 0.0 DU 145-Prostate carcinoma (brain metastasis) 0.0
MKN-45-Gastric carcinoma 0.0 MDA-MB-468-Breast adenocarcinoma 5.2
NCI-N87-Gastric carcinoma 1.4 SCC-4-Squamous cell carcinoma of
tongue 0.0 OVCAR-5-Ovarian carcinoma 2.1 SCC-9-Squamous cell
carcinoma of tongue 0.0 RL95-2-Uterine carcinoma 1.1
SCC-15-Squamous cell carcinoma of tongue 0.0 HelaS3-Cervical
adenocarcinoma 0.0 CAL 27-Squamous cell carcinoma of tongue 0.3
Clumn A - Rel. Exp.(%) Ag2060, Run 358962242
[0678]
103TABLE EJ Panel 4D Tissue Name A Tissue Name A Secondary Th1 act
0.8 HUVEC IL-1beta 0.7 Secondary Th2 act 0.8 HUVEC IFN gamma 0.0
Secondary Tr1 act 0.0 HUVEC TNF alpha + IFN gamma 0.0 Secondary Th1
rest 0.0 HUVEC TNF alpha + IL4 0.0 Secondary Th2 rest 0.0 HUVEC
IL-11 0.0 Secondary Tr1 rest 1.7 Lung Microvascular EC none 0.0
Primary Th1 act 0.0 Lung Microvascular EC TNF alpha + IL-1beta 0.3
Primary Th2 act 0.0 Microvascular Dermal EC none 0.0 Primary Tr1
act 0.0 Microsvasular Dermal EC TNF alpha + IL-1beta 0.0 Primary
Th1 rest 0.0 Bronchial epithelium TNF alpha + IL1beta 3.1 Primary
Th2 rest 0.0 Small airway epithelium none 2.5 Primary Tr1 rest 0.0
Small airway epithelium TNF alpha + IL-1beta 6.6 CD45RA CD4
lymphocyte act 0.0 Coronery artery SMC rest 0.0 CD45RO CD4
lymphocyte act 0.0 Coronery artery SMC TNF alpha + IL-1beta 0.0 CD8
lymphocyte act 0.0 Astrocytes rest 0.7 Secondary CD8 lymphocyte
rest 0.0 Astrocytes TNF alpha + IL-1beta 7.0 Secondary CD8
lymphocyte act 0.0 KU-812 (Basophil) rest 61.6 CD4 lymphocyte none
0.0 KU-812 (Basophil) PMA/ionomycin 100.0 2ry Th1/Th2/Tr1_anti-CD95
CH11 0.0 CCD1106 (Keratinocytes) none 0.0 LAK cells rest 0.0
CCD1106 (Keratinocytes) TNF alpha + IL-1beta 0.0 LAK cells IL-2 1.2
Liver cirrhosis 23.0 LAK cells IL-2 + IL-12 0.0 Lupus kidney 1.9
LAK cells IL-2 + IFN gamma 0.0 NCI-H292 none 1.0 LAK cells IL-2 +
IL-18 0.0 NCI-H292 IL-4 0.0 LAK cells PMA/ionomycin 0.0 NCI-H292
IL-9 0.0 NK Cells IL-2 rest 0.3 NCI-H292 IL-13 0.4 Two Way MLR 3
day 1.9 NCI-H292 IFN gamma 0.0 Two Way MLR 5 day 0.0 HPAEC none 0.0
Two Way MLR 7 day 0.0 HPAEC TNF alpha + IL-1beta 0.0 PBMC rest 0.0
Lung fibroblast none 7.7 PBMC PWM 0.0 Lung fibroblast TNF alpha +
IL-1beta 0.0 PBMC PHA-L 0.0 Lung fibroblast IL-4 6.5 Ramos (B cell)
none 0.0 Lung fibroblast IL-9 6.4 Ramos (B cell) ionomycin 0.0 Lung
fibroblast IL-13 8.1 B lymphocytes PWM 0.0 Lung fibroblast IFN
gamma 14.9 B lymphocytes CD40L and IL-4 0.0 Dermal fibroblast
CCD1070 rest 1.7 EOL-1 dbcAMP 0.0 Dermal fibroblast CCD1070 TNF
alpha 1.7 EOL-1 dbcAMP PMA/ionomycin 1.5 Dermal fibroblast CCD1070
IL-1beta 0.0 Dendritic cells none 0.0 Dermal fibroblast IFN gamma
6.9 Dendritic cells LPS 0.6 Dermal fibroblast IL-4 5.9 Dendritic
cells anti-CD40 0.0 IBD Colitis 2 1.4 Monocytes rest 0.0 IBD
Crohn's 18.7 Monocytes LPS 0.0 Colon 51.1 Macrophages rest 0.0 Lung
15.8 Macrophages LPS 0.0 Thymus 3.4 HUVEC none 0.6 Kidney 33.9
HUVEC starved 0.0 Coumn A - Rel. Exp.(%) Ag2060, Run 164295986
[0679]
104TABLE EK Panel 5 Islet Tissue Name A Tissue Name A
97457_Patient-02go_adipose 10.4 94709_Donor 2 AM - A_adipose 0.0
97476_Patient-07sk_skeletal muscle 5.7 94710_Donor 2 AM - B_adipose
0.0 97477_Patient-07ut_uterus 3.2 94711_Donor 2 AM - C_adipose 0.0
97478_Patient-07pl_placenta 1.0 94712_Donor 2 AD - A_adipose 2.1
99167_Bayer Patient 1 0.6 94713_Donor 2 AD - B_adipose 0.0
97482_Patient-08ut_uterus 1.0 94714_Donor 2 AD - C_adipose 0.0
97483_Patient-08pl_placenta 5.3 94742_Donor 3 U - A_Mesenchymal
Stem 0.0 Cells 97486_Patient-09sk_skeletal muscle 1.8 94743_Donor 3
U - B_Mesenchymal Stem 0.0 Cells 97487_Patient-09ut_uterus 5.6
94730_Donor 3 AM - A_adipose 0.0 97488_Patient-09pl_placenta 0.0
94731_Donor 3 AM - B_adipose 0.0 97492_Patient-10ut_uterus 2.6
94732_Donor 3 AM - C_adipose 0.0 97493_Patient-10pl_placenta 6.3
94733_Donor 3 AD - A_adipose 0.0 97495_Patient-11go_adipose 1.2
94734_Donor 3 AD - B_adipose 0.0 97496_Patient-11sk_skeletal muscle
2.6 94735_Donor 3 AD - C_adipose 0.0 97497_Patient-11ut_uterus 5.0
77138_Liver_HepG2untreated 0.0 97498_Patient-11pl_placenta 3.3
73556_Heart_Cardiac stromal cells 0.0 (primary)
97500_Patient-12go_adipose 2.2 81735_Small Intestine 100.0
97501_Patient-12sk_skeletal muscle 0.0 72409_Kidney_Proximal
Convoluted 1.2 Tubule 97502_Patient-12ut_uterus 2.1 82685_Small
intestine_Duodenum 17.4 97503_Patient-12pl_placenta 0.0
90650_Adrenal_Adrenocortical adenoma 1.4 94721_Donor 2 U -
A_Mesenchymal 0.0 72410_Kidney_HRCE 0.9 Stem Cells 94722_Donor 2 U
- B_Mesenchymal Stem 0.0 72411_Kidney_HRE 1.7 Cells 94723_Donor 2 U
- C_Mesenchymal Stem 0.0 73139_Uterus_Uterine smooth muscle cells
1.6 Cells Colum A - Rel. Exp.(%) Ag2060, Run 267095055
[0680]
105TABLE EL Panel CNS 1 Tissue Name A Tissue Name A BA4 Control
54.7 BA17 PSP 52.9 BA4 Control2 55.9 BA17 PSP2 26.6 BA4
Alzheimer's2 10.1 Sub Nigra Control 37.6 BA4 Parkinson's 32.8 Sub
Nigra Control2 27.4 BA4 Parkinson's2 45.1 Sub Nigra Alzheimer's2
20.0 BA4 Huntington's 32.1 Sub Nigra Parkinson's2 41.5 BA4
Huntington's2 8.9 Sub Nigra Huntington's 74.7 BA4 PSP 10.7 Sub
Nigra Huntington's2 55.5 BA4 PSP2 56.3 Sub Nigra PSP2 21.0 BA4
Depression 21.9 Sub Nigra Depression 10.6 BA4 Depression2 12.2 Sub
Nigra Depression2 12.7 BA7 Control 32.8 Glob Palladus Control 4.1
BA7 Control2 34.9 Glob Palladus Control2 12.2 BA7 Alzheimer's2 13.3
Glob Palladus Alzheimer's 17.4 BA7 Parkinson's 7.5 Glob Palladus
Alzheimer's2 3.6 BA7 Parkinson's2 23.8 Glob Palladus Parkinson's
40.6 BA7 Huntington's 41.5 Glob Palladus Parkinson's2 7.2 BA7
Huntington's2 24.7 Glob Palladus PSP 13.7 BA7 PSP 34.6 Glob
Palladus PSP2 7.4 BA7 PSP2 31.2 Glob Palladus Depression 7.5 BA7
Depression 20.7 Temp Pole Control 18.8 BA9 Control 28.9 Temp Pole
Control2 21.2 BA9 Control2 100.0 Temp Pole Alzheimer's 9.0 BA9
Alzheimer's 10.9 Temp Pole Alzheimer's2 4.6 BA9 Alzheimer's2 10.6
Temp Pole Parkinson's 18.0 BA9 Parkinson's 16.4 Temp Pole
Parkinson's2 23.0 BA9 Parkinson's2 39.8 Temp Pole Huntington's 32.5
BA9 Huntington's 28.1 Temp Pole PSP 11.7 BA9 Huntington's2 8.4 Temp
Pole PSP2 6.0 BA9 PSP 33.2 Temp Pole Depression2 13.8 BA9 PSP2 9.3
Cing Gyr Control 64.6 BA9 Depression 16.4 Cing Gyr Control2 35.6
BA9 Depression2 10.1 Cing Gyr Alzheimer's 35.4 BA17 Control 50.7
Cing Gyr Alzheimer's2 10.2 BA17 Control2 62.4 Cing Gyr Parkinson's
17.2 BA17 Alzheimer's2 33.9 Cing Gyr Parkinson's2 21.6 BA17
Parkinson's 27.5 Cing Gyr Huntington's 43.8 BA17 Parkinson's2 42.3
Cing Gyr Huntington's2 14.7 BA17 Huntington's 62.0 Cing Gyr PSP
30.1 BA17 Huntington's2 26.2 Cing Gyr PSP2 6.1 BA17 Depression 17.4
Cing Gyr Depression 10.3 BA17 Depression2 35.6 Cing Gyr Depression2
21.8 Colum A - Rel. Exp.(%) Ag2060, Run 171628579
[0681]
106TABLE EM general oncology screening panel_v_2.4 Tissue Name A
Tissue Name A Colon cancer 1 0.3 Bladder cancer NAT 2 1.2 Colon
cancer NAT 1 1.0 Bladder cancer NAT 3 0.5 Colon cancer 2 2.5
Bladder cancer NAT 4 7.8 Colon cancer NAT 2 1.8 Prostate
adenocarcinoma 1 100.0 Colon cancer 3 0.2 Prostate adenocarcinoma 2
3.6 Colon cancer NAT 3 9.0 Prostate adenocarcinoma 3 5.0 Colon
malignant cancer 4 0.2 Prostate adenocarcinoma 4 2.0 Colon normal
adjacent tissue 1.2 Prostate cancer NAT 5 4.9 4 Lung cancer 1 0.4
Prostate adenocarcinoma 6 4.5 Lung NAT 1 0.6 Prostate
adenocarcinoma 7 9.5 Lung cancer 2 6.2 Prostate adenocarcinoma 8
3.1 Lung NAT 2 2.4 Prostate adenocarcinoma 9 50.0 Squamous cell
carcinoma 3 0.5 Prostate cancer NAT 10 1.3 Lung NAT 3 0.5 Kidney
cancer 1 0.0 metastatic melanoma 1 1.1 KidneyNAT 1 0.5 Melanoma 2
0.2 Kidney cancer 2 0.1 Melanoma 3 1.6 Kidney NAT 2 0.7 metastatic
melanoma 4 0.2 Kidney cancer 3 0.1 metastatic melanoma 5 0.5 Kidney
NAT 3 0.6 Bladder cancer 1 3.2 Kidney cancer 4 0.0 Bladder cancer
NAT 1 0.0 Kidney NAT 4 0.2 Bladder cancer 2 16.7 Column A - Rel.
Exp.(%) Ag2060, Run 267145079
[0682] AI_comprehensive panel_v1.0 Summary: Ag2060 Highest
expression of this gene is seen in osteooarthritis cartilage sample
(CT=24.4). High expression of this gene is seen in normal and
osteooarthitis/rheumatoid arthritis bone and adjacent bone,
cartilage, synovium and synovial fluid samples. In addition,
moderate to low expression of this gene is also seen in normal
lung, COPD lung, emphysema, atopic asthma, asthma, allergy, Crohn's
disease (normal matched control and diseased), ulcerative colitis
(normal matched control and diseased), and psoriasis (normal
matched control and diseased). Therefore, therapeutic modulation of
this gene product may ameliorate symptoms/conditions associated
with autoimmune and inflammatory disorders including psoriasis,
allergy, asthma, inflammatory bowel disease, rheumatoid arthritis
and osteoarthritis.
[0683] Ardais Panel v.1.0 Summary: Ag2060 Highest expression of
this gene is seen in a lung cancer sample (CT=27.7). High
expression of this gene is seen in cancer and normal adjacent
samples from lung. Interestingly, expression of this gene is higher
in the normal adjacent lung compared to the corresponding cancer
samples. Therefore, this gene could be acting as a tumor suppressor
through a potential chemo repulsive effect on vascular endothelial
cells. Thus, therapeutic modulation of the activity of this gene or
its protein product may be useful in the treatment of lung
cancer.
[0684] CNS_neurodegeneration_v1.0 Summary: Ag2060 This panel
confirms the expression of this gene at low levels in the brains of
an independent group of individuals.
[0685] HASS Panel v1.0 Summary: Ag2060 Highest expression of this
gene is seen in adult glioma sample (CT=25). Moderate to low
expression of this gene is also seen in adult glioma, brain cancer
cell U87-MG cell line, primary astrocytes, renal proximal tubule
epithlial cell and melanocytes. Therefore, therapeutic modulation
of this gene or its protein product may be useful in the treatment
of brain cancer and brain related diseases.
[0686] Oncology_cell_line_screening_panel_v3.2 Summary: Ag2060
Highest expression of this gene is seen in a brain cancer PFSK-1
cell line (CT=29.7). Moderate to low expression of this gene is
also seen in number of cancer cell lines derived from brain,
breast, melanoma, leiomyosarcoma, pancreatic, myelogenous leukemia,
gastric, colon and lung cancers. Therefore, therapeutic modulation
of this gene or its protein product may be useful in the treatement
of brain, breast, melanoma, leiomyosarcoma, pancreatic, myelogenous
leukemia, gastric, colon and lung cancers.
[0687] Panel 1.3D Summary: Ag2060 Highest expression of this gene
is seen in brain hippocampus sample (CT=28.3). This gene is
expressed at moderate to high levels in all regions of the central
nervous system examined, including amygdala, hippocampus,
substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal
cord. This gene codes for semaphorin 3E protein. The semaphorin
family of molecules are largely involved in axon guidance and
modulation of CNS motility. Semaphorin 3E has been shown to be
involved in the chemo-repulsion and collapse of neuron growth cones
(Pozas E, Pascual M, Nguyen Ba-Charvet K T, Guijarro P, Sotelo C,
Chedotal A, Del Rio J A, Soriano E. Age-dependent effects of
secreted Semaphorins 3A, 3F, and 3E on developing hippocampal
axons: in vitro effects and phenotype of Semaphorin 3A (-/-) mice.
Mol Cell Neurosci 2001 July; 18(1):26-43.). Therefore, therapeutic
modulation of this gene product may be useful in the treatment of
central nervous system disorders such as Alzheimer's disease,
Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia
and depression.
[0688] Moderate to low expression of this gene is also seen in
number of cancer cell lines derived from brain, melanoma, breast,
ovarian, lung, renal, gastric and colon cancers. Therefore,
therapeutic modulation of this gene or its protein product may be
useful in the treatment of brain, melanoma, breast, ovarian, lung,
renal, gastric and colon cancers.
[0689] Among tissues with metabolic or endocrine function, this
gene is expressed at low levels in adrenal gland, thyroid,
pituitary gland, fetal skeletal muscle, and the gastrointestinal
tract. Therefore, therapeutic modulation of the activity of this
gene may prove useful in the treatment of endocrine/metabolically
related diseases, such as obesity and diabetes.
[0690] Panel 2.2 Summary: Ag2060 Highest expression of this gene is
seen in normal lung (CT=29.6). Moderate to low expression of this
gene is also seen in cancer and normal adjacent samples derived
from lung, stomach, liver, breast, thyroid, uterus, kidney, ovary,
prostate and colon. Interestingly, expression of this gene is
higher in normal samples compared to the corresponding cancer
samples. Therefore, therapeutic modulation of this gene or its
protein product may be useful in the treatment of lung, stomach,
liver, breast, thyroid, uterus, kidney, ovary, prostate and colon
cancers.
[0691] Panel 3D Summary: Ag2060 Highest expression of this gene is
seen in a brain cancer PFSK-1 cell line (CT=28.7). The expression
pattern of this gene correlates with the expression seen in panel
3.2.
[0692] Panel 4D Summary: Ag2060 Highest expression of this gene is
seen in activated basophils (CT=30.2). In addition, moderate to low
expression of this gene is also seen in activated astrocytes,
resting basophils, resting and activated lung fibroblasts,
activated dermal fibroblasts, liver cirrhosis, IBD Crohn's and
normal tissues represented by colon, lung and kidney. Therefore,
therapeutic modulation of the activity of this gene or its protein
product may be useful in the treatment of asthma, allergies,
hypersensitivity reactions, psoriasis, viral infections, liver
cirrhosis, and Crohn's disease.
[0693] Panel 5 Islet Summary: Ag2060 Moderate to low expression of
this gene is seen mainly in small intestine samples
(CTs=32-34.5).
[0694] Panel CNS.sub.--1 Summary: Ag2060 This panel confirms the
expression of this gene at low levels in the brains of an
independent group of individuals.
[0695] General oncology screening panel_v.sub.--2.4 Summary: Ag2060
Highest expression of this gene is seen in a prostate cancer sample
(CT=26.6). Moderate to low expression of this gene is also seen in
cancer and normal samples derived from lung, colon, prostate and
melanoma. Low expression of this gene is also seen in normal kidney
samples. Therefore, therapeutic modulation of this gene or its
protein product may be useful in the treatment of lung, colon,
prostate and melanoma cancers.
[0696] F. CG55023-01: Transforming Growth Factor Alpha
Precursor.
[0697] Expression of gene CG55023-01 was assessed using the
primer-probe sets Ag692, Ag264 and Ag264b, described in Tables FA,
FB and FC. Results of the RTQ-PCR runs are shown in Tables FD, FE,
FF, FG, FH, FI and FJ.
107TABLE FA Probe Name Ag692 Start SEQ ID Primers Sequences Length
Position No Forward 5'-cttgaagttctcacacctttgc-3' 22 207 165 Probe
TET-5'-tcataacagttactgcatcaacggtg-3'-TAMRA 26 237 166 Reverse
5'-tcatggtggaatgcacaag-3' 19 263 167
[0698]
108TABLE FB Probe Name Ag264 Start SEQ ID Primers Sequences Length
Position No Forward 5'-gtctatcttttattcaacgcaatgaca-3' 27 73 168
Probe TET-5'-agtcacggctgcctcttcggtca-3'-TAMRA 23 104 169 Reverse
5'-gggctgtgattggaggtgtt-3' 20 129 170
[0699]
109TABLE FC Probe Name Ag264b Start SEQ ID Primers Sequences Length
Position No Forward 5'-gtctatcttttattcaacgcaatgaca-3' 27 73 171
Probe TET-5'-cacggctgcctcttcggtcagtg-3'-TAMRA 23 101 172 Reverse
5'-gggctgtgattggaggtgtta-3' 21 128 173
[0700]
110TABLE FD Ardais Prostate 1.0 Tissue Name A Tissue Name A
145904_Prostate cancer(9E2) 2.7 153680_Prostate NAT(D69) 3.6
149776_Prostate cancer(AD5) 19.5 155799_Prostate cancer(EA8) 2.3
151135_Prostate NAT(B87) 4.6 145909_Prostate cancer(9E7) 2.3
151143_Prostate NAT(B8A) 5.0 151131_Prostate NAT(B85) 5.8
153653_Prostate cancer(D4E) 4.7 151139_Prostate NAT(B8E) 13.1
153661_Prostate cancer(D56) 8.8 153649_Prostate cancer(D4A) 17.6
153669_Prostate NAT(D5E) 5.6 153657_Prostate cancer(D52) 1.4
153677_Prostate NAT(D66) 0.5 153665_Prostate cancer(D5A) 5.7
153685_Prostate NAT(D6E) 2.2 153673_Prostate NAT(D62) 6.3
145905_Prostate NAT(A0C) 3.1 153681_Prostate NAT(D6A) 0.9
151128_Prostate cancer(B8C) 13.7 145910_Prostate NAT(9C3) 2.4
151136_Prostate cancer(B8B) 13.4 151132_Prostate cancer(B88) 8.1
151144_Prostate cancer(B8F) 2.7 151140_Prostate cancer(B95) 2.6
153654_Prostate cancer(D4F) 3.4 153650_Prostate cancer(D4B) 5.2
153662_Prostate cancer(D57) 2.2 153658_Prostate cancer(D53) 2.4
153670_Prostate NAT(D5F) 5.2 153666_Prostate cancer(D5B) 14.1
153678_Prostate NAT(D67) 7.5 153674_Prostate NAT(D63) 7.6
153686_Prostate NAT(D6F) 8.6 153682_Prostate NAT(D6B) 9.9
145906_Prostate NAT(A09) 2.6 149773_Prostate NAT(AD8) 0.0
151129_Prostate NAT(B93) 26.4 151133_Prostate NAT(B94) 4.1
151137_Prostate NAT(B86) 100.0 151141_Prostate NAT(B96) 23.0
151145_Prostate NAT(B91) 16.5 153651_Prostate cancer(D4C) 4.8
153655_Prostate cancer(D50) 20.4 153659_Prostate cancer(D54) 5.8
153663_Prostate cancer(D58) 4.1 153667_Prostate cancer(D5C) 28.9
153671_Prostate NAT(D60) 8.8 153675_Prostate NAT(D64) 4.9
153679_Prostate NAT(D68) 1.0 153683_Prostate NAT(D6C) 30.8
153687_Prostate NAT(D70) 2.1 149774_Prostate cancer(AD7) 1.7
145907_Prostate cancer(A0A) 2.6 151134_Prostate cancer(B92) 10.7
151130_Prostate cancer(B90) 7.3 151142_Prostate cancer(B89) 1.6
151138_Prostate cancer(B8D) 4.5 153652_Prostate cancer(D4D) 4.0
153648_Prostate cancer(D49) 4.2 153660_Prostate cancer(D55) 1.2
153656_Prostate cancer(D51) 1.8 153668_Prostate NAT(D5D) 56.3
153664_Prostate cancer(D59) 1.2 153676_Prostate NAT(D65) 9.5
153672_Prostate NAT(D61) 8.9 153684_Prostate NAT(D6D) 4.2 Column A
- el. Exp.(%) Ag692, Run 320985784
[0701]
111TABLE FE Oncology_cell_line_screening_panel_v3.1 Tissue Name A
Tissue Name A Daoy Medulloblastoma/Cerebellum 0.0 Ca Ski_Cervical
epidermoid carcinoma 46.7 (metastasis) TE671
Medulloblastom/Cerebellum 0.0 ES-2_Ovarian clear cell carcinoma 0.0
D283 Med Medulloblastoma/Cerebellum 0.0 Ramos/6 h stim_Stimulated
with 0.0 PMA/ionomycin 6 h PFSK-1 Primitive 0.0 Ramos/14 h
stim_Stimulated with 0.0 Neuroectodermal/Cerebellum PMA/ionomycin
14 h XF-498_CNS 0.0 MEG-01_Chronic myelogenous leukemia 7.3
(megokaryoblast) SNB-78_CNS/glioma 0.0 Raji_Burkitt's lymphoma 0.0
SF-268_CNS/glioblastoma 0.0 Daudi_Burkitt's lymphoma 0.0
T98G_Glioblastoma 0.0 U266_B-cell plasmacytoma/myeloma 0.0
SK-N-SH_Neuroblastoma (metastasis) 0.0 CA46_Burkitt's lymphoma 0.0
SF-295_CNS/glioblastoma 0.0 RL_non-Hodgkin's B-cell lymphoma 0.0
Cerebellum 0.0 JM1_pre-B-cell lymphoma/leukemia 0.0 Cerebellum 0.0
Jurkat_T cell leukemia 0.0 NCI-H292_Mucoepidermoid lung ca. 5.0
TF-1_Erythroleukemia 0.0 DMS-114_Small cell lung cancer 0.0 HUT
78_T-cell lymphoma 6.4 DMS-79_Small cell lung 0.0 U937_Histiocytic
lymphoma 0.0 cancer/neuroendocrine NCI-H146_Small cell lung 0.0
KU-812_Myelogenous leukemia 0.0 cancer/neuroendocrine
NCI-H526_Small cell lung 0.0 769-P_Clear cell renal ca. 0.0
cancer/neuroendocrine NCI-N417_Small cell lung 0.0 Caki-2_Clear
cell renal ca. 0.0 cancer/neuroendocrine NCI-H82_Small cell lung
0.0 SW 839_Clear cell renal ca. 0.0 cancer/neuroendocrine
NCI-H157_Squamous cell lung cancer 0.0 G401_Wilms' tumor 0.0
(metastasis) NCI-H1155_Large cell lung 0.0 Hs766T_Pancreatic ca.
(LN metastasis) 0.0 cancer/neuroendocrine NCI-H1299_Large cell lung
0.0 CAPAN-1_Pancreatic adenocarcinoma (liver 0.0
cancer/neuroendocrine metastasis) NCI-H727_Lung carcinoid 0.0
SU86.86_Pancreatic carcinoma (liver 6.7 metastasis) NCI-UMC-11_Lung
carcinoid 0.0 BxPC-3_Pancreatic adenocarcinoma 0.0 LX-1_Small cell
lung cancer 0.0 HPAC_Pancreatic adenocarcinoma 0.0 Colo-205_Colon
cancer 0.0 MIA PaCa-2_Pancreatic ca. 0.0 KM12_Colon cancer 0.0
CFPAC-1_Pancreatic ductal adenocarcinoma 0.0 KM20L2_Colon cancer
0.0 PANC-1_Pancreatic epithelioid ductal ca. 0.0 NCI-H716_Colon
cancer 0.0 T24_Bladder ca. (transitional cell) 0.0 SW-48_Colon
adenocarcinoma 0.0 5637_Bladder ca. 0.0 SW1116_Colon adenocarcinoma
0.0 HT-1197_Bladder ca. 100.0 LS 174T_Colon adenocarcinoma 0.0
UM-UC-3_Bladder ca. (transitional cell) 0.0 SW-948_Colon
adenocarcinoma 0.0 A204_Rhabdomyosarcoma 0.0 SW-480_Colon
adenocarcinoma 0.0 HT-1080_Fibrosarcoma 0.0 NCI-SNU-5_Gastric ca.
0.0 MG-63_Osteosarcoma (bone) 0.0 KATO III_Stomach 0.0
SK-LMS-1_Leiomyosarcoma (vulva) 0.0 NCI-SNU-16_Gastric ca. 0.0
SJRH30_Rhabdomyosarcoma (met to bone 0.0 marrow) NCI-SNU-1_Gastric
ca. 0.0 A431_Epidermoid ca. 0.0 RF-1_Gastric adenocarcinoma 0.0
WM266-4_Melanoma 0.0 RF-48_Gastric adenocarcinoma 0.0 DU
145_Prostate 0.0 MKN-45_Gastric ca. 0.0 MDA-MB-468_Breast
adenocarcinoma 0.0 NCI-N87_Gastric ca. 0.0 SSC-4_Tongue 1.5
OVCAR-5_Ovarian ca. 0.0 SSC-9_Tongue 26.8 RL95-2_Uterine carcinoma
16.0 SSC-15_Tongue 0.0 HelaS3_Cervical adenocarcinoma 30.6 CAL
27_Squamous cell ca. of tongue 0.0 Column A - Rel. Exp.(%) Ag264,
Run 223120410
[0702]
112TABLE FF Panel 1 Tissue Name A B C Tissue Name A B C Endothelial
cells 0.0 0.0 2.5 Renal ca. 786-0 0.0 0.0 2.4 Endothelial cells
(treated) 0.0 0.0 2.6 Renal ca. A498 0.0 0.0 2.3 Pancreas 0.0 0.0
3.5 Renal ca. RXF 393 0.0 0.0 3.1 Pancreatic ca. CAPAN 2 0.0 0.0
2.2 Renal ca. ACHN 0.0 0.0 3.5 Adrenal gland 0.0 0.0 3.6 Renal ca.
UO-31 0.0 0.0 2.5 Thyroid 0.0 0.0 3.7 Renal ca. TK-10 0.0 0.0 2.3
Salivary gland 0.0 0.0 2.8 Liver 0.0 0.0 2.6 Pituitary gland 0.0
0.0 2.4 Liver (fetal) 0.0 0.0 3.0 Brain (fetal) 0.0 0.0 3.2 Liver
ca. (hepatoblast) HepG2 0.0 0.0 2.2 Brain (whole) 0.0 0.0 2.5 Lung
0.0 0.0 4.5 Brain (amygdala) 0.0 0.0 2.7 Lung (fetal) 0.0 0.0 3.4
Brain (cerebellum) 0.0 0.0 3.2 Lung ca. (small cell) LX-1 0.0 0.0
3.0 Brain (hippocampus) 0.0 0.0 4.6 Lung ca. (small cell) NCI-H69
0.0 0.0 2.4 Brain (substantia nigra) 0.0 0.0 3.1 Lung ca. (s.cell
var.) SHP-77 0.0 0.0 2.0 Brain (thalamus) 0.0 0.0 4.0 Lung ca.
(large cell)NCI-H460 37.1 39.8 68.8 Brain (hypothalamus) 0.0 0.0
4.7 Lung ca. (non-sm. cell) A549 0.0 0.0 2.1 Spinal cord 0.0 0.0
2.9 Lung ca. (non-s. cell) NCI-H23 0.0 0.0 3.7 glio/astro U87-MG
0.0 0.0 3.3 Lung ca. (non-s. cell) HOP-62 0.0 0.0 3.2 gilo/astro
U-118-MG 0.0 0.0 2.4 Lung ca. (non-s. cl) NCI-H522 0.0 0.0 2.9
astrocytoma SW1783 0.0 0.0 3.1 Lung ca. (squam.) SW 900 0.0 0.0 3.4
neuro*; met SK-N-AS 0.0 0.0 3.0 Lung ca. (squam.) NCI-H596 0.0 0.0
2.9 astrocytoma SF-539 0.0 0.0 2.3 Mammary gland 0.0 0.0 2.9
astrocytoma SNB-75 0.0 0.0 2.6 Breast ca.* (pl. ef) MCF-7 0.0 0.0
2.1 glioma SNB-19 0.0 0.0 2.8 Breast ca.* (pl. ef) 0.0 0.0 2.9
MDA-MB-231 glioma U251 0.0 0.0 2.1 Breast ca.* (pl. ef) T47D 0.0
0.0 3.8 glioma SF-295 0.0 0.0 4.1 Breast ca. BT-549 0.0 0.0 2.0
Heart 0.0 0.0 4.5 Breast ca. MDA-N 0.0 0.0 2.4 Skeletal muscle 0.0
0.0 2.8 Ovary 0.0 0.0 3.0 Bone marrow 0.0 0.0 2.8 Ovarian ca.
OVCAR-3 0.0 0.0 6.9 Thymus 0.0 0.0 4.6 Ovarian ca. OVCAR-4 0.0 0.0
2.2 Spleen 0.0 0.0 2.2 Ovarian ca. OVCAR-5 0.0 0.0 2.7 Lymph node
0.0 0.0 2.4 Ovarian ca. OVCAR-8 0.0 0.0 3.0 Colon (ascending) 0.0
0.0 2.0 Ovarian ca. IGROV-1 0.0 0.0 2.6 Stomach 0.0 0.0 3.6 Ovarian
ca. (ascites) SK-OV-3 0.0 0.0 2.8 Small intestine 0.0 0.0 2.5
Uterus 0.0 0.0 2.3 Colon ca. SW480 0.0 0.0 2.4 Placenta 0.0 0.0 4.7
Colon ca.* SW620 (SW480 met) 0.0 0.0 2.7 Prostate 0.0 0.0 2.4 Colon
ca. HT29 0.0 0.0 2.5 Prostate ca.* (bone met) PC-3 100.0 100.0
100.0 Colon ca. HCT-116 0.0 0.0 2.8 Testis 0.0 10.0 5.3 Colon ca.
CaCo-2 0.0 0.0 4.0 Melanoma Hs688(A).T 0.0 0.0 2.4 Colon ca. HCT-15
0.0 0.0 2.0 Melanoma* (met) Hs688(B).T 0.0 0.0 3.7 Colon ca.
HCC-2998 0.0 0.0 2.6 Melanoma UACC-62 0.0 0.0 3.1 Gastric ca.*
(liver met) 0.0 0.0 3.1 Melanoma M14 0.0 0.0 2.4 NCI-N87 Bladder
0.0 0.0 2.8 Melanoma LOX IMVI 0.0 0.0 3.3 Trachea 0.0 0.0 2.7
Melanoma* (met) SK-MEL-5 0.0 0.0 2.5 Kidney 0.0 0.0 2.4 Melanoma
SK-MEL-28 0.0 0.0 2.6 Kidney (fetal) 0.0 0.0 3.3 Column A - Rel.
Exp. (%) Ag264, Run 87590466 Column B - Rel. Exp. (%) Ag264, Run
88794920 Column C - Rel. Exp. (%) Ag264b, Run 97806010
[0703]
113TABLE FG Panel 1.2 Tissue Name A B Tissue Name A B Endothelial
cells 2.9 0.0 Renal ca. 786-0 1.6 0.4 Heart (Fetal) 0.2 0.0 Renal
ca. A498 1.1 0.0 Pancreas 4.5 0.1 Renal ca. RXF 393 0.1 0.0
Pancreatic ca. CAPAN 2 2.3 0.4 Renal ca. ACHN 3.4 0.6 Adrenal Gland
1.6 0.1 Renal ca. UO-31 4.5 0.1 Thyroid 0.9 0.0 Renal ca. TK-10 8.0
2.2 Salivary gland 3.8 0.7 Liver 1.0 0.0 Pituitary gland 1.3 0.0
Liver (fetal) 1.1 0.3 Brain (fetal) 1.0 0.0 Liver ca. (hepatoblast)
HepG2 1.3 0.0 Brain (whole) 1.0 0.0 Lung 6.7 5.1 Brain (amygdala)
0.2 0.0 Lung (fetal) 1.5 0.9 Brain (cerebellum) 1.4 0.0 Lung ca.
(small cell) LX-1 6.5 1.2 Brain (hippocampus) 0.4 0.0 Lung ca.
(small cell) NCI-H69 0.9 0.1 Brain (thalamus) 0.6 0.0 Lung ca. (s.
cell var.) SHP-77 0.4 0.0 Cerebral Cortex 2.0 0.0 Lung ca. (large
cell)NCI-H460 41.8 36.3 Spinal cord 2.6 0.3 Lung ca. (non-sm. cell)
A549 3.3 0.8 glio/astro U87-MG 13.5 6.5 Lung ca. (non-s. cell)
NCI-H23 1.1 0.0 glio/astro U-118-MG 2.2 0.9 Lung ca. (non-s. cell)
HOP-62 6.8 3.7 astrocytoma SW1783 1.6 0.9 Lung ca. (non-s. cl)
NCI-H522 20.6 10.1 neuro*; met SK-N-AS 4.9 0.0 Lung ca. (squam.) SW
900 10.4 6.5 astrocytoma SF-539 0.5 0.0 Lung ca. (squam.) NCI-H596
1.5 0.0 astrocytoma SNB-75 0.1 0.0 Mammary gland 5.5 0.8 glioma
SNB-19 4.4 1.0 Breast ca.* (pl. ef) MCF-7 0.4 0.0 glioma U251 0.8
0.0 Breast ca.* (pl. ef) MDA-MB-231 2.2 0.8 glioma SF-295 4.0 0.0
Breast ca.* (pl. ef) T47D 4.0 1.9 Heart 5.3 3.4 Breast ca. BT-549
0.7 0.1 Skeletal Muscle 1.4 0.0 Breast ca. MDA-N 15.0 0.0 Bone
marrow 1.7 2.1 Ovary 0.2 0.0 Thymus 0.0 0.2 Ovarian ca. OVCAR-3
16.3 6.9 Spleen 0.6 0.0 Ovarian ca. OVCAR-4 0.5 0.0 Lymph node 0.8
0.0 Ovarian ca. OVCAR-5 6.9 2.6 Colorectal Tissue 0.1 0.0 Ovarian
ca. OVCAR-8 0.2 0.0 Stomach 3.7 1.8 Ovarian ca. IGROV-1 12.6 8.5
Small intestine 1.1 0.1 Ovarian ca. (ascites) SK-OV-3 3.1 0.9 Colon
ca. SW480 0.3 0.0 Uterus 0.4 0.0 Colon ca.* SW620 (SW480 met) 1.9
0.0 Placenta 6.5 4.7 Colon ca. HT29 2.2 0.3 Prostate 0.8 0.0 Colon
ca. HCT-116 3.4 1.4 Prostate ca.* (bone met) PC-3 100.0 100.0 Colon
ca. CaCo-2 1.2 0.2 Testis 8.7 8.0 Colon ca. Tissue (ODO3866) 0.3
0.0 Melanoma Hs688(A).T 0.5 0.0 Colon ca. HCC-2998 5.8 2.0
Melanoma* (met) Hs688(B).T 1.4 0.7 Gastric ca.* (liver met) NCI-N87
11.9 2.9 Melanoma UACC-62 1.0 0.0 Bladder 2.5 0.8 Melanoma M14 4.0
0.0 Trachea 1.2 0.0 Melanoma LOX IMVI 1.5 0.3 Kidney 2.3 0.0
Melanoma* (met) SK-MEL-5 5.1 0.0 Kidney (fetal) 2.5 0.2 Coumn A -
Rel. Exp. (%) Ag692, Run 114250175 Column B - Rel. Exp. (%) Ag692,
Run 117052376
[0704]
114TABLE FH Panel 2D Tissue Name A B Tissue Name A B Normal Colon
3.1 3.2 Kidney Margin 8120608 0.0 0.0 CC Well to Mod Diff (ODO3866)
0.0 0.3 Kidney Cancer 8120613 0.0 0.0 CC Margin (ODO3866) 0.0 1.0
Kidney Margin 8120614 0.0 0.0 CC Gr.2 rectosigmoid (ODO3868) 0.7
0.0 Kidney Cancer 9010320 0.0 0.0 CC Margin (ODO3868) 0.0 0.6
Kidney Margin 9010321 0.0 0.3 CC Mod Diff (ODO3920) 0.0 0.0 Normal
Uterus 0.0 0.0 CC Margin (ODO3920) 0.0 0.3 Uterus Cancer 064011 0.0
0.5 CC Gr. 2 ascend colon (ODO3921) 0.0 0.7 Normal Thyroid 0.2 0.0
CC Margin (ODO3921) 0.0 1.3 Thyroid Cancer 064010 0.0 0.3 CC from
Partial Hepatectomy 0.0 0.3 Thyroid Cancer A302152 0.3 0.0
(ODO4309) Mets Liver Margin (ODO4309) 0.0 0.0 Thyroid Margin
A302153 0.0 0.3 Colon mets to lung (OD04451-01) 0.3 1.0 Normal
Breast 2.6 2.4 Lung Margin (OD04451-02) 4.5 1.3 Breast Cancer
(OD04566) 0.0 0.0 Normal Prostate 6546-1 0.0 1.4 Breast Cancer
(OD04590-01) 0.0 0.0 Prostate Cancer (OD04410) 0.5 0.9 Breast
Cancer Mets 0.3 0.0 (OD04590-03) Prostate Margin (OD04410) 0.0 0.3
Breast Cancer Metastasis 0.0 0.3 (OD04655-05) Prostate Cancer
(OD04720-01) 0.4 0.3 Breast Cancer 064006 11.9 7.4 Prostate Margin
(OD04720-02) 1.7 2.7 Breast Cancer 1024 3.1 3.1 Normal Lung 061010
0.3 1.1 Breast Cancer 9100266 0.4 0.0 Lung Met to Muscle (ODO4286)
100.0 98.6 Breast Margin 9100265 0.0 0.3 Muscle Margin (ODO4286)
0.0 0.9 Breast Cancer A209073 22.7 16.2 Lung Malignant Cancer
(OD03126) 0.3 0.9 Breast Margin A209073 2.2 1.6 Lung Margin
(OD03126) 0.8 2.3 Normal Liver 0.0 0.7 Lung Cancer (OD04404) 81.2
100.0 Liver Cancer 064003 0.0 0.5 Lung Margin (OD04404) 3.7 3.8
Liver Cancer 1025 0.0 0.3 Lung Cancer (OD04565) 5.8 3.8 Liver
Cancer 1026 0.0 0.0 Lung Margin (OD04565) 0.0 0.7 Liver Cancer
6004-T 0.0 0.7 Lung Cancer (OD04237-01) 0.7 1.0 Liver Tissue 6004-N
0.0 0.1 Lung Margin (OD04237-02) 4.9 8.8 Liver Cancer 6005-T 0.0
0.2 Ocular Mel Met to Liver (ODO4310) 0.0 0.0 Liver Tissue 6005-N
0.0 0.0 Liver Margin (ODO4310) 0.0 0.5 Normal Bladder 0.7 0.0
Melanoma Mets to Lung (OD04321) 0.0 0.0 Bladder Cancer 1023 0.0 0.0
Lung Margin (OD04321) 8.8 7.5 Bladder Cancer A302173 50.7 48.6
Normal Kidney 0.0 1.0 Bladder Cancer (OD04718-01) 7.3 7.0 Kidney
Ca, Nuclear grade 2 1.6 1.6 Bladder Normal Adjacent 0.3 0.5
(OD04338) (OD04718-03) Kidney Margin (OD04338) 0.0 0.6 Normal Ovary
0.0 0.0 Kidney Ca Nuclear grade 1/2 0.0 1.8 Ovarian Cancer 064008
3.8 5.6 (OD04339) Kidney Margin (OD04339) 0.2 0.6 Ovarian Cancer
(OD04768-07) 0.0 0.0 Kidney Ca, Clear cell type (OD04340) 0.3 1.3
Ovary Margin (OD04768-08) 34.2 28.1 Kidney Margin (OD04340) 0.8 2.4
Normal Stomach 0.0 0.0 Kidney Ca, Nuclear grade 3 0.0 0.0 Gastric
Cancer 9060358 0.0 0.0 (OD04348) Kidney Margin (OD04348) 1.4 0.8
Stomach Margin 9060359 0.0 0.0 Kidney Cancer (OD04622-01) 0.3 0.3
Gastric Cancer 9060395 0.3 0.5 Kidney Margin (OD04622-03) 0.3 0.3
Stomach Margin 9060394 0.0 0.0 Kidney Cancer (OD04450-01) 0.0 0.0
Gastric Cancer 9060397 0.0 0.0 Kidney Margin (OD04450-03) 0.0 0.6
Stomach Margin 9060396 0.0 0.0 Kidney Cancer 8120607 0.3 0.0
Gastric Cancer 064005 0.0 0.2 Coumn A - Rel. Exp. (%) Ag264, Run
144872209 Column B - Rel. Exp. (%) Ag692, Run 145177146
[0705]
115TABLE FI Panel 4D Tissue Name A Tissue Name A Secondary Th1 act
2.2 HUVEC IL-1beta 0.3 Secondary Th2 act 2.8 HUVEC IFN gamma 1.3
Secondary Tr1 act 8.4 HUVEC TNF alpha + IFN gamma 0.0 Secondary Th1
rest 0.4 HUVEC TNF alpha + IL4 0.0 Secondary Th2 rest 3.5 HUVEC
IL-11 0.5 Secondary Tr1 rest 0.4 Lung Microvascular EC none 1.0
Primary Th1 act 0.4 Lung Microvascular EC TNF alpha + IL-1beta 0.4
Primary Th2 act 1.2 Microvascular Dermal EC none 2.1 Primary Tr1
act 1.4 Microsvasular Dermal EC TNF alpha + IL-1beta 0.4 Primary
Th1 rest 1.6 Bronchial epithelium TNF alpha + IL1beta 12.3 Primary
Th2 rest 2.5 Small airway epithelium none 15.6 Primary Tr1 rest 2.4
Small airway epithelium TNF alpha + IL-1beta 100.0 CD45RA CD4
lymphocyte act 1.4 Coronery artery SMC rest 3.6 CD45RO CD4
lymphocyte act 1.8 Coronery artery SMC TNF alpha + IL-1beta 0.6 CD8
lymphocyte act 0.5 Astrocytes rest 1.0 Secondary CD8 lymphocyte
rest 1.5 Astrocytes TNF alpha + IL-1beta 1.1 Secondary CD8
lymphocyte act 0.0 KU-812 (Basophil) rest 19.1 CD4 lymphocyte none
2.7 KU-812 (Basophil) PMA/ionomycin 48.3 2ry Th1/Th2/Tr1_anti-CD95
CH11 4.2 CCD1106 (Keratinocytes) none 14.6 LAK cells rest 0.0
CCD1106 (Keratinocytes) TNF alpha + IL-1beta 14.3 LAK cells IL-2
1.2 Liver cirrhosis 3.2 LAK cells IL-2 + IL-12 1.8 Lupus kidney 0.3
LAK cells IL-2 + IFN gamma 2.5 NCI-H292 none 8.5 LAK cells IL-2 +
IL-18 1.6 NCI-H292 IL-4 9.3 LAK cells PMA/ionomycin 3.0 NCI-H292
IL-9 13.2 NK Cells IL-2 rest 0.0 NCI-H292 IL-13 4.5 Two Way MLR 3
day 2.4 NCI-H292 IFN gamma 9.7 Two Way MLR 5 day 0.8 HPAEC none 0.4
Two Way MLR 7 day 0.0 HPAEC TNF alpha + IL-1beta 1.9 PBMC rest 0.8
Lung fibroblast none 0.9 PBMC PWM 4.6 Lung fibroblast TNF alpha +
IL-1beta 0.0 PBMC PHA-L 0.0 Lung fibroblast IL-4 1.0 Ramos (B cell)
none 0.8 Lung fibroblast IL-9 1.0 Ramos (B cell) ionomycin 0.8 Lung
fibroblast IL-13 1.0 B lymphocytes PWM 0.4 Lung fibroblast IFN
gamma 1.1 B lymphocytes CD40L and IL-4 0.7 Dermal fibroblast
CCD1070 rest 2.6 EOL-1 dbcAMP 5.6 Dermal fibroblast CCD1070 TNF
alpha 2.1 EOL-1 dbcAMP PMA/ionomycin 6.3 Dermal fibroblast CCD1070
IL-1beta 1.9 Dendritic cells none 0.0 Dermal fibroblast IFN gamma
1.1 Dendritic cells LPS 0.3 Dermal fibroblast IL-4 0.6 Dendritic
cells anti-CD40 0.9 IBD Colitis 2 0.6 Monocytes rest 0.4 IBD
Crohn's 0.4 Monocytes LPS 9.0 Colon 0.1 Macrophages rest 1.2 Lung
0.7 Macrophages LPS 0.1 Thymus 1.3 HUVEC none 0.0 Kidney 2.7 HUVEC
starved 0.7 Clumn A - Rel. Exp.(%) Ag692, Run 164318656
[0706]
116TABLE FJ Panel 5D Tissue Name A Tissue Name A
97457_Patient-02go_adipose 12.8 94709_Donor 2 AM - A_adipose 14.5
97476_Patient-07sk_skeletal muscle 0.0 94710_Donor 2 AM - B_adipose
14.2 97477_Patient-07ut_uterus 0.0 94711_Donor 2 AM - C_adipose
10.8 97478_Patient-07pl_placenta 1.8 94712_Donor 2 AD - A_adipose
33.4 97481_Patient-08sk_skeletal muscle 0.0 94713_Donor 2 AD -
B_adipose 48.6 97482_Patient-08ut_uterus 0.0 94714_Donor 2 AD -
C_adipose 42.9 97483_Patient-08pl_placenta 3.7 94742_Donor 3 U -
A_Mesenchymal Stem 19.6 Cells 97486_Patient-09sk_skeletal muscle
0.0 94743_Donor 3 U - B_Mesenchymal Stem 21.3 Cells
97487_Patient-09ut_uterus 0.0 94730_Donor 3 AM - A_adipose 5.4
97488_Patient-09pl_placenta 0.0 94731_Donor 3 AM - B_adipose 10.3
97492_Patient-10ut_uterus 0.0 94732_Donor 3 AM - C_adipose 4.0
97493_Patient-10pl_placenta 2.0 94733_Donor 3 AD - A_adipose 100.0
97495_Patient-11go_adipose 20.6 94734_Donor 3 AD - B_adipose 57.0
97496_Patient-11sk_skeletal muscle 1.3 94735_Donor 3 AD - C_adipose
51.8 97497_Patient-11ut_uterus 0.0 77138_Liver_HepG2untreated 16.3
97498_Patient-11pl_placenta 0.0 73556_Heart_Cardiac stromal cells
0.0 (primary) 97500_Patient-12go_adipose 19.8 81735_Small Intestine
0.0 97501_Patient-12sk_skeletal muscle 3.3 72409_Kidney_Proximal
Convoluted 3.4 Tubule 97502_Patient-12ut_uterus 0.0 82685_Small
intestine_Duodenum 0.0 97503_Patient-12pl_placenta 0.0
90650_Adrenal_Adrenocortical adenoma 0.0 94721_Donor 2 U -
A_Mesenchymal 57.0 72410_Kidney_HRCE 32.8 Stem Cells 94722_Donor 2
U - B_Mesenchymal Stem 50.7 72411_Kidney_HRE 68.8 Cells 94723_Donor
2 U - C_Mesenchymal Stem 56.6 73139_Uterus_Uterine smooth muscle
cells 0.0 Cells Clumn A - Rel. Exp.(%) Ag692, Run 258587750
[0707] Ardais Prostate 1.0 Summary: Ag692 Highest expression of
this gene is detected in normal prostate (CT=30). Moderate
expression of this gene is also seen in normal and cancer samples
derived from prostate.
[0708] Oncology_cell_line_screening_panel_v3.1 Summary: Ag264
Highest expression of this gene is seen in a bladder cancer cell
line (CT=29). Moderate to low expression of this gene is also seen
in a number of cancer cell line derived from tongue, pancreatic, T
cell lymphoma, cervical, uterine and lung cancers. Therefore,
therapeutic modulation of this gene or its protein product may be
useful in the treatment of tongue, pancreatic, T cell lymphoma,
cervical, uterine and lung cancers.
[0709] Panel 1 Summary: Ag264/Ag264b Results of three experiments
with this gene show reasonable concordance. The expression of this
gene is found to be highest in a sample derived from a prostate
cancer cell line (CTs=24-26). In addition, there is substantial
expression in a lung cancer cell line. Moreover, therapeutic
modulation of this gene, through the use of small molecule drugs,
protein therapeutics or antibodies might be of benefit in the
treatment of prostate or lung cancer.
[0710] Panel 1.2 Summary: Ag692 The expression of this gene was
assessed in two independent runs in this panel with excellent
concordance between the results. The expression of this gene is
found to be highest in a sample derived from a prostate cancer cell
line (CTs=23-24). In addition there is substantial expression in a
lung cancer cell line. This expression profile is consistent with
the expression seen in Panel 1. Thus, the expression of this gene
could be used to distinguish this prostate cell line sample from
the other samples in the panel. Moreover, therapeutic modulation of
this gene, through the use of small molecule drugs, protein
therapeutics or antibodies might be of benefit in the treatment of
prostate or lung cancer.
[0711] This gene also shows moderate expression in all CNS regions
examined. TGF alpha has numerous roles in the CNS, including
regulation of astrocyte reactivity, neuronal differentialtion and
survivial, and protection of motor neurons (Boillee S, Cadusseau J,
Coulpier M, Grannec G, Junier M P. Transforming growth factor
alpha: a promoter of motoneuron survival of potential biological
relevance. J Neurosci 2001 September 15;21(18):7079-88; Xian C J,
Zhou X F. Roles of transforming growth factor-alpha and related
molecules in the nervous system. Mol Neurobiol 1999
October-December;20(2-3):157-83; Junier M P. What role(s) for
TGFalpha in the central nervous system? Prog Neurobiol 2000
December;62(5):443-73). Because of its possible neuroprotective
effects, this molecule may be of use in the treatment of multiple
sclerosis, ALS, Alzheimer's, Parkinson's, or Huntington's diseases,
stroke, or brain or spinal cord trauma.
[0712] In addition, this gene is moderately expressed in pancreas,
adrenal, thyroid, pituitary, skeletal muscle, and adult and fetal
liver. Thus, this gene product may be an antibody target for the
treatment of metabolic and endocrine disease, including obesity and
Types 1 and 2 diabetes. Among metabolic tissues, this gene has
highest expression in heart (CT values=27-29), and is 79% identical
to mouse epigen protein. Epigen stimulates epithelial cell
proliferation (Strachan L., Murison J. G., Prestidge R. L., Sleeman
M. A., Watson J. D., Kumble K. D., 2001, Cloning and biological
activity of Epigen, a novel member of the epidermal growth factor
superfamily. J. Biol. Chem. 276:18265-18271.), suggesting that an
antibody to this gene product may be useful for prevention of
cardiomyocyte proliferation in diseases of cardiac hypertrophy.
[0713] Panel 2D Summary: Ag264/692 The expression of this gene was
assessed in two independent runs on panel 2D using two different
probe/primer pairs. The expression of this gene appears to be
highest in samples derived from lung cancer tissue (CTs=28-30). In
addition, there is substantial expression in samples derived from
two breast cancers, bladder cancer and a sample of normal ovarian
tissue. Thus, the expression of this gene could be used to
distinguish these lung cancer samples from other samples in the
panel. Moreover, therapeutic modulation of this gene, through the
use of small molecule drugs, antibodies or protein therapeutics may
be of benefit to the treatment of lung cancer, breast cancer or
bladder cancer.
[0714] Panel 4D Summary: Ag692 This gene, a TGF-alpha-like Epigen
protein homolog, is most highly expressed in small airway
epithelium activated with TNFalpha+IL-1beta (CT=28.71) and in
KU-812 basophil cells activated with phorbol ester and ionomycin
(CT=29.76). Epigne has been shown to stimulate the growth of
epithelial cells (Strachan L, Murison J G, Prestidge R L, Sleeman M
A, Watson J D, Kumble K D. Cloning and biological activity of
epigen, a novel member of the epidermal growth factor superfamily.
J Biol Chem. 2001 May 25;276(21):18265-71, PMID: 11278323).
Therefore, antibodies that block the action of this gene product
may be useful as therapeutics to reduce or eliminate the symptoms
in patients with asthma, emphysema, and allergy.
[0715] Panel 5D Summary: Ag692 Highest expression of this gene is
detected in differentiated adipose tissue (CT=31.8). Significant
expression of this gene is detected in undifferentiated mesenchymal
cells, midway and fully differentiated adipose tissues, adipose
samples from non-diabetic and diabetic patients, and kidney
samples. This gene codes for a TGF-alpha like epigen/epidermal
growth factor (EGF) protein homolog. EGF/TGF-alpha and of PGF2
alpha have been shown to act as differentiation inhibitors for
adipocyte precursors in primary culture (Serrero G, Lepak N., 1996,
Int J Obes Relat Metab Disord 20 Suppl 3:S58-64, PMID: 8680479). In
addition, Matsumoto et al. (2002, Biochem Biophys Res Commun
292(3):781-6) have shown that heparin binding epidermal growth
factor (HB-EGF) mRNA is abundantly expressed in human adipose
tissue, and is increased in the fat tissues of obese mice.
Therefore, therapeutic modulation of this gene or its protein
product may be useful in the treatment of metabolic related
diseases such as diabetes and obesity.
[0716] G. CG56136-01: IL1-Epsilon.
[0717] Expression of gene CG56136-01 was assessed using the
primer-probe sets Ag2460 and Ag349, described in Tables GA and GB.
Results of the RTQ-PCR runs are shown in Tables GC, GD, GE and
GF.
117TABLE GA Probe Name Ag2460 Start SEQ ID Primers Sequences Length
Position No Forward 5'-tcatagcagtcccgaggaa-3' 19 89 174 Probe
TET-5'-tcactattgccttaatctcatgccga-3'-TAMRA 26 125 175 Reverse
5'-ttctcaagggtctccacatg-3' 20 151 176
[0718]
118TABLE GB Probe Name Ag349 Start SEQ ID Primers Sequences Length
Position No Forward 5'-gggaaagccacagactcgaa-3' 20 352 177 Probe
TET-5'-cttctaccacagccagagtggcaggaact-3'-TAMRA 291 318 178 Reverse
5'-acccgagcctgtgaagtcct-3' 20 294 1179
[0719]
119TABLE GC Panel 1 Tissue Name A Tissue Name A Endothelial cells
0.0 Renal ca. 786-0 0.0 Endothelial cells (treated) 0.0 Renal ca.
A498 0.2 Pancreas 0.0 Renal ca. RXF 393 0.0 Pancreatic ca. CAPAN 2
0.0 Renal ca. ACHN 0.0 Adrenal gland 0.0 Renal ca. UO-31 0.0
Thyroid 0.5 Renal ca. TK-10 0.0 Salivary gland 25.5 Liver 0.0
Pituitary gland 0.0 Liver (fetal) 0.0 Brain (fetal) 0.0 Liver ca.
(hepatoblast) HepG2 0.0 Brain (whole) 0.0 Lung 0.0 Brain (amygdala)
0.0 Lung (fetal) 0.0 Brain (cerebellum) 0.0 Lung ca. (small cell)
LX-1 0.0 Brain (hippocampus) 0.0 Lung ca. (small cell) NCI-H69 1.5
Brain (substantia nigra) 0.0 Lung ca. (s.cell var.) SHP-77 0.0
Brain (thalamus) 0.0 Lung ca. (large cell) NCI-H460 1.3 Brain
(hypothalamus) 0.0 Lung ca. (non-sm. cell) A549 0.0 Spinal cord 0.0
Lung ca. (non-s.cell) NCI-H23 0.0 glio/astro U87-MG 59.0 Lung ca.
(non-s.cell) HOP-62 0.0 glio/astro U-118-MG 0.0 Lung ca. (non-s.cl)
NCI-H522 0.0 astrocytoma SW1783 0.0 Lung ca. (squam.) SW 900 0.0
neuro*; met SK-N-AS 0.0 Lung ca. (squam.) NCI-H596 0.0 astrocytoma
SF-539 0.0 Mammary gland 0.0 astrocytoma SNB-75 0.0 Breast ca.*
(pl.ef) MCF-7 0.0 glioma SNB-19 3.1 Breast ca.* (pl.ef) MDA-MB-231
0.0 glioma U251 0.0 Breast ca.* (pl. ef) T47D 0.0 glioma SF-295 0.0
Breast ca. BT-549 0.0 Heart 0.0 Breast ca. MDA-N 0.0 Skeletal
muscle 0.0 Ovary 0.0 Bone marrow 0.0 Ovarian ca. OVCAR-3 0.0 Thymus
66.4 Ovarian ca. OVCAR-4 0.0 Spleen 0.1 Ovarian ca. OVCAR-5 1.5
Lymph node 3.1 Ovarian ca. OVCAR-8 0.0 Colon (ascending) 29.9
Ovarian ca. IGROV-1 0.0 Stomach 77.4 Ovarian ca (ascites) SK-OV-3
0.0 Small intestine 0.0 Uterus 0.0 Colon ca. SW480 0.0 Placenta 0.0
Colon ca.* SW620 (SW480 met) 0.0 Prostate 0.0 Colon ca. HT29 0.0
Prostate ca.* (bone met) PC-3 0.0 Colon ca. HCT-116 0.0 Testis 0.0
Colon ca. CaCo-2 0.0 Melanoma Hs688(A).T 0.0 Colon ca. HCT-15 0.0
Melanoma* (met) Hs688(B).T 0.0 Colon ca. HCC-2998 0.0 Melanoma
UACC-62 0.0 Gastric ca.* (liver met) NCI-N87 0.0 Melanoma M14 0.0
Bladder 0.0 Melanoma LOX IMVI 0.0 Trachea 15.5 Melanoma* (met)
SK-MEL-5 0.0 Kidney 0.0 Melanoma SK-MEL-28 100.0 Kidney (fetal) 0.0
Column A - Rel. Exp.(%) Ag349, Run 97804233
[0720]
120TABLE GD Panel 1.3D Tissue Name A Tissue Name A Liver
adenocarcinoma 0.0 Kidney (fetal) 0.0 Pancreas 0.0 Renal ca. 786-0
0.0 Pancreatic ca. CAPAN 2 0.0 Renal ca. A498 9.6 Adrenal gland 0.0
Renal ca. RXF 393 0.0 Thyroid 5.6 Renal ca. ACHN 0.0 Salivary gland
19.8 Renal ca. UO-31 0.0 Pituitary gland 0.0 Renal ca. TK-10 0.0
Brain (fetal) 0.0 Liver 0.0 Brain (whole) 0.0 Liver (fetal) 0.0
Brain (amygdala) 0.0 Liver ca. (hepatoblast) HepG2 0.0 Brain
(cerebellum) 0.0 Lung 2.2 Brain (hippocampus) 0.0 Lung (fetal) 0.0
Brain (substantia nigra) 0.0 Lung ca. (small cell) LX-1 0.0 Brain
(thalamus) 0.0 Lung ca. (small cell) NCI-H69 0.0 Cerebral Cortex
0.0 Lung ca. (s.cell var.) SHP-77 0.0 Spinal cord 45.7 Lung ca.
(large cell) NCI-H460 0.0 glio/astro U87-MG 2.4 Lung ca. (non-sm.
cell) A549 0.0 glio/astro U-118-MG 2.5 Lung ca. (non-s.cell)
NCI-H23 0.0 astrocytoma SW1783 0.0 Lung ca. (non-s.cell) HOP-62 0.0
neuro*; met SK-N-AS 0.0 Lung ca. (non-s.cl) NCI-H522 0.0
astrocytoma SF-539 0.0 Lung ca. (squam.) SW 900 0.0 astrocytoma
SNB-75 0.0 Lung ca. (squam.) NCI-H596 0.0 glioma SNB-19 0.0 Mammary
gland 100.0 glioma U251 0.0 Breast ca.* (pl.ef) MCF-7 0.0 glioma
SF-295 0.0 Breast ca.* (pl.ef) MDA-MB-231 0.0 Heart (fetal) 0.0
Breast ca.* (pl.ef) T47D 0.0 Heart 0.0 Breast ca. BT-549 0.0
Skeletal muscle (fetal) 0.0 Breast ca. MDA-N 0.0 Skeletal muscle
0.0 Ovary 0.0 Bone marrow 0.0 Ovarian ca. OVCAR-3 0.0 Thymus 61.1
Ovarian ca. OVCAR-4 0.0 Spleen 0.0 Ovarian ca. OVCAR-5 0.0 Lymph
node 8.4 Ovarian ca. OVCAR-8 0.0 Colorectal 0.0 Ovarian ca. IGROV-1
0.0 Stomach 77.4 Ovarian ca.* (ascites) SK-OV-3 0.0 Small intestine
0.0 Uterus 0.0 Colon ca. SW480 0.0 Placenta 0.0 Colon ca.*
SW620(SW480 met) 0.0 Prostate 2.3 Colon ca. HT29 0.0 Prostate ca.*
(bone met) PC-3 0.0 Colon ca. HCT-116 0.0 Testis 0.0 Colon
ca.CaCo-2 0.0 Melanoma Hs688(A).T 0.0 Colon ca. tissue(ODO3866) 0.0
Melanoma* (met) Hs688(B).T 0.0 Colon ca. HCC-2998 0.0 Melanoma
UACC-62 0.0 Gastric ca.* (liver met) NCI-N87 0.0 Melanoma M14 0.0
Bladder 0.0 Melanoma LOX IMVI 0.0 Trachea 59.5 Melanoma* (met)
SK-MEL-5 0.0 Kidney 0.0 Adipose 0.0 Column A - Rel. Exp.(%) Ag2460,
Run 157914666
[0721]
121TABLE GE Panel 2D Tissue Name A Tissue Name A Normal Colon 0.0
Kidney Margin 8120608 0.0 CC Well to Mod Diff (ODO3866) 0.0 Kidney
Cancer 8120613 0.0 CC Margin (ODO3866) 0.0 Kidney Margin 8120614
0.0 CC Gr.2 rectosigmoid (ODO3868) 0.0 Kidney Cancer 9010320 0.0 CC
Margin (ODO3868) 0.0 Kidney Margin 9010321 0.0 CC Mod Diff
(ODO3920) 0.0 Normal Uterus 0.0 CC Margin (ODO3920) 0.0 Uterus
Cancer 064011 0.0 CC Gr.2 ascend colon (ODO3921) 0.0 Normal Thyroid
0.9 CC Margin (ODO3921) 0.0 Thyroid Cancer 064010 0.0 CC from
Partial Hepatectomy (ODO4309) 0.0 Thyroid Cancer A302152 0.0 Mets
Liver Margin (ODO4309) 0.0 Thyroid Margin A302153 0.0 Colon mets to
lung (OD04451-01) 0.0 Normal Breast 0.0 Lung Margin (OD04451-02)
0.0 Breast Cancer (OD04566) 0.0 Normal Prostate 6546-1 0.0 Breast
Cancer (OD04590-01) 0.0 Prostate Cancer (OD04410) 0.0 Breast Cancer
Mets (OD04590-03) 0.2 Prostate Margin (OD04410) 0.0 Breast Cancer
Metastasis (OD04655-05) 0.6 Prostate Cancer (OD04720-01) 0.0 Breast
Cancer 064006 0.0 Prostate Margin (OD04720-02) 0.0 Breast Cancer
1024 0.0 Normal Lung 061010 0.4 Breast Cancer 9100266 0.0 Lung Met
to Muscle (ODO4286) 0.0 Breast Margin 9100265 0.0 Muscle Margin
(ODO4286) 0.0 Breast Cancer A209073 0.0 Lung Malignant Cancer
(OD03126) 0.0 Breast Margin A209073 0.0 Lung Margin (OD03126) 0.0
Normal Liver 0.0 Lung Cancer (OD04404) 100.0 Liver Cancer 064003
0.0 Lung Margin (OD04404) 0.0 Liver Cancer 1025 0.1 Lung Cancer
(OD04565) 0.5 Liver Cancer 1026 0.0 Lung Margin (OD04565) 0.0 Liver
Cancer 6004-T 0.0 Lung Cancer (OD04237-01) 0.0 Liver Tissue 6004-N
0.0 Lung Margin (OD04237-02) 0.0 Liver Cancer 6005-T 0.0 Ocular Mel
Met to Liver (ODO4310) 0.0 Liver Tissue 6005-N 0.0 Liver Margin
(ODO4310) 0.0 Normal Bladder 0.0 Melanoma Mets to Lung (OD04321)
0.0 Bladder Cancer 1023 0.0 Lung Margin (OD04321) 0.0 Bladder
Cancer A302173 0.0 Normal Kidney 0.0 Bladder Cancer (OD04718-01)
0.0 Kidney Ca, Nuclear grade 2 (OD04338) 0.0 Bladder Normal
Adjacent 0.0 (OD04718-03) Kidney Margin (OD04338) 0.0 Normal Ovary
0.0 Kidney Ca Nuclear grade 1/2 (OD04339) 0.0 Ovarian Cancer 064008
0.0 Kidney Margin (OD04339) 0.0 Ovarian Cancer (OD04768-07) 0.0
Kidney Ca, Clear cell type (OD04340) 0.0 Ovary Margin (OD04768-08)
0.0 Kidney Margin (OD04340) 0.0 Normal Stomach 0.0 Kidney Ca,
Nuclear grade 3 (OD04348) 0.0 Gastric Cancer 9060358 0.0 Kidney
Margin (OD04348) 0.0 Stomach Margin 9060359 0.0 Kidney Cancer
(OD04622-01) 0.0 Gastric Cancer 9060395 0.0 Kidney Margin
(OD04622-03) 0.0 Stomach Margin 9060394 0.0 Kidney Cancer
(OD04450-01) 0.0 Gastric Cancer 9060397 0.0 Kidney Cancer
(OD04450-03) 0.0 Stomach Margin 9060396 0.0 Kidney Cancer 8120607
0.0 Gastric Cancer 064005 0.6 Column A - Rel. Exp.(%) Ag2460, Run
157914720
[0722]
122TABLE GF Panel 4D Tissue Name A Tissue Name A Secondary Th1 act
0.4 HUVEC IL-1beta 0.0 Secondary Th2 act 0.0 HUVEC IFN gamma 0.0
Secondary Tr1 act 0.5 HUVEC TNF alpha + IFN gamma 0.0 Secondary Th1
rest 2.3 HUVEC TNF alpha + IL4 0.0 Secondary Th2 rest 0.5 HUVEC
IL-11 0.0 Secondary Tr1 rest 3.7 Lung Microvascular EC none 0.0
Primary Th1 act 0.6 Lung Microvascular EC TNF alpha + IL-1beta 0.0
Primary Th2 act 1.1 Microvascular Dermal EC none 0.0 Primary Tr1
act 1.0 Microsvasular Dermal EC TNF alpha + IL-1beta 0.0 Primary
Th1 rest 15.0 Bronchial epithelium TNF alpha + IL-1beta 0.0 Primary
Th2 rest 9.2 Small airway epithelium none 2.1 Primary Tr1 rest 4.3
Small airway epithelium TNF alpha + IL-1beta 100.0 CD45RA CD4
lymphocyte act 0.9 Coronery artery SMC rest 0.0 CD45RO CD4
lymphocyte act 1.0 Coronery artery SMC TNF alpha + IL-1beta 0.0 CD8
lymphocyte act 1.1 Astrocytes rest 0.0 Secondary CD8 lymphocyte
rest 0.5 Astrocytes TNF alpha + IL-1beta 0.0 Secondary CD8
lymphocyte act 0.5 KU-812 (Basophil) rest 0.0 CD4 lymphocyte none
1.0 KU-812 (Basophil) PMA/ionomycin 0.0 2ry Th1/Th2/Tr1_anti-CD95
CH11 2.6 CCD1106 (Keratinocytes) none 0.0 LAK cells rest 0.7
CCD1106 (Keratinocytes) TNF alpha + IL-1beta 0.5 LAK cells IL-2 0.0
Liver cirrhosis 0.0 LAK cells IL-2 + IL-12 0.0 Lupus kidney 0.0 LAK
cells IL-2 + IFN gamma 0.5 NCI-H292 none 0.3 LAK cells IL-2 + IL-18
0.0 NCI-H292 IL-4 0.6 LAK cells PMA/ionomycin 0.0 NCI-H292 IL-9 1.1
LAK Cells IL-2 rest 0.0 NCI-H292 IL-13 0.0 Two Way MLR 3 day 0.0
NCI-H292 IFN gamma 0.0 Two Way MLR 5 day 0.0 HPAEC none 0.0 Two Way
MLR 7 day 0.8 HPAEC TNF alpha + IL-1beta 0.0 PBMC rest 2.1 Lung
fibroblast none 0.0 PBMC PWM 0.0 Lung fibroblast TNF alpha +
IL-1beta 0.0 PBMC PHA-L 2.0 Lung fibroblast IL-4 0.0 Ramos (B cell)
none 0.0 Lung fibroblast IL-9 0.0 Ramos (B cell) ionomycin 0.0 Lung
fibroblast IL-13 0.0 B lymphocytes PWM 1.8 Lung fibroblast IFN
gamma 0.0 B lymphocytes CD40L and IL-4 1.6 Dermal fibroblast
CCD1070 rest 0.0 EOL-1 dbcAMP 0.0 Dermal fibroblast CCD1070 TNF
alpha 0.8 EOL-1 dbcAMP PMA/ionomycin 0.0 Dermal fibroblast CCD1070
IL-1beta 0.0 Dendritic cells none 0.0 Dermal fibroblast IFN gamma
0.0 Dendritic cells LPS 0.0 Dermal fibroblast IL-4 0.0 Dendritic
cells anti-CD40 0.0 IBD Colitis 2 0.0 Monocytes rest 0.0 IBD
Crohn's 0.0 Monocytes LPS 1.1 Colon 0.0 Macrophages rest 0.6 Lung
0.0 Macrophages LPS 0.0 Thymus 0.0 HUVEC none 0.0 Kidney 9.9 HUVEC
starved 0.0 Column A - Rel. Exp.(%) Ag2460, Run 157914794
[0723] Panel 1 Summary: Ag349 Highest expression of this gene is
seen in a melanoma cell line (CT=28.7). There is also significant
expression in thymus. Panel 4D further discusses the role of this
gene in autoimmunity.
[0724] Panel 1.3D Summary: Ag2460 Expression of this gene is
limited to a few samples that are all derived from normal tissue.
Significant levels of expression are seen in mammary gland,
trachea, stomach, thymus, and spinal cord. Thus, expression of this
gene can be used to differentiate between these samples and other
samples on this panel.
[0725] Panel 2D Summary: Ag2460 Expression of this gene is limited
to a few samples, with highest expression in a lung cancer
(CT=27.5). Thus, expression of this gene could be used to
differentiate between this sample and other samples on this panel
and as a marker to detect the presence of lung cancer. Furthermore,
therapeutic modulation of the expression or function of this gene
may be effective in the treatment of lung cancer.
[0726] Panel 4D Summary: Ag2460 This gene encodes a homolog of the
IL-1 epsilon. Interleukin 1 (IL-1) is a member of a large family of
cytokines, which modulates immune and inflammatory responses
(Smith, D. E., Renshaw, B. R., Ketchem, R. R., Kubin, M., Garka, K.
E. and Sims, J. E., 2000, Four new members expand the interleukin-1
superfamily J. Biol. Chem. 275 (2), 1169-1175). IL-1 molecules such
as IL-1alpha, -beta, -delta, -gamma, and IL1-receptor agonist
(IL-1ra) are typically secreted by macrophages, mononuclear cells,
epithelial and endothelial cells. IL-1 molecules are first produced
as precursors of about 30 kDa and do not contain a signal sequence.
The IL-1 precursors are then proteolytically cleaved into their
secreted active forms (.about.17 kDa). Their immuno-modulatory
functions are mediated by two IL-1 receptors, which are members of
the immunoglobulin superfamily. The biological functions of IL-1
include: activation of vascular endothelial cells to secrete IL-6,
increase leukocyte adhesion and activate mononuclear phagocytes
which activate inflammatory leukocytes; tissue destruction, and
fever. Given the biological potency of the IL-1 family of proteins,
a need exists to identify new members of this family as well as
understand the biological function of its members. The high levels
of expression of this gene in small airway epithelium activated by
treatment with TNF-alpha+IL-1 beta(CT=28.9) indicate that
CG56136-01 may play a substantial role in mediating inflammation in
the lung. Thus, therapeutic targeting of CG56136-01 with a
monoclonal antibody is anticipated to limit or block the extent of
inflammation potential and thus the symptoms, caused by
pro-inflammatory cytokines such as IL-1 epsilon, when these
cytokines are induced in allergic, asthma and COPD patients.
Example D
Identification of Single Nucleotide Polymorphisms in NOVX Nucleic
Acid Sequences
[0727] 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.
[0728] 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 protein sequences. Programs used for these
analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and
other relevant programs.
[0729] 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.TM. program
SeqExtend or by identifying SeqCalling fragments mapping to the
appropriate regions of the genomic clones analyzed.
[0730] 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).
[0731] 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.
[0732] NOV1a SNP Data:
[0733] One polymorphic variant of NOV1a have been identified and
are shown in Table D1.
123TABLE D1 SNP Variants for CG50907-03. Nucleotides Amino Acids
Variant Position Initial Modified Position Initial Modified
13382241 135 G A 43 Gly Gly
[0734] NOV2a SNP Data:
[0735] Nine polymorphic variants of NOV2a have been identified and
are shown in Table D2.
124TABLE D2 SNP Variants for CG51896-04. Nucleotides Amino Acids
Variant Position Initial Modified Position Initial Modified
13379621 272 T C 8 Leu Pro 13376060 410 A G 54 His Arg 13376059 416
T C 56 Leu Pro 13374940 523 A G 92 Ser Gly 13375101 869 T C 207 Leu
Pro 13379747 967 G C 240 Ala Pro 13381632 2366 A G 706 Lys Arg
13381633 2921 T C 891 Leu Pro 13381634 3018 G A 923 Met Ile
[0736] NOV3a SNP Data:
[0737] Seven polymorphic variants of NOV3a have been identified and
are shown in Table D3.
125TABLE D3 SNP Variants for CG52324-01. Nucleotides Amino Acids
Variant Position Initial Modified Position Initial Modified
13382239 181 C T 23 Asp Asp 13376383 278 A G 56 Ile Val 13373853
426 C T 105 Pro Leu 13374241 485 G A 125 Glu Lys 13376382 591 A G
160 Lys Arg 13373851 661 T C 183 Arg Arg 13373849 756 A G 215 Glu
Gly
[0738] NOV4a SNP Data:
[0739] One polymorphic variant of NOV4a have been identified and
are shown in Table D4.
126TABLE D4 SNP Variants for CG53054-02. Nucleotides Amino Acids
Variant Position Initial Modified Position Initial Modified
13381656 685 C T 219 Arg Trp
[0740] NOV5a SNP Data:
[0741] One polymorphic variant of NOV5a have been identified and
are shown in Table D5.
127TABLE D5 SNP Variants for CG54818-01. Nucleotides Amino Acids
Variant Position Initial Modified Position Initial Modified
13381700 1874 G A 470 Glu Lys
[0742] NOV6a SNP Data:
[0743] Three polymorphic variants of NOV6a have been identified and
are shown in Table D6.
128TABLE D6 SNP Variants for CG55023-01. Nucleotides Amino Acids
Variant Position Initial Modified Position Initial Modified
13375083 80 T C 11 Leu Pro 13375081 383 T C 112 Ile Thr 13375082
482 A G 145 Asn Ser
[0744] NOV7a SNP Data:
[0745] Eighteen polymorphic variants of NOV7a have been identified
and are shown in Table D7.
129TABLE D7 SNP Variants for CG56136-01. Nucleotides Amino Acids
Variant Position Initial Modified Position Initial Modified
13374788 35 A G 12 Gln Arg 13374176 112 C T 38 Arg Cys 13374789 160
A G 54 Thr Ala 13374790 194 T C 65 Leu Pro 13376575 197 G A 66 Gly
Asp 13374179 206 G A 69 Gly Glu 13376735 240 G A 80 Gly Gly
13382221 269 A G 90 Lys Arg 13374181 273 T C 91 Asp Asp 13374870
287 A G 96 Tyr Cys 13374869 298 G T 100 Glu STOP 13374868 301 C T
101 Pro Ser 13376576 305 T C 102 Val Ala 13374177 340 A G 114 Arg
Gly 13374867 365 C T 122 Ala Val 13374866 374 G A 125 Gly Asp
13376577 376 T C 126 Trp Arg 13379530 400 G A 134 Gly Arg 13374865
404 G A 135 Gly Asp 13374864 431 T C 144 Leu Pro 13374863 442 A G
148 Asn Asp 13374862 461 T C 154 Leu Ser 13374861 463 A G 155 Thr
Ala 13374860 468 G A 156 Met Ile
Example E1
Expression of CG54818-03 Using Baculovirus Expression System
[0746] A 2.6 kb SalI-KpnI fragment containing the CG54818-03
sequence was subcloned into XhoI-KpnI digested pBlueBac4.5/V5-His
(CuraGen Corporation) insect expression vector to generate plasmid,
2080. Following standard procedures (Invitrogen pBlueBac protocol),
recombinant baculovirus was generated and plaque-purified. Fresh
Sf9 cells in adherent culture were infected with the recombinant
baculovirus. The culture media were harvested 5 days post-infection
and assayed for CG54818-03 protein expression by Western blot under
reducing conditions using an anti-V5 antibody. CG54818-03 is
expressed as a 104 kDa protein. (FIG. E1.)
Example E2
Expression of CG55023-06 in Human Embryonic Kidney 293 Cells
[0747] A 0.266 kb BamHI-XhoI fragment containing the CG55023-06
sequence was subcloned into BamHI-XhoI digested pCEP4/Sec to
generate plasmid 1939. The resulting plasmid 1939 was transfected
into 293 cells using the LipofectaminePlus reagent following the
manufacturer's instructions (Gibco/BRL). The cell pellet and
supernatant were harvested 72 h post transfection and examined for
CG55023-06 expression by Western blot (reducing conditions) using
an anti-V5 antibody. CG55023-06 is expressed as a 16 kDa protein
secreted by 293 cells (FIG. E2).
Example E3
Expression of CG55023-06 in Escherichia coli Strains E3381.s
[0748] A 0.266 kb BamHI-XhoI fragment containing the CG55023-06
sequence was subcloned into BamHI-XhoI digested pFLAG-CTS
(Invitrogen) to generate plasmid 1922. The resulting plasmid 1922
was transformed into E. coli using the standard transformation
protocol. The cell pellet and supernatant were harvested 2 h post
induction with IPTG and examined for CG55023-06 expression by
Western blot (reducing conditions) using an anti-FLAG antibody.
CG55023-06 is expressed as a 16 kDa protein. (FIG. E3)
Example E4
Expression of CG55023-06 Using Baculovirus Expression System
[0749] A 0.266 kb BamHI-XhoI fragment containing the CG55023-06
sequence was subcloned into the pMelV5His (CuraGen Corporation)
insect expression vector to generate plasmid 1940. Following
standard procedures (Invitrogen pBlueBac protocol), recombinant
baculovirus was generated and plaque-purified. Fresh Sf9 cells in
adherent culture were infected with the recombinant baculovirus.
The culture media was harvested after 5 days post-infection and
assayed for CG55023-06 protein expression by Western blot (reducing
conditions) using an anti-V5 antibody. CG55023-06 is expressed as a
16 kDa protein (FIG. E4).
Example E5
Expression of CG56136-03 in Human Embryonic Kidney 293 Cells
[0750] A 0.474 kb BamHI-XhoI fragment containing the CG56136-03
sequence was subcloned into BamHI-XhoI digested pCEP4/Sec to
generate plasmid 837. The resulting plasmid 1837 was transfected
into 293 cells using the LipofectaminePlus reagent following the
manufacturer's instructions (Gibco/BRL). The cell pellet and
supernatant were harvested 72 h post transfection and examined for
CG56136-03 expression by Western blot (reducing conditions) using
an anti-V5 antibody. CG56136-03 is expressed as a 30 kDa protein
secreted by 293 cells. (FIG. E5.)
Example F
PathCalling.TM. Data
[0751] See Above Example B for Description of PathCalling
Technology.
[0752] As shown in FIG. F1 below, data obtained from PathCalling
shows that CG54818-01, a Sema 3E protein, interacts with dickkopf
(DKK1) (AC094907) protein. Table F1 summarizes the amino acid
sequences of the bait and prey used to detect this novel
interaction.
130TABLE F1 Yeast Two-hybrid Interaction Information Interaction
Sema 3E Interaction DKK1 Interaction Number of Yeast Frame Domain
(aa) Domain (aa) Colonies Observed 1 (+) Bait: 506-595 Prey:
130-266 1
[0753] Both Sema 3E and DKK1 proteins are extracellular. In a
recent paper Sema 3E from chicken has been shown to inhibit growing
retinal axons (Steinbach K, Volkmer H, Schlosshauer B., 2002,
Semaphorin 3E/collapsin-5 inhibits growing retinal axons. Exp Cell
Res. 279(1):52-61. PMID:12213213). Sema 3E is highly expressed in
brain (See Table EG) and DKK1 is also known to regulate the spatial
patterning/morphogenesis of the mammalian central nervous system.
Thus, Sema 3E may play a role during CNS development.
[0754] In addition, human DKK-1 is shown to be a pro-apoptotic gene
(Shou J, Ali-Osman F, Multani A S, Pathak S, Fedi P, Srivenugopal K
S, 2002, Human Dkk-1, a gene encoding a Wnt antagonist, responds to
DNA damage and its overexpression sensitizes brain tumor cells to
apoptosis following alkylation damage of DNA. Oncogene
21(6):878-89, PMID: 11840333). DKK1 is a powerful inhibitor of the
Wnt signaling pathway. It binds and inhibits the function of
Wnt-co-receptor LRP6. Wnt proteins transmit myriad intercellular
signals crucial for the development and homeostasis of metazoan
animals from Hydra to human. Abnormal Wnt signaling causes a
growing number of diseases, including cancer and osteoporosis
(Wharton K A Jr., 2003, Runnin' with the Dvl: proteins that
associate with Dsh/Dvl and their significance to Wnt signal
transduction. Dev Biol. 2003 January 1;253(l):1-17. PMID:
12490194). Binding of Sema3E with DKK1 may relieve the
Wnt-inhibitory activity of DKK1, potentiating Wnt signaling.
[0755] The function of SEMA3E outside the nervous system is not
known. However, based on its expression in number of cancer samples
and cancer cell line (See Tables EC, EF, EG, EH, and EM) it may
play a role in tumorigenesis. The interaction between Sema3E and
DKK1 suggest a mechanism by which Sema3E may be linked to cancer.
The C-terminal domain of DKK1 binds Sema3E and interestingly, the
same region has been shown to bind LRP protein (Brott B K, Sokol S
Y., 2002, Regulation of Wnt/LRP signaling by distinct domains of
Dickkopf proteins. Mol Cell Biol. 2002 September;22(17):6100-10.
PMID 12167704). Therefore, binding of sema3E with DKK1 is likely to
prevent interaction between DKK1 and LRP resulting in a loss of
DKK1 antagonism. The region of sema3E that binds DKK1 is shared by
other class 3 semaphorins suggesting that one of the function of
this class of semaphorin may be to potentiate Wnt signaling.
OTHER EMBODIMENTS
[0756] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims, which follow. In particular,
it is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims. The choice of nucleic acid starting material, clone of
interest, or library type is believed to be a matter of routine for
a person of ordinary skill in the art with knowledge of the
embodiments described herein. Other aspects, advantages, and
modifications considered to be within the scope of the following
claims. The claims presented are representative of the inventions
disclosed herein. Other, unclaimed inventions are also
contemplated. Applicants reserve the right to pursue such
inventions in later claims.
* * * * *