U.S. patent application number 10/219664 was filed with the patent office on 2003-07-10 for atherosclerosis-associated genes.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Jones, Karen Anne, Murry, Lynn E., Volkmuth, Wayne, Walker, Michael G..
Application Number | 20030129176 10/219664 |
Document ID | / |
Family ID | 23370530 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129176 |
Kind Code |
A1 |
Jones, Karen Anne ; et
al. |
July 10, 2003 |
Atherosclerosis-associated genes
Abstract
The present invention relates to a combination comprising a
plurality of cDNAs which are differentially expressed in
cardiovascular diseases. The combination and compositions can be
used entirely or in part to diagnose, to stage, to treat, or to
monitor the progression or treatment of disorders associated with
atherosclerosis.
Inventors: |
Jones, Karen Anne; (Saffron
Walden, GB) ; Volkmuth, Wayne; (Calabasas, CA)
; Walker, Michael G.; (Sunnyvale, CA) ; Murry,
Lynn E.; (Fayetteville, AR) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
Palo Alto
CA
|
Family ID: |
23370530 |
Appl. No.: |
10/219664 |
Filed: |
August 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10219664 |
Aug 14, 2002 |
|
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09349015 |
Jul 7, 1999 |
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Current U.S.
Class: |
424/94.1 ;
435/183; 435/320.1; 435/325; 435/6.17; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 9/10 20180101; C07K 14/47 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/94.1 ; 435/6;
435/69.1; 435/183; 435/320.1; 435/325; 536/23.2 |
International
Class: |
A61K 038/43; C12Q
001/68; C07H 021/04; C12N 009/00; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1 A combination comprising a plurality of cDNAs wherein the cDNAs
are SEQ ID NOs:1-25 and the complements of SEQ ID NOs:1-25.
2. An isolated cDNA comprising a polynucleotide having the nucleic
acid sequence of SEQ ID No:8.
3. A method for detecting differential expression of one or more
cDNAs in a sample containing nucleic acids, the method comprising:
a) hybridizing the combination of claim 1 with nucleic acids of the
sample, thereby forming one or more hybridization complexes; b)
detecting the hybridization complexes; and c) comparing the
hybridization complexes with those of a standard, wherein
differences between the standard and sample hybridization complexes
indicate differential expression of cDNAs in the sample.
4. The method of claim 3, wherein the nucleic acids of the sample
are amplified prior to hybridization.
5. The method of claim 3, wherein the sample is from a subject with
a disorder associated with atherosclerosis.
6. The method of claim 3, wherein the sample is from aorta,
arteries, arterioles, endothelial cells, plaque, or blood.
7. The method of claim 3 wherein the combination is immobilized on
a substrate.
8. The method of claim 3 wherein differential expression is
diagnostic of angina pectoris, coronary artery disease, myocardial
infarction, hypertension, transient cerebral ischemia, mesenteric
ischemia, peripheral vascular disease, renal artery stenosis, or
stroke.
9. The method of claim 6 wherein the substrate is a microarray.
10. A method of using cDNAs to screen a sample to identify a ligand
which specifically binds a cDNA, the method comprising: a)
combining the combination of claim 1 with the sample under
conditions to allow specific binding; and b) detecting specific
binding between each cDNA and at least ligand, thereby identifying
a ligand that specifically binds to each cDNA.
11. The method of claim 10 wherein the ligands are DNA molecules,
proteins, RNA molecules, or transcription factors.
12. A vector containing the cDNA of claim 2.
13. A host cell containing the vector of claim 12.
14. A method for producing a protein, the method comprising the
steps of: a) culturing the host cell of claim 13 under conditions
for expression of protein; and b) recovering the protein from the
host cell culture.
15. A protein comprising a polypeptide having the amino acid of SEQ
ID NO:26.
16. A method for using a protein to screen a plurality of molecules
or compounds to identify at least one ligand which specifically
binds the protein, the method comprising: a) combining the protein
encoded by a cDNA of claim 1 with the plurality of molecules or
compounds under conditions to allow specific binding; and b)
detecting specific binding between the protein and a molecule or
compound, thereby identifying a ligand which specifically binds the
protein.
17. The method of claim 16 wherein the plurality of molecules or
compounds is selected from agonists, antagonists, antibodies, DNA
molecules, small molecule drugs, immunoglobulins, inhibitors,
mimetics, peptide nucleic acids, peptides, pharmaceutical agents,
proteins, RNA molecules, and ribozymes.
18. A method of using a protein to produce an antibody, the method
comprising: a) immunizing an animal with the protein encoded by a
cDNA of claim 1 under conditions to elicit an antibody response; b)
isolating animal antibodies; and c) screening the isolated
antibodies with the protein, thereby identifying an antibody which
specifically binds the protein.
19. A antibody produced by the method of claim 18.
20. A method for using an antibody to detect gene expression in a
sample, the method comprising: a) combining the antibody of claim
19 with a sample under conditions which allow the formation of
antibody:protein complexes; and b) detecting complex formation,
wherein complex formation indicates expression of the protein in
the sample.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a combination of isolated cDNAs
that are significantly co-expressed with one or more known
atherosclerosis-associa- ted genes. The invention also relates to
the use of the combination in the diagnosis, prognosis, treatment,
and evaluation of therapies for disorders associated with
atherosclerosis.
BACKGROUND OF THE INVENTION
[0002] Atherosclerosis is a disorder characterized by cellular
changes in the arterial intima and the formation of arterial
plaques containing intracellular and extracellular deposits of
lipids. The thickening of artery walls and the narrowing of the
arterial lumen underlies the pathologic condition in most cases of
coronary artery disease, aortic aneurysm, peripheral vascular
disease, and stroke. A number of metabolic pathways and a cascade
of molecular events is involved in the cellular morphogenesis,
proliferation, and cellular migration that results in atherogenesis
(Libby et al. (1997) Int J Cardiol 62 (S2):23-29).
[0003] The artery walls consist of three layers: the intima
(innermost), the media, and the adventitia (outermost). The intima
consists of a layer of endothelial cells lining the lumen of
arteries and arterioles. Endothelial cells form a barrier against
the indiscriminate entry of substances from the blood into the
artery. Specific transporter proteins expressed by endothelial
cells facilitate barrier function. Endothelial cells also secrete a
number of substances which help regulate downstream vascular
contractility blood coagulation, and other aspects of vascular
biology. The medial layer of the arterial wall contains smooth
muscle cells in a matrix of collagen and elastic fibers produced by
the smooth muscle cells. Contraction and relaxation of the smooth
muscle layer allows arteries and arterioles to modulate blood
pressure and blood flow. The outermost layer of the arterial wall,
the adventitia, is a mixture of collagen bundles, elastic fibers,
some smooth muscle cells, fibroblasts and nerve cells. The
adventitia provides structural integrity to the blood vessel and
acts as a support matrix for the media and intima.
[0004] Initiation of an atherosclerotic lesion often occurs
following vascular endothelial cell injury often attributable to
hypertension, diabetes mellitus, hyperlipidemia, fluctuating shear
stress, smoking, or transplant rejection. Nitric oxide and
superoxide anions are released and react to form cytodestructive
peroxynitrite radicals that cause injury to the endothelium and
myocytes of the intima and lead to expression of a variety of
molecules that produce local and systemic effects. These effects
include the release of mediators of inflammation such as cytokines,
complement components, prostaglandins, and downstream transcription
factors. Such mediators promote monocyte infiltration of the
vascular intima and lead to the upregulation of adhesion molecules
which encourages attachment of the monocytes to the damaged
endothelial cells. Simultaneously, components of the extracellular
matrix including collagens, fibrinogens, and matrix Gla protein are
induced and provide sites for monocyte attachment, and annexins,
plasminogen activator inhibitor 1, and nitric oxide synthases are
induced to counteract these effects.
[0005] Monocytes that infiltrate the lesion accumulate modified low
density lipoprotein through scavenger receptors such as CD36 and
macrophage scavenger receptor type I. The abundance of modified
lipids is a factor in atherogenesis and is influenced by modifying
enzymes such as lipoprotein lipase, carboxyl ester lipase, serum
amyloid P component, LDL-receptor related protein, microsomal
triglyceride transfer protein, and serum esterases such as
paraoxonase. Lipid metabolism is governed by cholesterol
biosynthesis enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A
synthase, and products of the apolipoprotein genes. Modified lipid
stabilization and accumulation is aided by perilipin and
alpha-2-macroglobulin.
[0006] As monocytes accumulate in the lesion, they can rupture and
release free cholesterol, cytokines, and procoagulants into the
surrounding environment. This is the process that leads to plaque
development; plaque consists of a mass of lipid-engorged monocytes
and a lipid-rich necrotic core covered by a fibrous cap. The
gradual progression of plaque growth is punctuated by thrombus
formation and leads to clinical symptoms such as unstable angina,
myocardial infarction, or stroke. Thrombus formation is initiated
by episodic plaque rupture which exposes flowing blood to tissue
factors, which induce coagulation, and collagen, which activates
platelets. After initiation of the atherosclerotic lesion, enzymes
that degrade extracellular matrix components (ECM) such as matrix
metalloproteinases and cathepsin K are up-regulated, and inhibitors
of ECM are down-regulated. This results in destabilization of the
atherosclerotic lesion and subsequent complications including
myocardial infarction, angina, and stroke. Further arterial
occlusion and infiltration increase with the expression of
coagulation factors and down-regulation of their inhibitors,
antithrombin III, and lipoprotein-associated coagulation
inhibitor.
[0007] Smooth muscle cells build up in the arterial media and
constitute one of the principal cell types in atherosclerotic and
restenotic lesions. They show a high degree of plasticity and are
able to shift between a differentiated, contractile phenotype and a
less differentiated, synthetic phenotype. This modulation occurs as
a response to factors secreted from cells at the site of vascular
injury and results in structural reorganization with a loss of
myofilaments and the formation of an extensive endoplasmic
reticulum and a large Golgi complex. Genes encoding secreted
protein, acidic and rich in cysteine (SPARC) and endothelin-1
contribute to these changes. At the same time, the expression of
cytoskeletal proteins such as calponin, myosin, desmin, and other
gene products in the cells is altered. As a result, the smooth
muscle cells lose their contractility and become able to migrate
from the media to the intima, to proliferate, and to secrete
extracellular matrix components which contribute to arterial
intimal thickening.
[0008] The initiation and progression of atherosclerotic lesion
development requires the interplay of various molecular pathways
Many genes that participate in these processes are known, and some
of them have been shown to have a direct role in atherosclerosis
pathogenesis by animal model experiments, in vitro assays, and
epidemiological studies (Krettek et al. (1997) Arterioscler Thromb
Vasc Biol 17:2897-2903; Fisher et al. (1997) Atherosclerosis
135:145-159; Shih et al. (1998) Circulation 95:2684-2693; and Bocan
et al. (1998) Atherosclerosis 139:21-30).
[0009] The present invention satisfies a need in the art by
providing a combination comprising a plurality of cDNAs that are
useful for diagnosis, prognosis, treatment, and evaluation of
therapies for disorders associated with atherosclerosis.
SUMMARY OF THE INVENTION
[0010] The invention provides a combination of isolated cDNAs that
are significantly co-expressed with one or more known
atherosclerosis-associa- ted genes. The combination comprises the
isolated cDNAs having the nucleic acid sequences of SEQ ID NOs:1-25
and the complements of SEQ ID NOs:1-25. In one embodiment, the
combination is placed on a substrate. In another embodiment, the
substrate is a microarray.
[0011] The invention also provides a method for using the
combination to detect gene expression in a sample containing
nucleic acids, the method comprising hybridizing the substrate
containing the combination to the nucleic acids of the sample under
conditions for formation of one or more hybridization complexes and
detecting hybridization complex formation, wherein complex
formation indicates gene expression in the sample. In one
embodiment, the sample is from artery or is obtained during
microsurgery to open a blocked artery. In another embodiment,
complex formation is compared to standards and is diagnostic of a
disorder associated with atherosclerosis.
[0012] The invention further provides a method of using a
combination to screen a plurality of molecules to identify at least
one ligand which specifically binds a cDNA of the combination, the
method comprising combining the substrate containing the
combination with molecules under conditions to allow specific
binding; and detecting specific binding, thereby identifying a
ligand which specifically binds at least one cDNA of the
combination. In one embodiment, the molecules are selected from DNA
molecules, peptides, proteins, RNA molecules, and transcription
factors.
[0013] The invention provides an isolated cDNA comprising a
polynucleotide having the nucleic acid sequence of SEQ ID NO:8 and
the complement of SEQ ID NO:8. In different embodiments, the cDNA
is used as a probe, in an expression vector, and in assays for
diagnosis, prognosis, and treatment of disorders associated with
atherosclerosis. The invention also provides a composition
comprising the cDNA and a labeling moiety. The invention further
provides a method for using the cDNA to screen a plurality of
molecules to identify a ligand which specifically binds the cDNA,
the method comprising combining the cDNA with a sample under
conditions to allow specific binding; recovering the bound cDNA;
and separating the ligand from the bound cDNA, thereby obtaining
purified ligand. In one embodiment, the molecules to be screened
are selected from DNA molecules, peptides, proteins, RNA molecules,
and transcription factors. The invention yet further provides a
method for using a cDNA to detect gene expression in a sample
containing nucleic acids, the method comprising hybridizing the
cDNA to nucleic acids of a sample under conditions for formation of
one or more hybridization complexes; and detecting hybridization
complex formation, wherein complex formation indicates gene
expression in the sample. In one embodiment, the cDNA is attached
to a substrate. In another embodiment, gene expression when
compared to standards is diagnostic of a disorder associated with
atherosclerosis.
[0014] The invention provides a vector containing the cDNA, and a
host cell containing the vector. The invention also provides a
method for producing a peptide or protein, the method comprising
culturing the host cell under conditions for expression of the
peptide or protein; and recovering the peptide or protein so
produced from cell culture.
[0015] The invention provides a purified peptide or protein
comprising an amino acid sequence expressed by a cDNA of the
invention. In one embodiment, the protein comprises the amino acid
sequence of SEQ ID NO:26. The invention additionally provides a
composition comprising the protein and a pharmaceutical carrier.
The invention also provides a method for using a peptide or protein
to screen a plurality of molecules to identify at least one ligand
which specifically binds the protein. In one embodiment, the
molecules to be screened are selected from agonists, antagonists,
antibodies, DNA molecules, transcription factors, RNA molecules,
and small drug molecules or compounds. The invention further
provides a method of using a peptide or protein to purify a
ligand.
[0016] The invention provides a method for using the peptide or
protein to produce an antibody which specifically binds the
protein. A method for preparing polyclonal antibodies comprises
immunizing a animal with peptide or protein under conditions to
elicit an antibody response, isolating animal antibodies, attaching
the peptide or protein to a substrate, contacting the substrate
with isolated antibodies under conditions to allow specific binding
to the peptide or protein, dissociating the antibodies from the
peptide or protein, thereby obtaining purified polyclonal
antibodies. A method for preparing monoclonal antibodies comprises
immunizing a animal with a peptide or protein under conditions to
elicit an antibody response, isolating antibody producing cells
from the animal, fusing the antibody producing cells with
immortalized cells in culture to form monoclonal antibody producing
hybridoma cells, culturing the hybridoma cells, and isolating
monoclonal antibodies from culture.
[0017] The invention provides purified antibodies which bind
specifically to a peptide or protein. The invention also provides a
method for using an antibody to detect expression of a peptide or
protein in a sample, the method comprising combining the antibody
with a sample under conditions for formation of antibody:peptide or
protein complexes, and detecting complex formation, wherein complex
formation indicates expression of the peptide or protein in the
sample. In one aspect, the amount of complex formation when
compared to standards is diagnostic of a disorder of the nervous
system.
[0018] The invention provides a method for immunopurification of a
protein comprising attaching an antibody to a substrate, exposing
the antibody to a sample containing protein under conditions to
allow antibody:protein complexes to form, dissociating the protein
from the complex, and collecting purified protein. The invention
also provides an array upon which a cDNA encoding a protein, the
protein, or an antibody which specifically binds the protein are
immobilized. The invention also provides a composition comprising a
cDNA, a protein, an antibody, or a ligand which has agonistic or
antagonistic activity.
[0019] The invention provides an antibody comprising an antigen
binding site, wherein the antigen binding site specifically binds
to the protein The invention also provides a method for treating a
disorder associated with the differential expression of a cDNA that
is coexpressed with one or more known atherosclerosis-associated
genes in a subject in need, the method comprising the step of
administering to the subject in need the antibody in an amount
effective for treating the disorder. The invention further provides
an immunoconjugate comprising the antigen binding site of the
antibody or joined to a therapeutic agent. The invention
additionally provides a method for treating a disorder associated
with the differential expression of a cDNA that is coexpressed with
one or more known atherosclerosis-associated genes in a subject in
need, the method comprising the step of administering to the
subject in need the immunoconjugate in an amount effective for
treating the disorder.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING, FIGURE AND TABLES
[0020] The Sequence Listing provides exemplary cDNAs associated
with atherosclerosis including polynucleotide sequences SEQ ID
NOs:1-25 and the polypeptide sequence, SEQ ID NO:26. Each sequence
is identified by a sequence identification number (SEQ ID NO).
[0021] FIG. 1 shows the cDNA having the nucleic acid sequence of
SEQ ID NO:8 which encodes the protein having the amino acid
sequence of SEQ ID NO:26. The alignment was produced using
MAcDNASIS,PRO software (Hitachi Software Engineering, South San
Francisco Calif.).
[0022] Table 4 shows the-co-expression values (-log p) between the
known atherosclerosis-associated genes (abbreviations as shown in
Table 3) and the cDNAs of the invention (SEQ IDs).
[0023] Table 5 summarizes the highly significantly coexpression
between each cDNA (SEQ ID) and two known atherosclerosis-associated
genes (Gene 1 and Gene 2). P-value and the function or importance
of the known atherosclerosis-associated gene are derived from
Tables 3 and 4.
[0024] Table 6 shows transcript images for several of the cDNAs
(SEQ ID) of the invention. Column 1 shows the SEQ ID; column 2, the
library name; column 3, the number of cDNAs in the library; column
4, the description of the sample from which the library was
constructed; column 5, transcript abundance; and column 6, percent
transcript abundance. These sets demonstrate differential
expression within experiments using cardiovascular tissues.
[0025] Table 7 shows microarray data for several of the cDNAs (SEQ
ID) of the invention. Column 1 shows the SEQ ID; column 2, the name
of the microarray (GEM) used for the experiment; column 3, the log2
(C5/Cy3 ratio); column 4, the description of the Cy3 sample; and
column 5, the description of the Cy5 sample. These data demonstrate
differential expression of the cDNAs in experiments using
cardiovascular samples.
DESCRIPTION OF THE INVENTION
[0026] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include the plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0027] Definitions
[0028] "Antibody" refers to intact immunoglobulin molecule, a
polyclonal antibody, a monoclonal antibody, a chimeric antibody, a
recombinant antibody, a humanized antibody, single chain
antibodies, a Fab fragment, an F(ab').sub.2 fragment, an Fv
fragment; and an antibody-peptide fusion protein.
[0029] "Antigenic determinant" refers to an antigenic or
immunogenic epitope, structural feature, or region of an
oligopeptide, peptide, or protein which is capable of inducing
formation of an antibody which specifically binds the protein.
Biological activity is not a prerequisite for immunogenicity.
[0030] "Array" refers to an ordered arrangement of at least two
cDNAs, proteins, or antibodies on a substrate. At least one of the
cDNAs, proteins, or antibodies represents a control or standard,
and the other represents a cDNA, protein, or antibody of diagnostic
or therapeutic interest. The arrangement of at least two and up to
about 40,000 cDNAs, proteins, or antibodies on the substrate
assures that the size and signal intensity of each labeled complex,
formed between each cDNA and at least one nucleic acid, each
protein and at least one ligand or antibody, or each antibody and
at least one protein to which the antibody specifically binds, is
individually distinguishable.
[0031] "Atherosclerosis-associated cDNA" refers to isolated
polynucleotide that exhibits a statistically significant
co-expression pattern with known atherosclerosis-associated genes.
The cDNAs are represented by SEQ ID NOs:1-25 of the Sequence
Listing and the complements of SEQ ID NOs:1-25. They may be of
recombinant or synthetic origin, used in its double-stranded or
single-stranded form, and combined with vitamins, minerals,
carbohydrates, lipids, proteins, other nucleic acids, a
pharmaceutical carrier or a labeling moiety to perform a particular
activity or form a useful composition.
[0032] An "atherosclerotic sample or tissue" may be taken using
needles, catheters, or scapels and include vessels including the
aorta, arteries, arterioles, endothelial cells, plaque, and blood.
The sample may contain nucleic acids, proteins, antibodies, and the
like. Additionally the sample may comprise the soluble fraction of
a cell preparation; an aliquot of media in which cells were grown;
a chromosome, an organelle, or membrane isolated or extracted from
a cell; genomic DNA, RNA, or cDNA in solution or bound to a
substrate.
[0033] A "combination" comprises at least two sequences selected
from SEQ ID NOs:1-25 as presented in the Sequence Listing and the
complements of SEQ ID NOs:1-25.
[0034] "Differential expression" refers to an increased or
up-regulated or a decreased or down-regulated expression as
detected by absence, presence, or at least two-fold change in the
amount of transcribed messenger RNA or translated protein in a
sample.
[0035] "Disorders associated with atherosclerosis" include angina
pectoris, coronary artery disease, myocardial infarction,
hypertension, transient cerebral ischemia, mesenteric ischemia,
peripheral vascular disease, renal artery stenosis, and stroke.
[0036] An "expression profile" is a representation of gene
expression in a sample. A nucleic acid expression profile is
produced using sequencing, hybridization, or amplification
technologies and mRNAs or cDNAs from a sample. A protein expression
profile, although time delayed, mirrors the nucleic acid expression
profile and uses two-dimensional polyacrylamide electrophoresis
(2D-PAGE) and mass spectrophotometry (MS) or western analysis,
enzyme-linked immunosorbent assays (ELISAs), fluorescence activated
cell sorting (FACS), radioimmunoassays (RIAs), or arrays and
labeling moieties or antibodies to detect expression in a sample.
The nucleic acids, proteins, or antibodies may be used in solution
or attached to a substrate, and their detection is based on methods
and labeling moieties well known in the art.
[0037] A "hybridization complex" is formed between a cDNA of the
invention and a nucleic acid of a sample when the purines of one
molecule hydrogen bond with the pyrimidines of the complementary
molecule, e.g., 5'-A-G-T-C-3' base pairs with its complete
complement, 3'-T-C-A-G-5'. The degree of complementarity and the
use of nucleotide analogs affect the efficiency and stringency of
hybridization reactions.
[0038] "Identity" as applied to sequences, refers to the
quantification (usually percentage) of nucleotide or residue
matches between at least two sequences aligned using a standardized
algorithm such as Smith-Waterman alignment (Smith and Waterman
(1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994)
Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997)
Nucleic Acids Res 25:3389-340). BLAST2 may be used in a
standardized and reproducible way to insert gaps in one of the
sequences in order to optimize alignment and to achieve a more
meaningful comparison between them.
[0039] "Similarity" as applied to proteins uses the same algorithms
but takes into account conservative substitutions of nucleotides or
residues.
[0040] "Isolated or purified" refers to a cDNA or protein that is
removed from its natural environment and that is separated from
other components with which it is naturally present.
[0041] "Genes known to be associated with atherosclerosis" include
human 22 kDa smooth muscle protein, calponin (CNN1), pro alpha 1
(I) collagen (COLIA1), collagen alpha-2 type I (COLIA2), collagen
alpha-6 type I (COL6A1), procollagen alpha 2(V) (COL5A2), collagen
VI alpha-2 (COL6A2), type VI collagen alpha 3 (COL6A3), pro-alpha-i
type 3 collagen (COL3A1), pro-alpha-1(V) collagen (COLSA1), matrix
Gla protein (MGP), cathepsin K (CSTK), fibrinogen beta chain gene
(FBG), pre-pro-von Willebrand factor (VWF), platelet endothelial
cell adhesion molecule (PECAM-1), antithrombin III variant (AT3),
lipoprotein lipase (LPL), alpha-2-macroglobulin (A2M),
apolipoprotein AI (APOA1), apolipoprotein AII (APOA1).sub.2,
apolipoprotein B-100 (APOB), lipoprotein apoCII (APOC2),
pre-apolipoprotein CIII (APOC3), apolipoprotein apo C-IV (APOC4),
macrophage scavenger receptor type I (MSR1), human antigen CD36
gene (CD36), serum amyloid P component (SAP), carboxyl ester lipase
gene (CEL), paraoxonase 1 (PONI), paraoxonase 2 (PON2), paraoxonase
3 (PON3), perilipin (PLIN), prostaglandin D2 synthase (PTGDS),
annexin II/lipocortin II(ANX2), annexin I/lipocortin (ANX1), and
secreted protein, acidic and rich in cysteine (SPARC).
[0042] "Labeling moiety" refers to any reporter molecule whether a
visible or radioactive label, stain or dye that can be attached to
or incorporated into a cDNA or protein. Visible labels and dyes
include but are not limited to anthocyanins, B glucuronidase,
BIODIPY, Coomassie blue, Cy3 and Cy5, digoxigenin, FITC, green
fluorescent protein, luciferase, spyro red, silver, and the like.
Radioactive markers include radioactive forms of hydrogen, iodine,
phosphorous, sulfur, and the like.
[0043] "Ligand" refers to any agent, molecule, or compound which
will bind specifically to a complementary site on a cDNA molecule,
a polynucleotide, an epitope or a protein. Such ligands stabilize
or modulate the activity of polynucleotides or proteins and may be
composed of inorganic or organic substances including nucleic
acids, proteins, carbohydrates, fats, and lipids.
[0044] "Markers for disorders associated with atherosclerosis"
refers to cDNAs, peptides or proteins, and antibodies which are
useful in the diagnosis, prognosis, treatment, selection or
evaluation of therapies for disorders associated with
atherosclerosis. These markers are differentially expressed in
samples from subjects predisposed to or manifesting one of these
disorders. The known atherosclerosis-associated genes and their
contribution and/or function to disorders associated with
atherosclerosis are listed in TABLE3.
[0045] "Probe" refers to a cDNA of the invention that hybridizes to
at least one nucleic acid in a sample. Where targets are single
stranded, probes are complementary single strands. Probes can be
labeled for use in hybridization reactions including Southern,
northern, in situ, dot blot, array, and like technologies or in
screening assays.
[0046] "Protein" refers to a polypeptide or any portion thereof. An
"oligopeptide" is an amino acid sequence from about five residues
to about 15 residues that is used as part of a fusion protein to
produce an antibody that specifically binds the protein.
[0047] "Specific binding" refers to a special and precise
interaction between two molecules which is dependent upon their
structure, particularly their molecular side groups. For example,
the intercalation of a regulatory protein into the major groove of
a DNA molecule, the hydrogen bonding along the backbone between two
single stranded nucleic acids, or the binding between an epitope of
a protein and an agonist, antagonist, or antibody.
[0048] "Substrate" refers to any rigid or semi-rigid support to
which cDNAs or proteins are bound and includes membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels,
capillaries or other tubing, plates, polymers, and microparticles
with a variety of surface forms including wells, trenches, pins,
channels and pores.
[0049] A "transcript image" (TI) is a profile of gene transcription
activity in a particular tissue at a particular time. TI provides
assessment of the relative abundance of expressed transcripts in
the cDNA libraries of an EST database as described in U.S. Pat. No.
5,840,484, incorporated herein by reference.
[0050] "Variant" refers to molecules that are recognized variations
of a polynucleotide or a protein. Splice variants may be determined
by BLAST score, wherein the score is at least 100, and most
preferably at least 400. Allelic variants have a high percent
identity to a polynucleotide and may differ by about three bases
per hundred bases. "Single nucleotide polymorphism" (SNP) refers to
a change in a single base as a result of a substitution, insertion
or deletion. The change may be conservative (purine for purine) or
non-conservative (purine to pyrimidine) and may or may not result
in a change in an encoded amino acid.
[0051] The Method
[0052] The present invention encompasses a method for identifying
cDNAs that are significantly co-expressed with known
atherosclerosis-associated genes. In particular, the method
identifies a combination of cDNAs useful in diagnosis, prognosis,
treatment, and evaluation of therapies for disorders associated
with atherosclerosis.
[0053] The method involves identifying cDNAs that are expressed in
a plurality of cDNA libraries. These cDNAs include genes of known
or unknown function whose expression patterns are compared with
genes having a known function and disease association to determine
whether a specified coexpression probability threshold is met.
Through this comparison, a subset of the cDNAs having a high
coexpression probability with the known genes can be
identified.
[0054] The cDNAs may originate from cDNA libraries derived from a
variety of sources including, but not limited to, eukaryotes such
as human, mouse, rat, dog, monkey, plant, and yeast; prokaryotes
such as bacteria; and viruses. The cDNAs can also be selected from
a variety of sequence types including, but not limited to,
expressed sequence tags (ESTs), assembled polynucleotide sequences,
full length gene coding regions, promoters, introns, enhancers, 5'
untranslated regions, and 3' untranslated regions. To have
statistically significant analytical results, the cDNAs need to be
expressed in at least three cDNA libraries.
[0055] The cDNA libraries used in the coexpression analysis of the
present invention can be obtained from adrenal gland, biliary
tract, bladder, blood cells, blood vessels, bone marrow, brain,
bronchus, cartilage, chromaffin system, colon, connective tissue,
cultured cells, embryonic stem cells, endocrine glands, epithelium,
esophagus, fetus, ganglia, heart, hypothalamus, immune system,
intestine, islets of Langerhans, kidney, larynx, liver, lung,
lymph, muscles, neurons, ovary, pancreas, penis, peripheral nervous
system, phagocytes, pituitary, placenta, pleurus, prostate,
salivary glands, seminal vesicles, skeleton, spleen, stomach,
testis, thymus, tongue, ureter, uterus, and the like. The number of
cDNA libraries selected can range from as few as 3 to greater than
10,000. Preferably, the number of the cDNA libraries is greater
than 500.
[0056] In a preferred embodiment, the cDNAs are assembled from
related sequences, such as assembled sequence fragments derived
from a single transcript. Assembly of the sequences can be
performed using sequences of various types including, but not
limited to, ESTs, extensions, or shotgun sequences. In a most
preferred embodiment, the cDNAs are derived from human sequences
that have been assembled using the algorithm disclosed in U.S. Ser.
No. 09/276,534, filed Mar. 25, 1999, incorporated herein by
reference.
[0057] Experimentally, differential expression of the cDNAs can be
evaluated by methods including, but not limited to, differential
display by spatial immobilization or by gel electrophoresis, genome
mismatch scanning, representational difference analysis, and
transcript imaging. Additionally, differential expression can be
assessed by microarray technology. These methods may be used alone
or in combination.
[0058] The known atherosclerosis-associated genes were selected
based on their function in pathways associated with atherogenesis,
their use as diagnostic or prognostic markers, their behavior in
model systems or their use as therapeutic targets.
[0059] The procedure for identifying a cDNA that exhibits a
statistically significant coexpression pattern with known
atherosclerosis-associated genes is as follows. First, the presence
or absence of a gene in a cDNA library is defined: a gene is
present in a cDNA library when at least one cDNA fragment
corresponding to that gene is detected in a cDNA sample taken from
the library, and a gene is absent from a library when no
corresponding cDNA fragment is detected in the sample.
[0060] Second, the significance of gene coexpression is evaluated
using a probability method to measure a due-to-chance probability
of the coexpression. The probability method can be the Fisher exact
test, the chi-squared test, or the kappa test. These tests and
examples of their applications are well known in the art and can be
found in standard statistics texts (Agresti (1990) Categorical Data
Analysis, John Wiley & Sons, New York N.Y.; Rice (1988)
Mathematical Statistics and Data Analysis, Duxbury Press, Pacific
Grove Calif.). A Bonferroni correction (Rice, supra, p. 384) can
also be applied in combination with one of the probability methods
for correcting statistical results of one gene vetsus multiple
other genes. In a preferred embodiment, the due-to-chance
probability is measured by a Fisher exact test, and the threshold
of the due-to-chance probability is set preferably to less than
0.001, more preferably to less than 0.00001.
[0061] To determine whether two genes, A and B, have similar
coexpression patterns, occurrence data vectors can be generated as
illustrated in Table 1. The presence of a gene occurring at least
once in a library is indicated by a one, and its absence from the
library, by a zero.
1TABLE 1 Occurrence data for genes A and B Library 1 Library 2
Library 3 . . . Library N gene A 1 1 0 . . . 0 gene B 1 0 1 . . .
0
[0062] For a given pair of genes, the occurrence data in Table 1
can be summarized in a 2.times.2 contingency table.
2TABLE 2 Contingency table for co-occurrences of genes A and B Gene
A present Gene A absent Total Gene B present 8 2 10 Gene B absent 2
18 20 Total 10 20 30
[0063] Table 2 presents co-occurrence data for gene A and gene B in
a total of 30 libraries. Both gene A and gene B occur 10 times in
the libraries. Table 2 summarizes and presents: 1) the number of
times gene A and B are both present in a library; 2) the number of
times gene A and B are both absent in a library; 3) the number of
times gene A is present, and gene B is absent; and 4) the number of
times gene B is present, and gene A is absent. The upper left entry
is the number of times the two genes co-occur in a library, and the
middle right entry is the number of times neither gene occurs in a
library. The off diagonal entries are the number of times one gene
occurs, and the other does not. Both A and B are present eight
times and absent 18 times. Gene A is present, and gene B is absent,
two times; and gene B is present, and gene A is absent, two times.
The probability ("p-value") that the above association occurs due
to chance as calculated using a Fisher exact test is 0.0003.
Associations are generally considered significant if a p-value is
less than 0.01 (Agresti, supra; Rice, supra).
[0064] This method of estimating the probability for co-expression
of two genes makes several assumptions. The method assumes that the
libraries are independent and are identically sampled. However, in
practical situations, the selected cDNA libraries are not entirely
independent, because more than one library may be obtained from a
single subject or tissue. Nor are they entirely identically
sampled, because different numbers of cDNAs may be sequenced from
each library. The number of cDNAs sequenced typically ranges from
5,000 to 10,000 cDNAs per library. In addition, because a Fisher
exact co-expression probability is calculated for each gene versus
45,233 other assembled genes, a Bonferroni correction for multiple
statistical tests is used.
[0065] The Invention
[0066] The present invention identifies 25
atherosclerosis-associated cDNAs that exhibit strong association
with genes known to be specifically expressed in atherosclerosis.
The results presented in Tables 4 and 5 show that the expression of
the 25 novel atherosclerosis-associated cDNAs have direct
association with the expression of known atherosclerosis-associated
genes as described in Table 3 and in the background of the
invention. Therefore, the novel atherosclerosis-associa- ted cDNAs
can potentially be used in diagnosis, prognosis, treatment or
evaluation of therapies fro disorders associated with
atherosclerosis. Further, the gene products of the 25 novel
atherosclerosis-associated cDNAs are either potential therapeutics
or targets for the development of therapeutics against disorders
associated with atherosclerosis.
[0067] Therefore, in one embodiment, the present invention
encompasses a combination comprising a plurality of cDNAs having
the nucleic acid sequences of SEQ ID NOs:1-25 or the complements of
SEQ ID NOs:1-25. These 25 cDNAs have been shown by the method of
the present invention to have statistically significant
co-expression with known atherosclerosis-associated genes and with
each other. The invention also encompasses a cDNA comprising a
polynucleotide having the nucleic acid sequence of SEQ ID NO:8 and
the complement thereof. As shown in FIG. 1, SEQ ID NO:8 encodes the
protein of SEQ ID NO:26. The invention further encompasses a
protein comprising the polypeptide having the amino acid sequence
of SEQ ID NO:26.
[0068] The protein encoded by SEQ ID NO:8 has 366 amino acids.
Motif analyses of SEQ ID NO:26 shows one potential cAMP- and
cGMP-dependent protein kinase phosphorylation site at residue S343,
two potential casein kinase II phosphorylation sites at residues
S179 and T351, and four potential protein kinase C phosphorylation
sites at residues T29, S85, T269, and T324. Additionally, SEQ ID
NO:26 contains a potential sugar transport protein signature
sequence from residues L201 to S217.
[0069] cDNAs and Their Uses
[0070] cDNAs can be prepared by a variety of synthetic or enzymatic
methods well known in the art. cDNAs can be synthesized, in whole
or in part, using chemical methods well known in the art (Caruthers
et al. (1980) Nucleic Acids Symp Ser (7):215-233). Alternatively,
cDNAs can be produced enzymatically or recombinantly, by in vitro
or in vivo transcription.
[0071] Nucleotide analogs can be incorporated into cDNAs by methods
well known in the art. The only requirement is that the
incorporated analog must base pair with native purines or
pyrimidines. For example, 2,6-diaminopurine can substitute for
adenine and form stronger bonds with thymidine than those between
adenine and thymidine. A weaker pair is formed when hypoxanthine is
substituted for guanine and base pairs with cytosine. Additionally,
cDNAs can include nucleotides that have been derivatized chemically
or enzymatically.
[0072] cDNAs can be synthesized on a substrate. Synthesis on the
surface of a substrate may be accomplished using a chemical
coupling procedure and a piezoelectric printing apparatus as
described by Baldeschweiler et al. (PCT publication WO95/251116).
Alternatively, the cDNAs can be synthesized on a substrate surface
using a self-addressable electronic device that controls when
reagents are added as described by Heller et al. (U.S. Pat. No.
5,605,662). cDNAs can be synthesized directly on a substrate by
sequentially dispensing reagents for their synthesis on the
substrate surface or by dispensing preformed DNA fragments to the
substrate surface. Typical dispensers include a micropipette
delivering solution to the substrate with a robotic system to
control the position of the micropipette with respect to the
substrate. There can be a multiplicity of dispensers so that
reagents can be delivered to the reaction regions efficiently.
[0073] cDNAs can be immobilized on a substrate by covalent means
such as by chemical bonding procedures or UV irradiation. In one
method, a cDNA is bound to a glass surface which has been modified
to contain epoxide or aldehyde groups. In another method, a cDNA is
placed on a polylysine coated surface and UV cross-linked to it as
described by Shalon et al. (WO95/35505). In yet another method, a
cDNA is actively transported from a solution to a given position on
a substrate by electrical means (Heller, supra). cDNAs do not have
to be directly bound to the substrate, but rather can be bound to
the substrate through a linker group. The linker groups are
typically about 6 to 50 atoms long to provide exposure of the
attached cDNA. Preferred linker groups include ethylene glycol
oligomers, diamines, diacids and the like. Reactive groups on the
substrate surface react with a terminal group of the linker to bind
the linker to the substrate. The other terminus of the linker is
then bound to the cDNA. Alternatively, polynucleotides, plasmids or
cells can be arranged on a filter. In the latter case, cells are
lysed, proteins and cellular components degraded, and the DNA is
coupled to the filter by UV cross-linking.
[0074] The cDNAs may be used for a variety of purposes. For
example, the combination of the invention may be used on an array.
The array, in turn, can be used in high-throughput methods for
detecting a related polynucleotide in a sample, screening a
plurality of molecules or compounds to identify a ligand,
diagnosing a disorder such as diabetes, or inhibiting or
inactivating a therapeutically relevant gene related to the
cDNA.
[0075] When the cDNAs of the invention are employed on an array,
the cDNAs are arranged in an ordered fashion so that each cDNA is
present at a specified location. Because the cDNAs are at specified
locations on the substrate, the hybridization patterns and
intensities, which together create a unique, can be interpreted in
terms of expression levels of particular genes and can be
correlated with a particular metabolic process, condition,
disorder, disease, stage of disease, or treatment.
[0076] Hybridization
[0077] The cDNAs or fragments or complements thereof may be used in
various hybridization technologies. The cDNAs may be labeled using
a variety of reporter molecules by either PCR, recombinant, or
enzymatic techniques. For example, a commercially available vector
containing the cDNA is transcribed in the presence of an
appropriate polymerase, such as T7 or SP6 polymerase, and at least
one labeled nucleotide. Commercial kits are available for labeling
and cleanup of such cDNAs. Radioactive (Amersham Biosciences (APB),
Piscataway N.J.), fluorescent (Qiagen-Operon, Alameda Calif.), and
chemiluminescent labeling (Promega, Madison Wis.) are well known in
the art.
[0078] A cDNA may represent the complete coding region of an mRNA
or be designed or derived from unique regions of the mRNA or
genomic molecule, an intron, a 3' untranslated region, or from a
conserved motif. The cDNA is at least 18 contiguous nucleotides in
length and is usually single stranded. Such a cDNA may be used
under hybridization conditions that allow binding only to an
identical sequence, a naturally occurring molecule encoding the
same protein, or an allelic variant. Discovery of related human and
mammalian sequences may also be accomplished using a pool of
degenerate cDNAs and appropriate hybridization conditions.
Generally, a cDNA for use in Southern or northern hybridizations
may be from about 400 to about 6000 nucleotides long. Such cDNAs
have high binding specificity in solution-based or substrate-based
hybridizations. An oligonucleotide, a fragment of the cDNA, may be
used to detect a polynucleotide in a sample using PCR.
[0079] The stringency of hybridization is determined by G+C content
of the cDNA, salt concentration, and temperature. In particular,
stringency is increased by reducing the concentration of salt or
raising the hybridization temperature. In solutions used for some
membrane based hybridizations, addition of an organic solvent such
as formamide allows the reaction to occur at a lower temperature.
Hybridization may be performed with buffers, such as 5.times.saline
sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at
60.degree. C., that permit the formation of a hybridization complex
between nucleic acid sequences that contain some mismatches.
Subsequent washes are performed with buffers such as 0.2.times.SSC
with 0.1% SDS at either 45.degree. C. (medium stringency) or
65.degree.-68.degree. C. (high stringency). At high stringency,
hybridization complexes will remain stable only where the nucleic
acids are completely complementary. In some membrane-based
hybridizations, preferably 35% or most preferably 50%, formamide
may be added to the hybridization solution to reduce the
temperature at which hybridization is performed. Background signals
may be reduced by the use of detergents such as Sarkosyl or TRITON
X-100 (Sigma-Aldrich, St. Louis Mo.) and a blocking agent such as
denatured salmon sperm DNA. Selection of components and conditions
for hybridization are well known to those skilled in the art and
are reviewed in Ausubel et al. (1997, Short Protocols in Molecular
Biology, John Wiley & Sons, New York N.Y., Units 2.8-2.11,
3.18-3.19 and 4.6-4.9).
[0080] Dot-blot, slot-blot, low density and high density arrays are
prepared and analyzed using methods known in the art. cDNAs from
about 18 consecutive nucleotides to about 5000 consecutive
nucleotides in length are contemplated by the invention and used in
array technologies. The preferred number of cDNAs on an array is at
least about 100,000, a more preferred number is at least about
40,000, an even more preferred number is at least about 10,000, and
a most preferred number is at least about 600 to about 800. The
array may be used to monitor the expression level of large numbers
of genes simultaneously and to identify genetic variants,
mutations, and SNPs. Such information may be used to determine gene
function; to understand the genetic basis of a disorder; to
diagnose a disorder; and to develop and monitor the activities of
therapeutic agents being used to control or cure a disorder. (See,
e.g., U.S. Pat. No. 5,474,796; WO95/11995; WO95/35505; U.S. Pat.
No. 5,605,662; and U.S. Pat. No. 5,958,342.)
[0081] Screening and Purification Assays Using cDNAs
[0082] A cDNA may be used to screen a library or a plurality of
molecules or compounds for a ligand which specifically binds the
cDNA. Ligands may be DNA molecules, RNA molecules, peptide nucleic
acid molecules, peptides, proteins such as transcription factors,
promoters, enhancers, repressors, and other proteins that regulate
replication, transcription, or translation of the polynucleotide in
the biological system. The assay involves combining the cDNA or a
fragment thereof with the molecules or compounds under conditions
that allow specific binding and detecting the bound cDNA to
identify at least one ligand that specifically binds the cDNA.
[0083] In one embodiment, the cDNA may be incubated with a library
of isolated and purified molecules or compounds and binding
activity determined by methods such as a gel-retardation assay
(U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional
assay. In another embodiment, the cDNA may be incubated with
nuclear extracts from biopsied and/or cultured cells and tissues.
Specific binding between the cDNA and a molecule or compound in the
nuclear extract is initially determined by gel shift assay. Protein
binding may be confirmed by raising antibodies against the protein
and adding the antibodies to the gel-retardation assay where
specific binding will cause a supershift in the assay.
[0084] In another embodiment, the cDNA may be used to purify a
molecule or compound using affinity chromatography methods well
known in the art. In one embodiment, the cDNA is chemically reacted
with cyanogen bromide groups on a polymeric resin or gel. Then a
sample is passed over and reacts with or binds to the cDNA. The
molecule or compound which is bound to the cDNA may be released
from the cDNA by increasing the salt concentration of the
flow-through medium and collected.
[0085] The cDNA may be used to purify a ligand from a sample. A
method for using a cDNA to purify a ligand would involve combining
the cDNA or a fragment thereof with a sample under conditions to
allow specific binding, recovering the bound cDNA, and using an
appropriate agent to separate the cDNA from the purified
ligand.
[0086] Protein Production and Uses
[0087] The full length cDNAs or fragments thereof may be used to
produce purified proteins using recombinant DNA technologies
described herein and taught in Ausubel (supra; Units 16.1-16.62).
One of the advantages of producing proteins by these procedures is
the ability to obtain highly-enriched sources of the proteins
thereby simplifying purification procedures.
[0088] The proteins may contain amino acid substitutions, deletions
or insertions made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues involved. Such substitutions may be
conservative in nature when the substituted residue has structural
or chemical properties similar to the original residue (e.g.,
replacement of leucine with isoleucine or valine) or they may be
nonconservative when the replacement residue is radically different
(e.g., a glycine replaced by a tryptophan). Computer programs
included in LASERGENE software (DNASTAR, Madison Wis.) and
algorithms included in RasMol software (University of
Massachusetts, Amherst Mass.) may be used to help determine which
and how many amino acid residues in a particular portion of the
protein may be substituted, inserted, or deleted without abolishing
biological or immunological activity.
[0089] Expression of Encoded Proteins
[0090] Expression of a particular cDNA may be accomplished by
cloning the cDNA into a vector and transforming this vector into a
host cell. The cloning vector used for the construction of cDNA
libraries in the LIFESEQ databases (Incyte Genomics, Palo Alto
Calif.) may also be used for expression. Such vectors usually
contain a promoter and a polylinker useful for cloning, priming,
and transcription. An exemplary vector may also contain the
promoter for .beta.-galactosidase, an amino-terminal methionine and
the subsequent seven amino acid residues of .beta.-galactosidase.
The vector may be transformed into competent E. coli cells.
Induction of the isolated bacterial strain with
isopropylthiogalactoside (IPTG) using standard methods will produce
a fusion protein that contains an N terminal methionine, the first
seven residues of .beta.-galactosidase, about 15 residues of
linker, and the protein encoded by the cDNA.
[0091] The cDNA may be shuttled into other vectors known to be
useful for expression of protein in specific hosts.
Oligonucleotides containing cloning sites and fragments of DNA
sufficient to hybridize to stretches at both ends of the cDNA may
be chemically synthesized by standard methods. These primers may
then be used to amplify the desired fragments by PCR. The fragments
may be digested with appropriate restriction enzymes under standard
conditions and isolated using gel electrophoresis. Alternatively,
similar fragments are produced by digestion of the cDNA with
appropriate restriction enzymes and filled in with chemically
synthesized oligonucleotides. Fragments of the coding sequence from
more than one gene may be ligated together and expressed.
[0092] Signal sequences that dictate secretion of soluble proteins
are particularly desirable as component parts of a recombinant
sequence. For example, a chimeric protein may be expressed that
includes one or more additional purification-facilitating domains.
Such domains include, but are not limited to, metal-chelating
domains that allow purification on immobilized metals, protein A
domains that allow purification on immobilized immunoglobulin, and
the domain utilized in the FLAGS extension/affinity purification
system (Immunex, Seattle Wash.). The inclusion of a
cleavable-linker sequence such as ENTEROKINASEMAX (Invitrogen, San
Diego Calif.) between the protein and the purification domain may
also be used to recover the protein.
[0093] Suitable host cells may include, but are not limited to,
mammalian cells such as Chinese Hamster Ovary (CHO) and human 293
cells, insect cells such as Sf9 cells, plant cells such as
Nicotiana tabacum, yeast cells such as Saccharomyces cerevisiae,
and bacteria such as E. coli. For each of these cell systems, a
useful vector may also include an origin of replication and one or
two selectable markers to allow selection in bacteria as well as in
a transformed eukaryotic host. Vectors for use in eukaryotic host
cells may require the addition of 3' poly(A) tail if the cDNA lacks
poly(A).
[0094] Additionally, the vector may contain promoters or enhancers
that increase gene expression. Many promoters are known and used in
the art. Most promoters are host specific and exemplary promoters
includes SV40 promoters for CHO cells; T7 promoters for bacterial
hosts; viral promoters and enhancers for plant cells; and PGH
promoters for yeast. Adenoviral vectors with the rous sarcoma virus
enhancer or retroviral vectors with long terminal repeat promoters
may be used to drive protein expression in mammalian cell lines.
Once homogeneous cultures of recombinant cells are obtained, large
quantities of secreted soluble protein may be recovered from the
conditioned medium and analyzed using chromatographic methods well
known in the art. An alternative method for the production of large
amounts of secreted protein involves the transformation of
mammalian embryos and the recovery of the recombinant protein from
milk produced by transgenic cows, goats, sheep, and the like.
[0095] In addition to recombinant production, proteins or portions
thereof may be produced manually, using solid-phase techniques
(Stewart et al. (1969) Solid-Phase Peptide Synthesis, W H Freeman,
San Francisco Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154),
or using machines such as the 431A peptide synthesizer (Applied
Biosystems (ABI), Foster City Calif.). Proteins produced by any of
the above methods may be used as pharmaceutical compositions to
treat disorders associated with null or inadequate expression of
the genomic sequence.
[0096] Screening and Purification Assays Using Proteins
[0097] A protein or a portion thereof encoded by the cDNA may be
used to screen a library or a plurality of molecules or compounds
for a ligand with specific binding affinity or to purify a molecule
or compound from a sample. The protein or portion thereof employed
in such screening may be free in solution, affixed to an abiotic or
biotic substrate, or located intracellularly. For example, viable
or fixed prokaryotic host cells that are stably transformed with
recombinant nucleic acids that have expressed and positioned a
protein on their cell surface can be used in screening assays. The
cells are screened against a library or a plurality of ligands and
the specificity of binding or formation of complexes between the
expressed protein and the ligand may be measured. The ligands may
be agonists, antagonists, antibodies, DNA molecules, enhancers,
small drug molecules, immunoglobulins, inhibitors, mimetics,
peptide nucleic acid molecules, peptides, pharmaceutical agents,
proteins, and regulatory proteins, repressors, RNA molecules,
ribozymes, transcription factors, or any other test molecule or
compound that specifically binds the protein. An exemplary assay
involves combining the mammalian protein or a portion thereof with
the molecules or compounds under conditions that allow specific
binding and detecting the bound protein to identify at least one
ligand that specifically binds the protein.
[0098] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding the protein specifically compete with a test compound
capable of binding to the protein or oligopeptide or fragment
thereof. One method for high throughput screening using very small
assay volumes and very small amounts of test compound is described
in U.S. Pat. No. 5,876,946. Molecules or compounds identified by
screening may be used in a model system to evaluate their toxicity,
diagnostic, or therapeutic potential.
[0099] The protein may be used to purify a ligand from a sample. A
method for using a protein to purify a ligand would involve
combining the protein or a portion thereof with a sample under
conditions to allow specific binding, recovering the bound protein,
and using an appropriate chaotropic agent to separate the protein
from the purified ligand.
[0100] Production of Antibodies
[0101] A protein encoded by a cDNA of the invention may be used to
produce specific antibodies. Antibodies may be produced using an
oligopeptide or a portion of the protein with inherent
immunological activity. Methods for producing antibodies include:
1) injecting an animal, usually goats, rabbits, or mice, with the
protein, or an antigenically-effective portion or an oligopeptide
thereof, to induce an immune response; 2) engineering hybridomas to
produce monoclonal antibodies; 3) inducing in vivo production in
the lymphocyte population; or 4) screening libraries of recombinant
immunoglobulins. Recombinant immunoglobulins may be produced as
taught in U.S. Pat. No. 4,816,567.
[0102] Antibodies produced using the proteins of the invention are
useful for the diagnosis of prepathologic disorders as well as the
diagnosis of chronic or acute diseases characterized by
abnormalities in the expression, amount, or distribution of the
protein. A variety of protocols for competitive binding or
immunoradiometric assays using either polyclonal or monoclonal
antibodies specific for proteins are well known in the art.
Immunoassays typically involve the formation of complexes between a
protein and its specific binding molecule or compound and the
measurement of complex formation. Immunoassays may employ a
two-site, monoclonal-based assay that utilizes monoclonal
antibodies reactive to two noninterfering epitopes on a specific
protein or a competitive binding assay (Pound (1998) Immunochemical
Protocols, Humana Press, Totowa N.J.).
[0103] Immunoassay procedures may be used to quantify expression of
the protein in cell cultures, in subjects with a particular
disorder or in model animal systems under various conditions.
Increased or decreased production of proteins as monitored by
immunoassay may contribute to knowledge of the cellular activities
associated with developmental pathways, engineered conditions or
diseases, or treatment efficacy. The quantity of a given protein in
a given tissue may be determined by performing immunoassays on
freeze-thawed detergent extracts of biological samples and
comparing the slope of the binding curves to binding curves
generated by purified protein.
[0104] Antibody Arrays
[0105] In an alternative to yeast two hybrid system analysis of
proteins, an antibody array can be used to study protein-protein
interactions and phosphorylation. A variety of protein ligands are
immobilized on a membrane using methods well known in the art. The
array is incubated in the presence of cell lysate until
protein:antibody complexes are formed. Proteins of interest are
identified by exposing the membrane to an antibody specific to the
protein of interest. In the alternative, a protein of interest is
labeled with digoxigenin (DIG) and exposed to the membrane; then
the membrane is exposed to anti-DIG antibody which reveals where
the protein of interest forms a complex. The identity of the
proteins with which the protein of interest interacts is determined
by the position of the protein of interest on the membrane.
[0106] Antibody arrays can also be used for high-throughput
screening of recombinant antibodies. Bacteria containing antibody
genes are robotically-picked and gridded at high density (up to
18,342 different double-spotted clones) on a filter. Up to 15
antigens at a time are used to screen for clones to identify those
that express binding antibody fragments. These antibody arrays can
also be used to identify proteins which are differentially
expressed in samples (de Wildt et al. (2000) Nature Biotechnol
18:989-94).
[0107] Labeling of Molecules for Assay
[0108] A wide variety of reporter molecules and conjugation
techniques are known by those skilled in the art and may be used in
various cDNA, polynucleotide, protein, peptide or antibody assays.
Synthesis of labeled molecules may be achieved using commercial
kits for incorporation of a labeled nucleotide such as
.sup.32P-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as
.sup.35S-methionine. Polynucleotides, cDNAs, proteins, or
antibodies may be directly labeled with a reporter molecule by
chemical conjugation to amines, thiols and other groups present in
the molecules using reagents such as BIODIPY or FITC (Molecular
Probes, Eugene Oreg.).
[0109] The proteins and antibodies may be labeled for purposes of
assay by joining them, either covalently or noncovalently, with a
reporter molecule that provides for a detectable signal. A wide
variety of labels and conjugation techniques are known and have
been reported in the scientific and patent literature including,
but not limited to U.S. Pat. No. 3,817,837; U.S. Pat. No.
3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No. 3,996,345; U.S.
Pat. No. 4,277,437; U.S. Pat. No. 4,275,149; and U.S. Pat. No.
4,366,241.
[0110] Diagnostics
[0111] The cDNAs, or fragments thereof, may be used to detect and
quantify differential gene expression; absence, presence, or excess
expression of mRNAs; or to monitor mRNA levels during therapeutic
intervention. Disorders associated with atherosclerosis include
angina pectoris, coronary artery disease, myocardial infarction,
hypertension, transient cerebral ischemia, mesenteric ischemia,
peripheral vascular disease, renal artery stenosis, and stroke.
These cDNAs can also be utilized as markers of treatment efficacy
against the disorders noted above and other disorders, conditions,
and diseases over a period ranging from several days to months. The
diagnostic assay may use hybridization or amplification technology
to compare gene expression in a biological sample from a patient to
standard samples in order to detect altered gene expression.
Qualitative or quantitative methods for this comparison are well
known in the art.
[0112] For example, the cDNA may be labeled by standard methods and
added to a biological sample from a patient under conditions for
hybridization complex formation. After an incubation period, the
sample is washed and the amount of label (or signal) associated
with hybridization complexes is quantified and compared with a
standard value. If the amount of label in the patient sample is
significantly altered in comparison to the standard value, then the
presence of the associated condition, disease or disorder is
indicated.
[0113] In order to provide a basis for the diagnosis of a
condition, disease or disorder associated with gene expression, a
normal or standard expression profile is established. This may be
accomplished by combining a biological sample taken from normal
subjects, either animal or human, with a probe under conditions for
hybridization or amplification. Standard hybridization may be
quantified by comparing the values obtained using normal subjects
with values from an experiment in which a known amount of a
purified target sequence is used. Standard values obtained in this
manner may be compared with values obtained from samples from
patients who are symptomatic for a particular condition, disease,
or disorder. Deviation from standard values toward those associated
with a particular condition is used to diagnose that condition.
[0114] Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies and in
clinical trial or to monitor the treatment of an individual
patient. Once the presence of a condition is established and a
treatment protocol is initiated, diagnostic assays may be repeated
on a regular basis to determine if the level of expression in the
patient begins to approximate that which is observed in a normal
subject. The results obtained from successive assays may be used to
show the efficacy of treatment over a period ranging from several
days to months.
[0115] Gene Expression Profiles
[0116] A gene expression profile comprises a plurality of cDNAs and
a plurality of detectable hybridization complexes, wherein each
complex is formed by hybridization of one or more probes to one or
more complementary nucleic acids in a sample. The cDNAs of the
invention are used as elements on a array to analyze gene
expression profiles. In one embodiment, the array is used to
monitor the progression of disease. Researchers or clinicians can
catalog the differences in gene expression between healthy and
diseased tissues or cells. By analyzing changes in patterns of gene
expression, disease can be diagnosed at earlier stages before the
patient is symptomatic. The invention can be used to formulate a
prognosis and to design a treatment regimen. The invention can also
be used to monitor the efficacy of treatment. For treatments with
known side effects, the array is employed to improve the treatment
regimen. A dosage is established that causes a change in genetic
expression patterns indicative of successful treatment. Expression
patterns associated with the onset of undesirable side effects are
avoided. This approach may be more sensitive and rapid than waiting
for the patient to show inadequate improvement, or to manifest side
effects, before altering the course of treatment.
[0117] Experimentally, expression profiles can also be evaluated by
methods including, but not limited to, differential display by
spatial immobilization or by gel electrophoresis, labeling with
radionuclide and quantification using a scintillation counter,
genome mismatch scanning, representational difference analysis,
transcript imaging, quantitative PCR, and by protein or antibody
arrays. Expression profiles produced by these methods may be
contrasted with expression profiles produced using normal or
diseased tissues. Of note is the correspondence between mRNA and
protein expression has been discussed by Zweiger (2001, Transducing
the Genome. McGraw-Hill, San Francisco, Calif.) and Glavas et al.
(2001; T cell activation upregulates cyclic nucleotide
phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342)
among others.
[0118] In another embodiment, animal models which mimic a human
disease can be used to produce expression profiles associated with
a particular condition, disorder or disease; or treatment of the
condition, disorder or disease. Novel treatment regimens may be
tested in these animal models using arrays to establish and then
follow expression profiles over time. In addition, arrays may be
used with cell cultures or tissues removed from animal models to
rapidly screen large numbers of candidate drug molecules, looking
for ones that produce an expression profile similar to those of
known therapeutic drugs, with the expectation that molecules with
the same expression profile will likely have similar therapeutic
effects. Thus, the invention provides the means to rapidly
determine the molecular mode of action of a drug.
[0119] Assays Using Antibodies
[0120] Antibodies directed against antigenic determinants of a
protein encoded by a cDNA of the invention may be used in assays to
quantify the amount of protein found in a particular human cell.
Such assays include methods utilizing the antibody and a label to
detect expression level under normal or disease conditions. The
antibodies may be used with or without modification, and labeled by
joining them, either covalently or noncovalently, with a labeling
moiety.
[0121] Protocols for detecting and measuring protein expression
using either polyclonal or monoclonal antibodies are well known in
the art. Examples include, but are not limited to, western
analysis, ELISA, RIA, FACS, and arrays. Such immunoassays typically
involve the formation of complexes between the protein and its
specific antibody and the measurement of such complexes. These
assays are specifically described in Pound (supra).
[0122] Therapeutics
[0123] The cDNAs and fragments thereof can be used in gene therapy.
cDNAs can be delivered ex vivo to target cells, such as cells of
bone marrow. Once stable integration and transcription and or
translation are confirmed, the bone marrow may be reintroduced into
the subject. Expression of the protein encoded by the cDNA may
correct a disorder associated with mutation of a normal sequence,
reduction or loss of an endogenous target protein, or overepression
of an endogenous or mutant protein. Alternatively, cDNAs may be
delivered in vivo using vectors such as retrovirus, adenovirus,
adeno-associated virus, herpes simplex virus, and bacterial
plasmids. Non-viral methods of gene delivery include cationic
liposomes, polylysine conjugates, artificial viral envelopes, and
direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et
al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med
76(3-4):184-192; Weiss et al. (1999) Cell Mol Life Sci
55(3):334-358; Agrawal (1996) Antisense Therapeutics, Humana Press,
Totowa N.J.; and August et al. (1997) Gene Therapy (Advances in
Pharmacology, Vol. 40), Academic Press, San Diego Calif.).
[0124] In addition, expression of a particular protein can be
regulated through the specific binding of a fragment of a cDNA to a
genomic sequence or an mRNA which encodes the protein or directs
its transcription or translation. The cDNA can be modified or
derivatized to any RNA-like or DNA-like material including peptide
nucleic acids, branched nucleic acids, and the like. These
sequences can be produced biologically by transforming an
appropriate host cell with a vector containing the sequence of
interest.
[0125] Molecules which regulate the activity of the cDNA or encoded
protein are useful as therapeutics for diabetes mellitus, obesity,
hypertension, atherosclerosis, polycystic ovarian syndrome, and
cancers including breast, prostate, and colon. Such molecules
include agonists which increase the expression or activity of the
polynucleotide or encoded protein, respectively; or antagonists
which decrease expression or activity of the polynucleotide or
encoded protein, respectively. In one aspect, an antibody which
specifically binds the protein may be used directly as an
antagonist or indirectly as a delivery mechanism for bringing a
pharmaceutical agent to cells or tissues which express the
protein.
[0126] Additionally, any of the proteins, or their ligands, or
complementary nucleic acid sequences may be administered as
pharmaceutical compositions or in combination with other
appropriate therapeutic agents. Selection of the appropriate agents
for use in combination therapy may be made by one of ordinary skill
in the art, according to conventional pharmaceutical principles.
The combination of therapeutic agents may act synergistically to
affect the treatment or prevention of the conditions and disorders
associated with an immune response. Using this approach, one may be
able to achieve therapeutic efficacy with lower dosages of each
agent, thus reducing the potential for adverse side effects.
Further, the therapeutic agents may be combined with
pharmaceutically-acceptable carriers including excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Further
details on techniques for formulation and administration used by
doctors and pharmacists may be found in the latest edition of
Remington's Pharmaceutical Sciences (Mack Publishing, Easton
Pa.).
[0127] Model Systems
[0128] Animal models may be used as bioassays where they exhibit a
phenotypic response similar to that of humans and where exposure
conditions are relevant to human exposures. Mammals are the most
common models, and most infectious agent, cancer, drug, and
toxicity studies are performed on rodents such as rats or mice
because of low cost, availability, lifespan, reproductive
potential, and abundant reference literature. Inbred and outbred
rodent strains provide a convenient model for investigation of the
physiological consequences of underexpression or overexpression of
genes of interest and for the development of methods for diagnosis
and treatment of diseases. A mammal inbred to overexpress a
particular gene (for example, secreted in milk) may also serve as a
convenient source of the protein expressed by that gene.
[0129] Transgenic Animal Models
[0130] Transgenic rodents that overexpress or underexpress a gene
of interest may be inbred and used to model human diseases or to
test therapeutic or toxic agents. (See, e.g., U.S. Pat. No.
5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the
introduced gene may be activated at a specific time in a specific
tissue type during fetal or postnatal development. Expression of
the transgene is monitored by analysis of phenotype, of
tissue-specific mRNA expression, or of serum and tissue protein
levels in transgenic animals before, during, and after challenge
with experimental drug therapies.
[0131] Embryonic Stem Cells
[0132] Embryonic (ES) stem cells isolated from rodent embryos
retain the potential to form embryonic tissues. When ES cells such
as the mouse 129/SvJ cell line are placed in a blastocyst from the
C57BL/6 mouse strain, they resume normal development and contribute
to tissues of the live-born animal. ES cells are preferred for use
in the creation of experimental knockout and knockin animals. The
method for this process is well known in the art and the steps are:
the cDNA is introduced into a vector, the vector is transformed
into ES cells, transformed cells are identified and microinjected
into mouse cell blastocysts, blastocysts are surgically transferred
to pseudopregnant dams. The resulting chimeric progeny are
genotyped and bred to produce heterozygous or homozygous
strains.
[0133] Knockout Analysis
[0134] In gene knockout analysis, a region of a gene is
enzymatically modified to include a non-natural intervening
sequence such as the neomycin phosphotransferase gene (neo;
Capecchi (1989) Science 244:1288-1292). The modified gene is
transformed into cultured ES cells and integrates into the
endogenous genome by homologous recombination. The inserted
sequence disrupts transcription and translation of the endogenous
gene.
[0135] Knockin Analysis
[0136] ES cells can be used to create knockin humanized animals or
transgenic animal models of human diseases. With knockin
technology, a region of a human gene is injected into animal ES
cells, and the human sequence integrates into the animal cell
genome. Transgenic progeny or inbred lines are studied and treated
with potential pharmaceutical agents to obtain information on the
progression and treatment of the analogous human condition.
[0137] As described herein, the uses of the cDNAs, provided in the
Sequence Listing of this application, and their encoded proteins
are exemplary of known techniques and are not intended to reflect
any limitation on their use in any technique that would be known to
the person of average skill in the art. Furthermore, the cDNAs
provided in this application may be used in molecular biology
techniques that have not yet been developed, provided the new
techniques rely on properties of nucleotide sequences that are
currently known to the person of ordinary skill in the art, e.g.,
the triplet genetic code, specific base pair interactions, and the
like. Likewise, reference to a method may include combining more
than one method for obtaining or assembling full length cDNA
sequences that will be known to those skilled in the art. It is
also to be understood that this invention is not limited to the
particular methodology, protocols, and reagents described, as these
may vary. It is also understood that the terminology used herein is
for the purpose of describing particular embodiments only, and is
not intended to limit the scope of the present invention which will
be limited only by the appended claims. The examples below are
provided to illustrate the subject invention and are not included
for the purpose of limiting the invention.
EXAMPLES
[0138] It is to be understood that this invention is not limited to
the particular devices, machines, materials and methods described.
Although particular embodiments are described, equivalent
embodiments may be used to practice the invention. The described
embodiments are provided to illustrate the invention and are not
intended to limit the scope of the invention which is limited only
by the appended claims.
[0139] I cDNA Library Construction
[0140] The cDNA library SMCCNOS01 was selected as an example to
demonstrate the construction of cDNA libraries from which the cDNAs
co-expressed with known atherosclerosis-associated genes were
derived. The SMCCNOS01 subtracted coronary artery smooth muscle
cell library was constructed using 7.56.times.10.sup.6 clones from
the SMCCNOT02 library and was subjected to two rounds of
subtraction hybridization for 48 hours with 6.12.times.10.sup.6
clones from SMCCNOT01.
[0141] The SMCCNOT02 library was constructed using RNA isolated
from coronary artery smooth muscle cells removed from a 3-year-old
Caucasian male. The cells were treated for 20 hours with TNF.alpha.
and IL-1.beta. at 10 ng/ml each. The SMCCNOT01 was constructed
using RNA isolated from untreated coronary artery smooth muscle
cells from the same donor. Subtractive hybridization conditions
were based on the methodologies of Swaroop et al. (1991; Nucleic
Acids Res 19:1954) and Bonaldo et al. (1996; Genome Research
6:791).
[0142] For both cDNA libraries, SMCCNOT01 and SMCCNOT02, the frozen
coronary artery smooth muscle cells (50-100 mg) were homogenized in
GTC buffer (4.0M guanidine thiocyanate, 0.1M Tris-HCl pH 7.5, 1%
2-mercaptoethanol). Two volumes of binding buffer (0.4M LiCl, 0.1M
Tris-HCl pH 7.5, 0.02M EDTA) were added, and the resulting mixture
was vortexed at 13,000 rpm. The supernatant was removed and
combined with oligo d(T).sub.25 bound streptavidin particles (MPG).
After rotation at room temperature, the mRNA-oligo d(T).sub.25
bound streptavidin particles were separated from the supernatant,
washed twice with hybridization buffer 1 (0.15M NaCl, 0.01M
Tris-HCl pH 8.0, 1 mM EDTA, 0.1% lauryl sarcosinate) using magnetic
separation at each step to remove the supernatant from the
particles. Bound mRNA was eluted from the particles with release
solution and heated to 65.degree. C. The supernatant containing
eluted mRNA was magnetically separated from the particles and used
to construct the cDNA libraries.
[0143] The RNA was used according to the recommended protocols in
the SUPERSCRIPT plasmid system (Invitrogen). The cDNAs were
fractionated on a SEPHAROSE CLIB column (APB), and those cDNAs
exceeding 400 bp were ligated into pINCY plasmid (Incyte Genomics,
Palo Alto Calif.). Recombinant plasmids were transformed into
DH5.alpha. competent cells or ELECTROMAX cells (Invitrogen).
[0144] II Isolation and Sequencing of cDNA Clones
[0145] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 plasmid kit (Qiagen, Valencia Calif.). The
recommended protocol was employed except for the following changes:
1) the bacteria were inoculated into 1 ml of sterile TERRIFIC BROTH
(BD Biosciences, San Jose Calif.) with carbenicillin at 25 mg/l and
glycerol at 0.4%; 2) the cells were cultured for 19 hours and then
lysed with 0.3 ml of lysis buffer; 3) following isopropanol
precipitation, the plasmid DNA pellet was resuspended in 0.1 ml
distilled water, and 4) the samples were transferred to a 96-well
block for storage at 4.degree. C.
[0146] The cDNAs were prepared using a MICROLAB 2200 system
(Hamilton, Reno Nev.) in combination with the DNA ENGINE thermal
cycler (MJ Research, Watertown Mass.). cDNAs were sequenced by the
method of Sanger et al. (1975, J Mol Biol 94:441-446) using PRISM
377 (ABI) or MEGABACE 1000 sequencing systems (APB).
[0147] Most of the sequences were sequenced using standard
protocols and kits (ABI) at solution volumes of
0.25.times.-1.0.times.concentrations. In the alternative, some of
the sequences disclosed herein were sequenced using solutions and
dyes from APB.
[0148] III Selection, Assembly, and Characterization of
Sequences
[0149] The sequences used for co-expression analysis were assembled
from EST sequences, 5' and 3' longread sequences, and full length
coding sequences. The cDNAs claimed herein were expressed in at
least three libraries.
[0150] The assembly process is described as follows. EST sequence
chromatograms were processed and verified. Quality scores were
obtained using PHRED (Ewing et al. (1998) Genome Res 8:175-185;
Ewing and Green (1998) Genome Res 8:186-194), and edited sequences
were loaded into a relational database management system (RDBMS).
The sequences were clustered using BLAST with a product score of
50. All clusters of two or more sequences created a bin which
represents one transcribed gene.
[0151] Assembly of the component sequences within each bin was
performed using a modification of Phrap, a publicly available
program for assembling DNA fragments (Green, P. University of
Washington, Seattle Wash.). Bins that showed 82% identity from a
local pair-wise alignment between any of the consensus sequences
were merged.
[0152] Bins were annotated by screening the consensus sequence in
each bin against public databases, such as GBpri and GenPept from
NCBI. The annotation process involved a FASTn screen against the
GBpri database in GenBank. Those hits with a percent identity of
greater than or equal to 75% and an alignment length of greater
than or equal to 100 base pairs were recorded as homolog hits. The
residual unannotated sequences were screened by FASTx against
GenPept. Those alignments with an E value of less than or equal to
10.sup.-8 were recorded as homologs.
[0153] Sequences were then reclustered using BLASTn and
Cross-Match, a program for rapid amino acid and nucleic acid
sequence comparison and database search (Green, supra),
sequentially. Any BLAST alignment between a sequence and a
consensus sequence with a score greater than 150 was realigned
using cross-match. The sequence was added to the bin whose
consensus sequence gave the highest Smith-Waterman score (Smith et
al. (1992) Protein Engineering 5:35-51) amongst local alignments
with at least 82% identity. Non-matching sequences were moved into
new bins, and assembly processes were repeated.
[0154] IV Coexpression Analyses of Atherosclerosis-Associated
Genes
[0155] Known atherosclerosis-associated genes were selected to
identify the cDNAs that are closely associated with
atherosclerosis. The known atherosclerosis-associated genes which
were used in this analysis all occur within the LIFESEQ Gold
database (Incyte Genomics), and brief descriptions of their
functions as they have been reported in the literature are listed
in Table 3.
3TABLE 3 Descriptions of Known Atherosclerosis-Associated Genes
GENE DESCRIPTION AND REFERENCES Human 22kDa Smooth muscle
cell-specific gene which is down-regulated during smooth smooth
muscle muscle cell dedifferentiation as part of atherogenic process
(Sobue et al. (1998) protein (SM22) Horm Res 50(S2):15-24; Sobue et
al. (1999) Mol Cell Biochem 190:105-18) calponin (CNN1) Calponin is
smooth muscle-specific and may mediate smooth muscle contractility
through binding of the amino-terminal end of the myosin regulatory
light chain. Involved in phenotypic modulation of smooth muscle
cells, a feature of atherosclerosis (Szymanski et al.(1999)
Biochemistry 38:3778-84) pro alpha 1(I) Member of family of fibrous
structural proteins. Most abundant structural collagen (COL1A1)
component of the extracellular matrix. Secreted as procollagen and
converted to collagen by matrix metalloproteinases. Collagens are
important in atherosclerosis for promoting platelet aggregation and
for providing sites for platelet adhesion to the vessel wall (Wen
et al. (1999) Arterioscler Thromb Vasc Biol 19:519-24) collagen
alpha-2 type see COL1A1 above I (COL1A2) COL6A1 see COL1A1 above
procollagen alpha see COL1A1 above 2(V) (COL5A2) collagen VI
alpha-2 see COL1A1 above (COL6A2) type VI collagen see COL1A1 above
alpha3 (COL6A3) pro-alpha-1 type 3 see COL1A1 above collagen
(COL3A1) pro-alpha-1 (V) see COL1A1 above collagen (COL3A1) matrix
Gla protein Role in active calcification of vascular smooth muscle
cells, suggested by (MGP) expression study on VSMC in vitro
differentiation study. Calcifying phenotype associated with high
MGP levels. MGP knockout mice develop to term, but die up to 2
months after birth due to extensive calcification of the arteries,
causing blood vessel rupture (Luo et al. (1997) Nature 386:78-81;
Mori et al. (1998) FEES Lett 433:19-22) cathepsin K (CTSK)
Nonmetalloenzyme, potent elastase present in advanced
atherosclerotic plaques. Contributes to the breakdown of components
of vascular extracellular matrix, reducing tensile strength,
increasing plaque vulnerability (Sukhova et al.(1998) J Clin Invest
102:576-83) fibrinogen beta chain Component of fibrin in the
extracellular matrix. Fibrin deposition is an integral gene (FGB)
part of advanced atherosclerotic lesion development. Variation at
the beta fibrinogen locus associated with peripheral
atherosclerosis (Sueishi et al. (1998) Semin Thromb Hemost
24:255-260; Fowkes et al.(1992) Lancet 339:693-696) pre-pro-von
Blood glycoprotein involved in normal hemostasis. Mediates adhesion
of Willebrand factor platelets to sites of vascular damage. Also
acts as a cofactor in factor VIII (VWF) activity in blood
coagulation. Increased levels of VWF are found in atherosclerosis
and in several of its major risk factors, including
hypercholesterolemia, diabetes, obesity, hypertension. Levels serve
as a predictor of adverse clinical outcome following vascular
surgery, possibly as an indicator of thrombus formation (Sadler
(1998) Annu Rev Biochem 67:395-424 Blann et al. (1994) Eur J Vase
Surg 8:10-15; Kessler et al. (1998) Diabetes Metab 24:327-36;
Folsom et al. (1997) Circulation 96:1102-1108) platelet endothelial
Signaling molecule in the migration of cells as part of the
pathophysiology of cell adhesion vascular occulsive diseases such
as atherosclerosis. Analysis of molecule endothelial/monocyte
co-cultures indicates oxidative stress induces (PECAM-1)
transendothelial migration of monocytes as a result of
phosphorylation of PECAM-1 (Rattan et al. (1997) Am J Physiol
273:E453-61) antithrombin III ATIII is the sole blood component
through which heparin exerts its anti- variant (AT3) coagulation
effect. Deficiency in ATIII causes recurrent venous thrombosis and
pulmonary embolism and can be inherited in autosomal dominant
fashion (Hultin et al. (1988) Thromb Haemost 59:468-73; Lane et al.
(1996) Blood Rev 10:59-74) lipoprotein lipase Hydrolyses
triglyceride in chylomicrons and therefore regulates metabolism of
(LPL) circulating lipoproteins. Appears to have an atherogenic
effect on the arterial wall due to its ability to alter the
properties of LDL. Increased activity of LPL is found in
atherosclerotic arteries when compared to normal. Expressed by
macrophages in atherosclerotic lesions. Mutations in LPL
responsible for familial hypercholesterolemia and premature
atherosclerosis (Fisher et al. (1997) Atherosclerosis 135:145-159;
Goldberg (1996) J Lipid Res 37:693-707; Gerdes et al. (1997)
Circulation 96:733-740) alpha-2-macro- Foam cell formation--retains
LDL cholesterol in the lipid core of globulin (A2M) atherosclerotic
plaque (Llorente et al. (1998) Rev Esp Cardiol 51:633-641)
apolipoprotein AI Participates in reverse cholesterol transport
from tissues to the liver. Promotes (APOA1) cholesterol efflux from
tissues and acts as a cofactor for lecithin cholesterol
acyltransferase (LCAT). Mutations in ApoA1 and of ApoAI/CIII/AIV
gene cluster assoc with atherosclerosis. Transgenic mice expressing
high plasma APOAI levels are protected from fatty streak
development with a high atherogenic diet (Gordon et al. (1989)
Circulation 79:8-15; Rubin et al. (1991) Nature 353:265-7;
Karathanasis et al. (1987) Proc Natl Acad Sci 84:7198- 7202)
apolipoprotein AII Major component of HDL. Appears to have an
opposite effect to that of (APOA2) APOAI, though exact function
unknown. APOAII may have ability to convert HDL from an anti- to a
pro-inflammatory particle, with paraoxonase having a role in this
transformation process. Plasma APOAII levels significantly
associated with plasma free fatty acid levels. Transgenic mice
expressing varying levels of APOAII show increased atherosclerotic
lesions than wt when fed an atherogenic diet. Possible interaction
between diet/genotype and atherogenic potential (Escola-Gil et al.
(1998) J Lipid Res 39:457-462; Warden et al. (1993) Proc Natl Acad
Sci 90:10886-10890) apolipoprotein B-100 Main apolipoprotein of
chylomicrons and low density lipoproteins. Mutations in (APOB)
APOB100 underly familial defective apolipoprotein B-100 in which
patients suffer from premature atherosclerosis. Mutations result in
defect in binding of LDL to LDL receptor, and accumulation of
plasma LDL. High-expressing APOB transgenic mice exibit elevated
VLDL-LDL cholesterol and atherogenic lesions (Callow et al. (1995)
J Clin Invest 96:1639-1646; Brasaemle et al.(1997) J Biol Chem
272:9378-9387) lipoprotein apoCII Role in lipoprotein metabolism.
Cofactor in the activity of lipoprotein lipase the (APOC2) enzyme
that hydrolyzes triglycerides in plasma and transfers the fatty
acids to tissues. Mutations in APOC2 responsible for
hyperlipoproteinemia 1B, similar to lipoprotein lipase deficiency
(Cox et al. (1978) N Engl J Med 299:1421-1424; Arimoto et al.
(1998) J Lipid Res 39:143-151) pre-apolipoprotein Inhibits
lipoprotein lipase and hepatic lipase, decreases uptake of lymph
CIII (APOC3) chylomicrons by hepatic cells. APOA3 possibly delays
breakdown of triglyceride rich particles. SstI RFLP in apoCIII is
associated with plasma triglyceride and apoCIII levels and
hyperlipidemic phenotypes (Henderson et al.(1987) Hum Genet
75:62-65) apolipoprotein apoC- APOC4 is a lipid-binding protein
that has the potential to alter lipid metabolism. IV (APOC4) Human
APOC4 transgenic mice are hypertriglyceridaemic compared to normal
controls (Allan et al. (1996) J Lipid Res 37:1510-1518) macrophage
Mediates binding, internalisation and processing of
negatively-charged macro- scavenger receptor molecules. Implicated
in the pathological deposition of cholesterol in arterial type I
(MSR1) walls during atherogenesis (Han et al. (1998) Hum Mol Genet
7:1039-1046) Human antigen Acts as a scavenger receptor for
oxidised LDL. Transient regulation under CD36 gene (CD36) control
of M-CSF during monocyte-macrophage differentiation increases foam
cell accumulation, Possible role in atherogenesis: increased M-CSF
levels detected in atherosclerotic lesions in rabbits and humans
(Huh et al. (1996) Blood 87:2020-2028; Aitman et al. (1999) Nat
Genet 21:76-83) serum amyloid P Plasma glycoprotein expressed in
atherosclerotic lesions. Interacts with component (SAP)
lipoproteins in specific manner (Li et al. (1995) Arterioscler
Thromb Vasc Biol 15:252-257; Li et al. (1998) Biochem Biophys Res
Commun 244:249-252) carboxyl ester lipase CEL gene expression
increases in presence of oxidised and native LDL in gene (CEL)
vitro. It is expressed in the vessel wall and in aortic extracts -
may interact with cholesterol to modulate progression of
atherosclerosis (Li et al. (1998) Biochem J 329:675-679)
paraoxonase 1 Serum esterase exclusively associated with
high-density lipoproteins; it might (PON1) confer protection
against coronary artery disease by destroying pro- inflammatory
oxidized lipids in oxidized low-density lipoproteins. PON1 gln192-
to-arg polymorphism associated with coronary artery disease.
Association between PON1 genetic variation and plasma LDL, HDL and
non-HDL and apoB levels in genetically isolated Alberta Hutterite
population. When fed on a high-fat, high-cholesterol diet,
PON1-null mice were more susceptible to atherosclerosis than
wild-type (Serrato et al. (1995) J Clin Invest 96:3005-3008;
Boright et al. (1998) Atherosclerosis 139:131-136; Shih et al.
(1998) Nature 394:284-287) paraoxonase 2 Serum esterase exclusively
associated with high-density lipoproteins; it might (PON2) confer
protection against coronary artery disease by destroying pro-
inflammatory oxidized lipids in oxidized low-density lipoproteins.
Common polymorphism at codon 311 (cys-ser) in PON2 associated with
CHD alone and synergistically with the 192 polymorphism in PON1 in
Asian Indians. Association between genetic variation in PON2 and
plasma cholesterol and apolipoprotein A1 in genetically isolated
Alberta Hutterite population (Sanghera et al. (1998) Am J Hum Genet
62:36-44; Boright supra) paraoxonase 3 Serum esterase exclusively
associated with high-density lipoproteins; it might (PON3) confer
protection against coronary artery disease by destroying pro-
inflammatory oxidized lipids in oxidized low-density lipoproteins.
Other members PON2, 3 associated with CHD and cholesterol levels
(Laplaud et al. (1998) Clin Chem Lab Med 36:431-441) perilipin
(PLIN) Lipid storage droplets of steroidogenic cells are surrounded
by perilipins, family of phosphorylated proteins encoded by a
singlegene, detected in adipocytes and steroidogenic cells.
Possible role in lipid metabolism (Brasaemle et al. (1997) J Biol
Chem 272:9378-9387) Prostaglandin D2 Catalyses conversion of PGH2
to PGD2, a prostaglandin important in smooth synthase (PTGDS)
muscle contraction/relaxation and potent inhibitor of platelet
aggregation. Northern analysis shows strong specific expression in
heart. Immunocyto- chemical localization to myocardial and atrio
endocardial cells, and accumulates in end-stage atherosclerotic
plaques. High plasma levels detected in severe angina patients
(Eguchi et al. (1997) Proc Natl Acad Sci 94:14689-14694) Annexin
Inhibits phospholipase A2 activity and the production of
arachidonic acid, the II/lipocortinII(ANX2) precursor of the
inflammatory mediators prostaglandins and leukotrienes. ANX2 is an
important anti-inflammatory molecule that binds plasminogen and t-
PA and is suspected of having a role in atherogenesis. Binding of
plasminoger to ANX2 is specifically inhibited by the excess
atherogenic Lp(a) (Hajjar et al. (1998) J Investig Med 46:364-369)
Annexin I/lipocortin(ANX1) Inhibits phospholipase A2 activity and
production of arachidonic acid, the precursor of the inflammatory
mediators prostaglandins and leukotrienes. ANXI is an important
anti-inflammatory molecule (Wallner et al. (1986) Nature 320:77-81)
Secreted protein, Extracellular glycoprotein secreted by
endothelial cells which has a suspected acidic and rich in role in
calcification of atherosclerotic plaques. Interacts with PDGF-B
cysteine (SPARC) containing dimers and inhibits binding to its
receptors. Expression of SPARC and PDGF is minimal in most adult
tissues, but is enhanced following injury and advanced
atherosclerotic lesions. Selective expression of SPARC causes
rounding of adherent endothelial cells and influences extravasation
of macromolecules (Raines et al. (1992) Proc Natl Acad Sci
89:1281-1285; Goldblum et al. (1994) Proc Natl Acad Sci
91:3448-3452)
[0156] From a total of 45,233 assembled gene sequences, 25 cDNAs
were identified (SEQ ID NOs:1-25 of the Sequence Listing) that show
strong association with the known atherosclerosis-associated
genes.
[0157] Initially, the degree of association was measured by
probability values using a cutoff p-value less than 0.00001. The
sequences were further examined to ensure that the genes that
passed the probability test had strong association with known
atherosclerosis-associated genes. Details of the co-expression
patterns for the known genes and co-expressed cDNAs are presented
in Table 4. The entries in Table 4 are the negative log of the
p-value (-log p) for the coexpression of the two genes. Table 5
summarizes the highly significant co-expression relationships
between each cDNA, two marker genes and their functions.
[0158] V Atherosclerosis-Associated cDNAs
[0159] Using the co-expression analysis method, cDNAs comprising
the polynucleotides of SEQ ID NOs:1-25 and their complements, were
identified by their highly significant co-expression with known
atherosclerosis-associated genes.
[0160] BLAST and other motif searches were performed for SEQ ID
NOs:1-25 according to Example VII. SEQ ID NO:8 was determined to be
full length and translated as shown in FIG. 1.
[0161] VI Transcript Imaging
[0162] Transcript images were performed for several of the cDNAs of
the invention using the LIFESEQ GOLD database (July 02 release,
Incyte Genomics). This process allowed assessment of the relative
abundance of the expressed polynucleotides in all of the cDNA
libraries, but those in the cardiovascular category are
specifically emphasized. Criteria for transcript imaging can be
selected from category, number of cDNAs per library, library
description, disease indication, clinical relevance of sample, and
the like.
[0163] All sequences and cDNA libraries in the LIFESEQ database
have been categorized by system, organ/tissue and cell type. For
each category, the number of libraries in which the sequence was
expressed were counted and shown over the total number of libraries
in that category. For each library, the number of cDNAs were
counted and shown over the total number of cDNAs in that library.
In some transcript images, all normalized or subtracted libraries,
which have high copy number sequences removed prior to processing,
and all mixed or pooled tissues, which are considered non-specific
in that they contain more than one tissue type or more than one
subject's tissue, can be excluded from the analysis. Treated and
untreated cell lines and/or fetal tissue data can also be excluded
where clinical relevance is emphasized. Conversely, fetal tissue
can be emphasized wherever elucidation of inherited disorders or
differentiation of particular adult or embryonic stem cells into
tissues or organs such as heart, kidney, nerves or pancreas would
be aided by removing clinical samples from the analysis. Transcript
imaging can also be used to support data from other methodologies
such as guilt-by-association and hybridization analyses.
[0164] The transcript images for SEQ ID NOs:3-6, 8,13, 15-20, and
22 are shown in Table 6. The first column shows library name; the
second column, the number of cDNAs sequenced in that library; the
third column, the description of the library; the fourth column,
absolute abundance of the transcript in the library; and the fifth
column, percentage abundance of the transcript in the library. In
some cases, the normal library shows differential expression of the
cDNA; in others, the induced or diseased library shows differential
expression. For example, for SEQ ID NO:8, the endothelial cells
treated with growth factors (VEGF and EF) show 2-8.times.higher
expression than untreated endothelial cells in the same
experiment.
[0165] These data confirm the differential expression of SEQ ID
NOs:3-6, 8,13, 15-20, and 22 under conditions that correlate with
disorders associated with atherosclerosis. VII Homology Searching
for Atherosclerosis-Associated cDNAs and Polypeptides The
polynucleotide sequences, SEQ ID NO:1-25, and polypeptide sequence,
SEQ ID NO:26, were queried against databases derived from sources
such as GenBank and SwissProt. These databases, which contain
previously identified and annotated sequences, were searched for
regions of similarity using BLAST (Altschul, supra). BLAST searched
for matches and reported only those that satisfied the probability
thresholds of 10-25 or less for nucleotide sequences and 10-8 or
less for polypeptide sequences.
[0166] The polypeptide sequence was also analyzed for known motif
patterns using MOTIFS, SPSCAN, BLIMPS, and HMM-based protocols.
MOTIFS (Genetics Computer Group, Madison Wis.) searches polypeptide
sequences for patterns that match those defined in the Prosite
Dictionary of Protein Sites and Patterns (Bairoch, supra) and
displays the patterns found and their corresponding literature
abstracts. SPSCAN (Genetics Computer Group) searches for potential
signal peptide sequences using a weighted matrix method (Nielsen et
al. (1997) Protein Engineering 10:1-6). Hits with a score of 5 or
greater were considered. BLIMPS uses a weighted matrix analysis
algorithm to search for sequence similarity between the polypeptide
sequences and those contained in BLOCKS, a database consisting of
short amino acid segments, or blocks of 3-60 amino acids in length,
compiled from the PROSITE database (Henikoff; supra; Bairoch,
supra), and those in PRINTS, a protein fingerprint database based
on non-redundant sequences obtained from sources such as SwissProt,
GenBank, PIR, and NRL-3D (Attwood et al. (1997) J Chem Inf Comput
Sci 37:417-424). For the purposes of the present invention, the
BLIMPS searches reported matches with a cutoff score of 1000 or
greater and a cutoff probability value of 1.0.times.10.sup.-3.
HMM-based protocols were based on a probabilistic approach and
searched for consensus primary structures of gene families in the
protein sequences (Eddy, supra; Sonnhammer, supra). More than 500
known protein families with cutoff scores ranging from 10 to 50
bits were selected for use in this invention.
[0167] VIII Hybridization Technologies: Selection of Sequences,
Microarray Preparation and Use
[0168] SEQ ID NO:1-25 are represented among the template sequences
in the LIFESEQ GOLD database (Incyte Genomics). Several of these
sequences, specifically SEQ ID NOs:1, 2, 10, 12, 18, and 32-34 have
been used on microarrays in experiments investigating differential
gene expression in cardiovascular samples. Table 7 presents the
results of these experiments; results were significant if the log2
Cy/Cy5 ratio exceeded .+-.1.00 in either the normal or the induced
or disease state.
[0169] Exemplary Experimental Materials and Protocols
[0170] Activation of the vascular endothelium is considered to be a
central event in a wide range of both physiological and
pathophysiological processes, such as vascular tone regulation,
coagulation and thrombosis, atherosclerosis, and inflammation.
[0171] HAEC, human aortic endothelial cells, are primary cells
derived from the endothelium of a human aorta. They have been used
as an experimental model for investigating the role of the
endothelium in human vascular biology in vitro. HAECs were grown to
85% confluency, split into two samples, one of which was then
treated with growth factors, LDL, cytokines, O2, and the like for
variable time periods.
[0172] ECV304, HUAEC, and HUVEC are endothelial cell lines derived
from the endothelium of the human umbilical artery or vein. This
cell model has been extensively used to study the functional
biology of human endothelial cells. These cells were also grown to
about 85% confluency, split into samples, one of which was then
treated with growth factors, LDL, cytokines, O2, and the like for
variable time periods.
[0173] The experimental treatments and time of exposure are shown
by the tissue description in Table 7.
[0174] Exemplary Activators and Inducers
[0175] TNF-.alpha. is a pleiotropic cytokine that is known to play
a central role in the mediation of inflammatory responses through
activation of multiple signal transduction pathways. TNF-.alpha. is
produced by activated lymphocytes, macrophages, and other white
blood cells, and is known to activate endothelial cells. Monitoring
the endothelial cells' response to TNF-.alpha. at the level of the
mRNA expression can provide information necessary for better
understanding of both TNF-.alpha. signaling pathways and
endothelial cell biology.
[0176] PMA is a broad activator of the protein kinase C-dependent
pathways. lonomycin is a calcium ionophore that permits the entry
of calcium in the cell, hence increasing the cytosolic calcium
concentration. The combination of PMA and ionomycin activates two
of the major signaling pathways used by mammalian cells to interact
with their environment. In T cells, the combination of PMA and
ionomycin mimics the type of secondary signaling events elicited
during optimal B cell activation.
[0177] Microarrays
[0178] The HUMAN GENOME GEM series 1-5 microarrays (Incyte
Genomics) contain 45,320 array elements which represent 22,632
annotated clusters and 22,688 unannotated clusters. For the UNIGEM
series microarrays (Incyte Genomics), Incyte clones were mapped to
non-redundant Unigene clusters (Unigene database (build 46), NCBI;
Shuler (1997) J Mol Med 75:694-698), and the 5' clone with the
strongest BLAST alignment (at least 90% identity and 100 bp
overlap) was chosen, verified, and used in the construction of the
microarray. The UNIGEM V 2.0 microarray (Incyte Genomics) contains
8,502 array elements which represent 8,372 annotated genes and 130
unannotated clusters.
[0179] To construct microarrays, cDNAs were amplified from
bacterial cells using primers complementary to vector sequences
flanking the cDNA insert. Thirty cycles of PCR increased the
initial quantity of cDNAs from 1-2 ng to a final quantity greater
than 5 .mu.g. Amplified cDNAs were then purified using
SEPHACRYL-400 columns (APB). Purified cDNAs were immobilized on
polymer-coated glass slides. Glass microscope slides (Corning,
Corning N.Y.) were cleaned by ultrasound in 0.1% SDS and acetone,
with extensive distilled water washes between and after treatments.
Glass slides were etched in 4% hydrofluoric acid (VWR Scientific
Products, West Chester Pa.), washed thoroughly in distilled water,
and coated with 0.05% aminopropyl silane (Sigma-Aldrich) in 95%
ethanol. Coated slides were cured in a 110.degree. C. oven. cDNAs
were applied to the coated glass substrate using a procedure
described in U.S. Pat. No. 5,807,522. One microliter of the cDNA at
an average concentration of 100 ng/.mu.l was loaded into the open
capillary printing element by a high-speed robotic apparatus which
then deposited about 5 nl of cDNA per slide.
[0180] Microarrays were UV-crosslinked using a STRATALINKER
UV-crosslinker (Stratagene), and then washed at room temperature
once in 0.2% SDS and three times in distilled water. Non-specific
binding sites were blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (Tropix, Bedford Mass.) for 30
minutes at 60.degree. C. followed by washes in 0.2% SDS and
distilled water as before.
[0181] Isolation and Labeling of Sample cDNAs
[0182] Cells were harvested and lysed in 1 ml of TRIZOL reagent
(5.times.10.sup.6 cells/ml; Invitrogen). The lysates were vortexed
thoroughly and incubated at room temperature for 2-3 minutes and
extracted with 0.5 ml chloroform. The extract was mixed, incubated
at room temperature for 5 minutes, and centrifuged at
16,000.times.g for 15 minutes at 4.degree. C. The aqueous layer was
collected, and an equal volume of isopropanol was added. Samples
were mixed, incubated at room temperature for 10 minutes, and
centrifuged at 16,000.times.g for 20 minutes at 4.degree. C. The
supernatant was removed, and the RNA pellet was washed with 1 ml of
70% ethanol, centrifuged at 16,000.times.g at 4.degree. C., and
resuspended in RNAse-free water. The concentration of the RNA was
determined by measuring the optical density at 260 nm.
[0183] Poly(A) RNA was prepared using an OLIGOTEX mRNA kit (Qiagen)
with the following modifications: OLIGOTEX beads were washed in
tubes rather than spin columns, resuspended in elution buffer, and
then loaded onto spin columns to recover the mRNA. To obtain
maximum yield, the mRNA was eluted twice.
[0184] Each poly(A) RNA sample was reverse transcribed using MMLV
reverse-transcriptase, 0.05 pg/.mu.l oligo-d(T) primer (21mer),
1.times.first strand buffer, 0.03 units/ul RNAse inhibitor, 500 uM
dATP, 500 uM dGTP, 500 uM dTTP, 40 uM dCTP, and 40 uM either
dCTP-Cy3 or dCTP-Cy5 (APB). The reverse transcription reaction was
performed in a 25 ml volume containing 200 ng poly(A) RNA using the
GEMBRIGHT kit (Incyte Genomics). Specific control poly(A) RNAs
(YCFRO6, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25,
YCFR44, YCFR26) were synthesized by in vitro transcription from
non-coding yeast genomic DNA (W. Lei, unpublished). As quantitative
controls, control mRNAs (YCFRO6, YCFR45, YCFR67, and YCFR85) at
0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse
transcription reaction at ratios of 1:100,000, 1:10,000, 1:1000,
1:100 (w/w) to sample mRNA, respectively. To sample differential
expression patterns, control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25,
YCFR44, YCFR26) were diluted into reverse transcription reaction at
ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA.
Reactions were incubated at 37.degree. C. for 2 hr, treated with
2.5 ml of 0.5M sodium hydroxide, and incubated for 20 minutes at
85.degree. C. to the stop the reaction and degrade the RNA.
[0185] cDNAs were purified using two successive CHROMA SPIN 30 gel
filtration spin columns (Clontech). Cy3- and Cy5-labeled reaction
samples were combined as described below and ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml
of 100% ethanol. The cDNAs were then dried to completion using a
SpeedVAC system (Savant Instruments, Holbrook N.Y.) and resuspended
in 14 .mu.l 5.times.SSC, 0.2% SDS.
[0186] Hybridization and Detection
[0187] Hybridization reactions contained 9 .mu.l of sample mixture
containing 0.2 .mu.g each of Cy3 and Cy5 labeled cDNA synthesis
products in 5.times.SSC, 0.2% SDS hybridization buffer. The mixture
was heated to 65.degree. C. for 5 minutes and was aliquoted onto
the microarray surface and covered with an 1.8 cm.sup.2 coverslip.
The microarrays were transferred to a waterproof chamber having a
cavity just slightly larger than a microscope slide. The chamber
was kept at 100% humidity internally by the addition of 140 .mu.l
of 5.times.SSC in a corner of the chamber. The chamber containing
the microarrays was incubated for about 6.5 hours at 60.degree. C.
The microarrays were washed for 10 min at 45.degree. C. in low
stringency wash buffer (1.times.SSC, 0.1% SDS), three times for 10
minutes each at 45.degree. C. in high stringency wash buffer
(0.1.times.SSC), and dried.
[0188] Reporter-labeled hybridization complexes were detected with
a microscope equipped with an Innova 70 mixed gas 10 W laser
(Coherent, Santa Clara Calif.) capable of generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of
Cy5. The excitation laser light was focused on the microarray using
a 20.times.microscope objective (Nikon, Melville N.Y.). The slide
containing the microarray was placed on a computer-controlled X-Y
stage on the microscope and raster-scanned past the objective. The
1.8 cm.times.1.8 cm microarray used in the present example was
scanned with a resolution of 20 micrometers.
[0189] In two separate scans, the mixed gas multiline laser excited
the two fluorophores sequentially. Emitted light was split, based
on wavelength, into two photomultiplier tube detectors (PMT R1477;
Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the
two fluorophores. Appropriate filters positioned between the
microarray and the photomultiplier tubes were used to filter the
signals. The emission maxima of the fluorophores used were 565 nm
for Cy3 and 650 nm for Cy5. Each microarray was typically scanned
twice, one scan per fluorophore using the appropriate filters at
the laser source, although the apparatus was capable of recording
the spectra from both fluorophores simultaneously.
[0190] The sensitivity of the scans was calibrated using the signal
intensity generated by a cDNA control species. Samples of the
calibrating cDNA were separately labeled with the two fluorophores
and identical amounts of each were added to the hybridization
mixture. A specific location on the microarray contained a
complementary DNA sequence, allowing the intensity of the signal at
that location to be correlated with a weight ratio of hybridizing
species of 1:100,000.
[0191] The output of the photomultiplier tube was digitized using a
12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog
Devices, Norwood, Mass.) installed in an IBM-compatible PC
computer. The digitized data were displayed as an image where the
signal intensity was mapped using a linear 20-color transformation
to a pseudocolor scale ranging from blue (low signal) to red (high
signal). The data was also analyzed quantitatively. Where two
different fluorophores were excited and measured simultaneously,
the data were first corrected for optical crosstalk (due to
overlapping emission spectra) between the fluorophores using each
fluorophore's emission spectrum.
[0192] A grid was superimposed over the fluorescence signal image
such that the signal from each spot was centered in each element of
the grid. The fluorescence signal within each element was then
integrated to obtain a numerical value corresponding to the average
intensity of the signal. The software used for signal analysis was
the GEMTOOLS gene expression analysis program (Incyte Genomics).
Significance was defined as signal to background ratio exceeding
2.times.and area hybridization exceeding 40%.
[0193] IX Further Characterization of Differentially Expressed
cDNAs and Proteins
[0194] Clones were aligned against the LIFESEQ Gold 5.1 database
(Incyte Genomics) and an Incyte template and its sequence variants
were chosen for each clone. The template and variant sequences were
aligned against the GenBank nucleotide sequence databases using
BLASTn (vers. 2.0, NCBI) to acquire annotation. The template and
variant sequences were translated into amino acid sequences which
were aligned against GenPept and other protein databases using
BLASTp (vers. 2.0, NCBI) to acquire annotation and
characterization, i.e., structural motifs. Table 3 shows the
GenBank annotations (where available) for SEQ ID NOs:1-25 of this
invention as produced by BLAST analysis.
[0195] Percent sequence identity can be determined electronically
for two or more amino acid or nucleic acid sequences using the
MEGALIGN program, a component of LASERGENE software (DNASTAR). The
percent identity between two amino acid sequences is calculated by
dividing the length of sequence A, minus the number of gap residues
in sequence A, minus the number of gap residues in sequence B, into
the sum of the residue matches between sequence A and sequence B,
times one hundred. Gaps of low or of no homology between the two
amino acid sequences are not included in determining percentage
identity.
[0196] Sequences with conserved protein motifs may be searched
using the BLOCKS search program. This program analyses sequence
information contained in the Swiss-Prot and PROSITE databases and
is useful for determining the classification of uncharacterized
proteins translated from genomic or cDNA sequences (Bairoch, supra;
Attwood, supra). PROSITE database is a useful source for
identifying functional or structural domains that are not detected
using motifs due to extreme sequence divergence. Using weight
matrices, these domains are calibrated against the SWISS-PROT
database to obtain a measure of the chance distribution of the
matches.
[0197] The PRINTS database can be searched using the BLIMPS search
program to obtain protein family "fingerprints". The PRINTS
database complements the PROSITE database by exploiting groups of
conserved motifs within sequence alignments to build characteristic
signatures of different protein families. For both BLOCKS and
PRINTS analyses, the cutoff scores for local similarity were:
[0198] >1300=strong, 1000-1300=suggestive; for global similarity
were: p<exp-3; and for strength (degree of correlation) were:
>1300=strong, 1000-1300=weak.
[0199] X Other Hybridization Technologies and Analyses
[0200] Other hybridization technologies utilize a variety of
substrates such as nylon membranes, capillary tubes, etc. Arranging
cDNAs on polymer coated slides is described in Example V; sample
cDNA preparation and hybridization and analysis using polymer
coated slides is described in examples VI and VII,
respectively.
[0201] The cDNAs are applied to a membrane substrate by one of the
following methods. A mixture of cDNAs is fractionated by gel
electrophoresis and transferred to a nylon membrane by capillary
transfer. Alternatively, the cDNAs are individually ligated to a
vector and inserted into bacterial host cells to form a library.
The cDNAs are then arranged on a substrate by one of the following
methods. In the first method, bacterial cells containing individual
clones are robotically picked and arranged on a nylon membrane. The
membrane is placed on LB agar containing selective agent
(carbenicillin, kanamycin, ampicillin, or chloramphenicol depending
on the vector used) and incubated at 37.degree. C. for 16 hr. The
membrane is removed from the agar and consecutively placed colony
side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH),
neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in
2.times.SSC for 10 min each. The membrane is then UV irradiated in
a STRATALINKER UV-crosslinker (Stratagene).
[0202] In the second method, cDNAs are amplified from bacterial
vectors by thirty cycles of PCR using primers complementary to
vector sequences flanking the insert. PCR amplification increases a
starting concentration of 1-2 ng nucleic acid to a final quantity
greater than 5 .mu.g. Amplified nucleic acids from about 400 bp to
about 5000 bp in length are purified using SEPHACRYL-400 beads
(APB). Purified nucleic acids are arranged on a nylon membrane
manually or using a dot/slot blotting manifold and suction device
and are immobilized by denaturation, neutralization, and UV
irradiation as described above.
[0203] Hybridization probes derived from cDNAs of the Sequence
Listing are employed for screening cDNAs, mRNAs, or genomic DNA in
membrane-based hybridizations. Probes are prepared by diluting the
cDNAs to a concentration of 40-50 ng in 45 IL TE buffer, denaturing
by heating to 100.degree. C. for five min and briefly centrifuging.
The denatured cDNA is then added to a REDIPRIME tube (APB), gently
mixed until blue color is evenly distributed, and briefly
centrifuged. Five microliters of [.sup.32P]dCTP is added to the
tube, and the contents are incubated at 37.degree. C. for 10 min.
The labeling reaction is stopped by adding 5 .mu.l of 0.2M EDTA,
and probe is purified from unincorporated nucleotides using a
PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to
100.degree. C. for five min and then snap cooled for two min on
ice.
[0204] Membranes are pre-hybridized in hybridization solution
containing 1% Sarkosyl and Ix high phosphate buffer (0.5 M NaCl,
0.1 M Na.sub.2HPO.sub.4, 5 mM EDTA, pH 7) at 55.degree. C. for two
hr. The probe, diluted in 15 ml fresh hybridization solution, is
then added to the membrane. The membrane is hybridized with the
probe at 55.degree. C. for 16 hr. Following hybridization, the
membrane is washed for 15 min at 25.degree. C. in imM Tris (pH
8.0), 1% Sarkosyl, and four times for 15 min each at 25.degree. C.
in 1 mM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR
film (Eastman Kodak, Rochester N.Y.) is exposed to the membrane
overnight at -70.degree. C., developed, and examined.
[0205] XI Expression of the Encoded Protein
[0206] Expression and purification of a protein encoded by a cDNA
of the invention is achieved using bacterial or virus-based
expression systems. For expression in bacteria, cDNA is subcloned
into a vector containing an antibiotic resistance gene and an
inducible promoter that directs high levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage
promoter in conjunction with the lac operator regulatory element.
Recombinant vectors are transformed into bacterial hosts, such as
BL21(DE3). Antibiotic resistant bacteria express the protein upon
induction with IPTG. Expression in eukaryotic cells is achieved by
infecting Spodoptera frugiperda (Sf9) insect cells with recombinant
baculovirus, Autographica californica nuclear polyhedrosis virus.
The polyhedrin gene of baculovirus is replaced with the cDNA by
either homologous recombination or bacterial-mediated transposition
involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong polyhedrin promoter drives high levels of
transcription.
[0207] For ease of purification, the protein is synthesized as a
fusion protein with glutathione-S-transferase (GST; APB) or a
similar alternative such as FLAG. The fusion protein is purified on
immobilized glutathione under conditions that maintain protein
activity and antigenicity. After purification, the GST moiety is
proteolytically cleaved from the protein with thrombin. A fusion
protein with FLAG, an 8-amino acid peptide, is purified using
commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak, Rochester N.Y.).
[0208] XII Production of Antibodies
[0209] A denatured protein from a reverse phase HPLC separation is
obtained in quantities up to 75 mg. This denatured protein is used
to immunize mice or rabbits following standard protocols. About 100
.mu.g is used to immunize a mouse, while up to 1 mg is used to
immunize a rabbit. The denatured protein is radioiodinated and
incubated with murine B-cell hybridomas to screen for monoclonal
antibodies. About 20 mg of protein is sufficient for labeling and
screening several thousand clones.
[0210] In another approach, the amino acid sequence translated from
a cDNA of the invention is analyzed using PROTEAN software
(DNASTAR) to determine antigenic determinants of the protein. The
optimal sequences for immunization are usually at the C-terminus,
the N-terminus, and those intervening, hydrophilic regions of the
protein that are likely to be exposed to the external environment
when the protein is in its natural conformation. Typically,
oligopeptides about 15 residues in length are synthesized using an
431 peptide synthesizer (ABI) using Fmoc-chemistry and then coupled
to keyhole limpet hemocyanin (KLH; Sigma-Aldrich) by reaction with
M-maleimidobenzoyl-N-hydroxysuccinimide ester. If necessary, a
cysteine may be introduced at the N-terminus of the peptide to
permit coupling to KLH. Rabbits are immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. The
resulting antisera are tested for antipeptide activity by binding
the peptide to plastic, blocking with 1% BSA, reacting with rabbit
antisera, washing, and reacting with radioiodinated goat
anti-rabbit IgG.
[0211] Hybridomas are prepared and screened using standard
techniques. Hybridomas of interest are detected by screening with
radioiodinated protein to identify those fusions producing a
monoclonal antibody specific for the protein. In a typical
protocol, wells of 96 well plates (FAST, Becton-Dickinson, Palo
Alto Calif.) are coated with affinity-purified, specific
rabbit-anti-mouse (or suitable anti-species Ig) antibodies at 10
mg/ml. The coated wells are blocked with 1% BSA and washed and
exposed to supernatants from hybridomas. After incubation, the
wells are exposed to radiolabeled protein at 1 mg/ml. Clones
producing antibodies bind a quantity of labeled protein that is
detectable above background.
[0212] Such clones are expanded and subjected to 2 cycles of
cloning at 1 cell/3 wells. Cloned hybridomas are injected into
pristane-treated mice to produce ascites, and monoclonal antibody
is purified from the ascitic fluid by affinity chromatography on
protein A (APB). Monoclonal antibodies with affinities of at least
10.sup.8 M.sup.-, preferably 10.sup.9 to 1010 M.sup.-1 or stronger,
are made by procedures well known in the art.
[0213] XIII Purification of Naturally Occurring Protein Using
Antibodies
[0214] Naturally occurring or recombinant protein is purified by
immunoaffinity chromatography using antibodies specific for the
protein. An immunoaffinity column is constructed by covalently
coupling the antibody to CNBr-activated SEPHAROSE resin (APB).
Media containing the protein is passed over the immunoaffinity
column, and the column is washed using high ionic strength buffers
in the presence of detergent to allow preferential absorbance of
the protein. After coupling, the protein is eluted from the column
using a buffer of pH 2-3 or a high concentration of urea or
thiocyanate ion to disrupt antibody/protein binding, and the
protein is collected.
[0215] XIV Screening for Molecules That Specifically Bind the cDNA
or Protein
[0216] The cDNA or fragments thereof and the protein or portions
thereof are labeled with .sup.32P-dCTP, Cy3-dCTP, Cy5-dCTP (APB),
or BIODIPY or FITC (Molecular Probes), respectively. Candidate
molecules or compounds previously arranged on a substrate are
incubated in the presence of labeled nucleic or amino acid. After
incubation under conditions for either a cDNA or a protein, the
substrate is washed, and any position on the substrate retaining
label, which indicates specific binding or complex formation, is
assayed. The binding molecule is identified by its arrayed position
on the substrate. Data obtained using different concentrations of
the nucleic acid or protein are used to calculate affinity between
the labeled nucleic acid or protein and the bound molecule. High
throughput screening using very small assay volumes and very small
amounts of test compound is fully described in U.S. Pat. No.
5,876,946.
[0217] All patents and publications mentioned in the specification
are incorporated herein by reference. Various modifications and
variations of the described method and system of the invention will
be apparent to those skilled in the art without departing from the
scope and spirit of the invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
that are obvious to those skilled in the field of molecular biology
or related fields are intended to be within the scope of the
following claims.
4TABLE 4 Co-expression of 25 cDNAs with known atherosclerosis genes
(- log p). SEQ ID CNN1 COL1A1 COL1A2 COL6A1 COL5A2 COL6A2 COL6A3
COL3A1 COL5A1 MGP CTSK FGB VWF 1 0 1 0 0 0 0 0 0 0 0 0 4 1 2 1 1 3
2 2 2 4 3 4 5 3 1 6 3 0 1 0 1 1 1 0 0 1 1 1 4 0 4 1 0 1 1 1 0 1 1 1
0 1 7 0 5 5 9 12 13 3 14 11 12 4 10 13 1 3 6 6 4 7 9 4 13 5 6 8 14
6 1 12 7 5 4 5 5 2 7 5 6 5 10 1 2 1 8 1 1 0 2 1 1 0 1 0 1 0 1 1 9 1
1 4 2 1 6 4 4 5 6 1 0 10 10 3 4 3 3 3 4 4 3 1 4 1 0 5 11 1 1 1 1 1
1 1 0 1 1 2 1 1 12 11 4 7 9 2 9 11 10 7 11 3 0 5 13 2 6 5 6 9 8 4 6
7 1 3 1 1 14 0 0 0 0 0 0 0 1 0 1 1 4 1 15 17 7 13 17 2 25 12 11 6
21 6 3 7 16 13 22 27 27 15 33 24 34 15 20 18 0 8 17 2 1 3 3 0 2 6 3
1 9 1 0 7 18 6 5 6 7 3 7 10 6 2 14 5 0 5 19 0 1 1 2 1 1 0 1 0 0 1 7
0 20 8 7 8 12 3 9 11 10 3 11 6 1 8 21 0 2 4 2 4 2 5 5 3 2 1 0 4 22
1 1 0 1 3 1 2 1 1 1 1 4 1 23 1 1 0 1 0 1 0 1 0 0 0 7 0 24 0 1 2 0 0
0 0 1 0 1 0 8 1 25 48 11 17 24 3 26 18 17 13 20 6 1 8 SEQ ID PECAM1
AT3 LPL A2M APOA1 APOA2 APOB APOC2 APOC3 APOC4 MSR1 CD36 SAP CEL 1
0 5 0 1 3 4 2 2 5 4 0 0 6 0 2 7 0 1 4 0 1 0 0 0 0 2 3 0 1 3 0 8 1 1
5 4 3 3 5 7 0 0 8 1 4 0 4 1 2 4 5 4 4 6 2 0 1 3 0 5 2 0 4 9 1 0 0 1
0 0 4 2 0 2 6 6 1 5 7 0 1 1 0 1 0 7 3 0 2 7 4 3 3 8 1 1 2 0 2 1 2 7
2 0 8 0 0 3 3 0 0 0 2 0 0 1 1 1 0 9 8 0 5 6 1 1 1 2 0 0 5 6 0 1 10
3 0 4 4 0 0 0 0 0 0 9 5 0 0 11 1 2 1 0 3 1 2 2 1 0 0 2 3 6 12 2 1 3
6 2 0 1 0 0 0 4 3 0 3 13 0 0 1 1 3 2 1 2 1 0 1 2 1 3 14 0 5 1 1 4 5
5 5 3 1 0 0 2 1 15 5 1 1 13 1 1 1 0 1 0 10 1 1 0 16 7 0 3 12 1 0 0
1 1 0 9 5 0 1 17 5 0 6 7 0 0 0 1 0 0 5 9 0 1 18 4 0 6 10 1 0 1 0 0
0 8 5 1 1 19 1 8 1 3 5 8 4 6 8 9 1 1 11 0 20 2 0 12 10 1 1 0 1 0 0
9 14 0 0 21 4 0 8 5 0 1 0 1 1 0 5 11 1 1 22 4 9 0 2 6 8 7 6 4 3 1 1
7 0 23 0 6 0 1 6 7 7 4 8 1 0 1 7 1 24 0 7 0 2 8 10 10 7 8 4 0 0 5 1
25 2 0 2 20 1 0 3 0 0 0 10 4 0 1 SEQ ID PON1 PON2 PON3 PLIN PTGDS
ANX2 ANX1 SPARC SM22 1 5 1 2 0 0 2 0 1 0 2 1 6 1 1 2 3 4 8 3 3 5 2
2 0 0 1 0 1 0 4 2 0 1 0 0 1 0 1 1 5 1 1 1 6 4 6 3 9 9 6 1 4 0 11 7
6 4 11 14 7 1 4 5 2 1 11 7 7 8 8 1 5 0 1 23 1 1 4 2 9 0 7 1 6 3 3 2
7 4 10 0 1 0 15 2 2 1 2 4 11 6 2 7 4 0 0 2 1 0 12 1 3 1 3 3 6 3 7 9
13 2 3 2 2 1 1 3 4 2 14 2 2 1 1 0 0 0 1 0 15 0 2 1 1 12 7 9 14 27
16 1 2 2 7 6 12 9 22 20 17 0 1 0 6 2 3 1 6 3 18 1 2 3 8 6 3 10 6 11
19 5 1 2 0 1 0 1 0 1 20 0 0 0 19 4 4 2 7 10 21 0 1 0 8 0 2 2 3 3 22
4 2 2 1 2 1 3 1 1 23 4 1 3 1 0 0 0 1 0 24 3 1 1 0 2 1 1 1 0 25 1 1
1 3 11 9 6 9 30
[0218]
5TABLE 5 Summary of coexpressed genes and their function in
atherosclerosis-associated disorders SEQ ID Pvalue Gene 1 Gene
function from Table 3 Pvalue Gene 2 Gene function from Table 3 1 6
SAP associated with amyloid P 5 APOC3 associated with plasma in
lesions triglyceride and hyperlipidemia 2 8 SPARC calcification of
plaques 7 PECAM- implicated in migration of cells under O2 stress 3
8 AT3 deficiency causes recurrent 8 SAP associated with amyloid P
in venous thrombosis lesions 4 7 FGB fibrin deposition in plaque 6
APOC3 apolipoprotein CIII 5 14 COL6A2 promoting platelet
aggregation 12 CTSK present in advanced plaques 6 14 SM22
downregulated during 14 MGP calcificaton associated with
atherogenesis high expression 7 11 ANX2 role in atherogenesis 10
MGP calcificaton associated with high expression 8 23 PTDGS
accumulates in end-stage 15 AGT associated with higher total
atherosclerotic plaques cholesterol levels 9 10 VWF Increased
levels found in 8 PECAM- implicated in migration of atherosclerosis
cells under O2 stress 10 15 PLIN functions in lipid metabolism 9
MSR1 deposits cholesterol during atherogenesis 11 7 PON3 associated
with coronary heart 6 CEL implicated in the progression disease and
cholesterol levels of atherosclerosis 12 11 COL6A3 promoting
platelet aggregation 11 MGP calcificaton associated with high
expression 13 9 COL5A2 promoting platelet aggregation 8 COL6A2
promoting platelet aggregation 14 5 APOA/B accumulation of plasma
LDL in 5 AT3 deficiency causes recurrent atherogenesis venous
thrombosis 15 27 SM22 downregulated during 25 COL6A2 promoting
platelet aggregation atherogenesis 16 34 COL3A1 promoting platelet
aggregation 22 SPARC calcification of plaques 17 9 MGP calcificaton
associated with 9 CD36 receptor for oxidised LDL high expression 18
11 SM22 downregulated during 10 A2M retains LDL in core of plaque
atherogenesis 19 11 SAP associated with amyloid P in 9 APOC4 alters
lipid metabolism toward lesions hypertriglyceridaemia 20 19 PLIN
functions in lipid metabolism 14 CD36 receptor for oxidised LDL 21
11 CD36 receptor for oxidised LDL 8 PLIN functions in lipid
metabolism 22 9 AT3 deficiency causes recurrent 8 APOA2 associated
with increased venous thrombosis atherosclerotic lesions 23 8 APOC3
associated with plasma 7 SAP associated with amyloid P in
triglyceride and hyperlipidemia lesions 24 10 APOA/B accumulation
of plasma LDL in 8 FGB fibrin deposition in plaque atherogenesis 25
48 CNN1 modulation of SMC, a feature 26 COL6A2 promoting platelet
aggregation of atherosclerosis
[0219]
6TABLE 6 Cardiovascular transcript images for the coexpressed cDNAs
SEQ ID Library cDNAs Description of Sample Abundance % Abundance 3
SMCRUNT01 3472 renal vein, smooth muscle cells, 57M, Untx 1 0.0288
ENDVUNT01 5215 microvascular, dermal, endothelial cells, 22F, Untx
1 0.0192 HEAANOT01 12578 heart, coronary artery, CAD, 46M 1 0.0080
4 MONOTXN05 2709 periph blood, monocytes, 42F, t/IL-10, LPS, NORM 1
0.0369 MONOTXT02 3554 periph blood, monocytes, 42F, t/IL-10, LPS 1
0.0281 MCLRUNT01 6149 periph blood mononuclear cells, 60M,
untreated 1 0.0163 5 ARTANOT06 6311 aorta, adventitia, 48M 2 0.0317
HEAONOT02 3482 heart, aorta, 10M 1 0.0287 HEAONOT04 4002 heart,
aorta, 12F 1 0.0250 ARTANOT07 5716 aorta, adventitia, 65F 1 0.0175
6 HEAONOE01 3639 heart, aorta, 39M, 5RP 10 0.2748 HEAONOT03 3720
heart, aorta, aw/cerebral agenesis, 27F 3 0.0806 HEAPNOT01 3502
heart, coronary artery, plaque, pool 2 0.0571 8 ENDVTXT01 1882
microvascular, dermal, endothelial cells, 22F, t/bFGF, EF 3 0.1594
ENDVTXT02 1876 microvascular, dermal, endothelial cells, 22F,
t/VEGE, EF 1 0.0533 ENDVUNT01 5215 microvascular, dermal,
endothelial cells, 22F, Untx 1 0.0192 13 SMCCNOS01 3494 coronary
artery, smooth muscle cells, 3M, t/TNF, IL-1, SUB 1 0.0286
SMCANOT01 7327 aortic smooth muscle line, M 1 0.0136 15 SMCRTXT01
3453 renal vein, smooth muscle cells, 57M t/TNF, IL1 2 0.0579
SMCRUNT01 3472 renal vein, smooth muscle cells, 57M, Untx 1 0.0288
16 SMCCNOT01 4266 coronary artery, smooth muscle cells, 3M 5 0.1172
SMCCNOT02 3980 coronary artery, smooth muscle cells, 3M, t/TNF,
IL-1 2 0.0503 SMCCNOS01 3494 coronary artery, smooth muscle cells,
3M, t/TNF, IL-1, SUB 1 0.0286 17 ENDIUNT01 3582 iliac artery,
endothelial cells, F, control, untreated 26 0.7259 ENDITXT01 3464
iliac artery, endothelial cells, F, t/1% oxygen 24 hr 21 0.6062
ENDINOT02 3208 iliac artery, endothelial cells, F, t/TNF, IL-1 20
hr 10 0.3117 18 ENDVTXT01 1882 microvascular, dermal, endothelial
cells, 22F, t/bFGF, EF 1 0.0531 ENDVNOT01 4955 microvascular,
dermal, endothelial cells, 18F, untreated 1 0.0202 19 ENDATXP01
1877 aorta, endothelial cells, t/TNF, TIGR 1 0.0533 ENDVTXT02 1876
microvascular, dermal, endothelial cells, 22F, t/VEGF, EF 1 0.0533
ENDVTXT01 1882 microvascular, dermal, endothelial cells, 22F,
t/bFGF, EF 1 0.0531 ENDVNOT01 4955 microvascular, dermal,
endothelial cells, 18F, untreated 1 0.0202 20 HEAANOT01 12578
heart, coronary artery, CAD, 46M 13 0.1034 HEAONOT05 3959 heart,
aorta, 17F 3 0.0758 HEAONOT04 4002 heart, aorta, 12F 1 0.0250 22
ENDITXT01 3464 iliac artery, endothelial cells, F, t/1% oxygen 24
hr 1 0.0289 HEAONOE01 3639 heart, aorta, 39M, 5RP 1 0.0275
HEAANOT01 12578 heart, coronary artery, CAD, 46M 1 0.0080
[0220]
7TABLE 7 Microarray data from cardiovascular experiments SEQ ID GEM
Log2 (Cy5/Cy3) Cy3 Sample Cy5 Sample 1 LG1 -1.01 ECV304 Line, Untx
ECV304 Line, t/PMA + Iono 2 HG1 -1.03 HUVEC Cells, Untx, HUVEC
Cells, t/TNFa 10 ng/mL Nrml 1 hr, Nrml 2 HG1 -1.03 HUAEC Cells,
Untx, HUAEC Cells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2 HG1 -1.03
HUVEC Cells, Untx, HUVEC Cells, t/IL1b + TNFa Nrml 10 ng/mL, 10
ng/mL 2 4hr, 4 hr, Nrml 2 HG1 -1.07 HUVEC Cells, Untx, HUVEC Cells,
t/PMA + TNFa Nrml, 24 hr 10 nM, 10 ng/mL 24 hr, 1 hr, Nrml 2 HG1
-1.07 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 48 hr,
Nrml 2 HG1 -1.10 HUVEC Cells, Untx HUVEC Cells, t/IL4 10 ng/mL 24
hr 2 HG1 -1.10 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml
2 hr, Nrml 2 HG1 -1.12 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa
48 hr 10 ng/mL, 10 ng/mL 24 hr, 1 hr 2 HG1 -1.12 HUVEC Cells, Untx
HUVEC Cells, t/IL10 + TNFa 10 ng/mL, 10 ng/mL 24 hr,4 hr 2 HG1
-1.13 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL Nrml 4 hr,
Nrml 2 HG1 -1.14 HUVEC Cells, Untx, HUVEC Cells, t/CHX + TNFa Nrml
10 mcg/mL, 10 ng/mL 30 min, 24 hr, Nrml 2 HG1 -1.14 HUVEC Cells,
Untx, HUVEC Cells, t/PMA 10 nM Nrml, 24 hr 24 hr, Nrml 2 HG1 -1.15
HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2
HG1 -1.17 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 24
hr, Nrml 2 HG1 -1.18 HUVEC Cells, Untx, HUVEC Cells, t/8ClcAMP +
TNFa Nrml, 24 hr 7.5 microM, 10 ng/mL 24 hr, 4 hr, Nrml 2 HG1 -1.19
HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2
HG1 -1.21 HUVEC Cells, Untx, HUVEC Cells, t/TL1b + TNFa Nrml 10
ng/mL, 10 ng/mL 2 4hr, 24 hr, Nrml 2 HG1 -1.22 HIAEC Cells, Untx,
HIAEC Cells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2 HG1 -1.22 HUVEC
Cells, Untx, HUVEC Cells, t/TNFa + TNFa Nrml, 24 hr .1 ng/ml, 10
ng/ml 24 hr, 24 hr, Nrml 2 HG1 -1.23 HUVEC Cells, Untx HUVEC Cells,
t/TNFa 10 ng/mL 4 hr" 2 HG1 -1.26 HUVEC Cells, Untx, HUVEC Cells,
t/8ClcAMP + TNFa Nrml, 24 hr 7.5 microM, 10 ng/mL 24 hr, 24 hr,
Nrml 2 HG1 -1.28 HUVEC Cells, Untx HUVEC Cells, t/TNFa .1 ng/mL 4
hr 2 HG1 -1.29 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml
4 hr, Nrml 2 HG1 -1.29 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10
ng/mL Nrml 4 hr, Nrml 2 HG1 -1.30 HUVEC Cells, Untx HUVEC Cells,
t/IL10 + TNFa 10 ng/mL, 10 ng/mL 24 hr, 24 hr 2 HG1 -1.31 HAEC
Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml Nrml 10 hr, Nrml 2 HG1
-1.31 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml Nrml 24 hr,
Nrml 2 HG1 -1.34 HUVEC Cells, Untx, HUVEC Cells, t/lL4 + TNFa Nrml,
24 hr 10 ng/ml, 10 ng/ml 24 hr, 4 hr, Nrml 2 HG1 -1.36 HAEC Cells,
Untx, HAEC Cells, t/TNFa 10 ng/ml Nrml 4 hr, Nrml 2 HG1 -1.40 HUVEC
Cells, Untx, HUVEC Cells, t/IL4 + TNFa Nrml, 24 hr 10 ng/ml, 10
ng/ml 24 hr, 24 hr, Nrml 2 HG1 -1.43 HUVEC Cells, Untx HUVEC Cells,
t/TNFa 10 ng/mL 24 hr 2 HG1 -1.44 HUVEC Cells, Untx, HUVEC Cells,
t/IL1b 10 ng/mL Nrml 24 hr, Nrml 2 HG1 -1.48 HUVEC Cells, Untx
HUVEC Cells, t/TNFa 10 ng/mL 24 hr" 2 HG1 -1.52 HUAEC Cells, Untx,
HUAEC Cells, t/TNFa 10 ng/mL Nrml 8 hr, Nrml 2 HG1 -1.53 HUVEC
Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 4 hr, Nrml 2 HG1
-1.53 HUVEC Cells, Untx HUVEC Cells, t/CHX + TNFa 10 mcg/ml, 10
ng/ml 30 min, 23.5 hr 2 HG1 -1.53 HUVEC Cells, Untx HUVEC Cells,
t/TNFa 1 ng/mL 24 hr 2 HG1 -1.55 HAEC Cells, Untx, HAEC Cells,
t/TNFa 10 ng/ml Nrml 8 hr, Nrml 2 HG1 -1.58 HUVEC Cells, Untx,
HUVEC Cells, t/PD98059 + TNFa Nrml, 24 hr 50 microM, 10 ng/mL 24
hr,24 hr, Nrml 2 HG1 -1.59 HUVEC Cells, Untx, HUVEC Cells, t/TNFa
10 ng/mL Nrml 3 d, Nrml 2 HG1 -1.59 HAEC Cells, Untx, HAEC Cells,
t/TNFa 10 ng/ml Nrml 6 hr, Nrml 2 HG1 -1.61 HUVEC Cells, Untx,
HUVEC Cells, t/IL1b + TNFa Nrml 10 ng/mL, 10 ng/mL 24 hr, 1 hr,
Nrml 2 HG1 -1.62 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/ml
Nrml, 24 hr 24 hr, Nrml 2 HG1 -1.72 HUVEC Cells, Untx, HUVEC Cells,
t/TNFa 10 ng/mL Nrml 8 hr, Nrml 2 HG1 -1.80 HUVEC Cells, Untx,
HUVEC Cells, t/PD98059 + TNFa Nrml, 24 hr 50 microM, 10 ng/mL 24
hr, 4 hr, Nrml 2 HG1 -1.83 HIAEC Cells, Untx, HIAEC Cells, t/TNFa
10 ng/mL Nrml 8 hr, Nrml 2 HG1 -2.07 HUVEC Cells, Untx, HUVEC
Cells, t/IL4 + TNFa 48 hr 10 ng/mL, 10 ng/mL 24 hr, 24 hr 2 HG1
-2.15 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 8 hr,
Nrml 2 HG1 -2.18 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa 48 hr
10 ng/mL, 10 ng/mL 24 hr,4 hr 10 UG1 3.38 HUVEC Cells, Untx, HUVEC
Cells, t/CHX + TNFa Nrml 10 mcg/mL, 10 ng/mL 30 min, 4 hr, Nrml 10
UG1 3.16 HUVEC Cells, Untx, HUVEC Cells, t/CHX + TNFa Nrml 10
mcg/mL, 10 ng/mL 30 min, 1 hr, Nrml 10 UG1 2.85 HUAEC Cells, Untx,
HUAEC Cells, t/TNFa 10 ng/mL Nrml 2 hr, Nrml 10 UG1 2.68 HPAEC
Cells, Untx, HPAEC Cells, t/TNFa 10 ng/mL Nrml 1 hr, Nrml 10 UG1
2.64 HPAEC Cells, Untx, HPAEC Cells, t/TNFa 10 ng/mL Nrml 2 hr,
Nrml 10 UG1 2.60 HUVEC Cells, Untx, HUVEC Cells, t/PMA + TNFa Nrml,
24 hr 100 nM, 10 ng/mL 24 hr, 1 hr, Nrml 10 UG1 2.55 HUVEC Cells,
Untx, HUVEC Cells, t/PMA + TNFa Nrml, 24 hr 100 nM, 10 ng/mL 24 hr,
24 hr, Nrml 10 UG1 2.47 HUVEC Cells, Untx, HUVEC Cells, t/Dex +
TNFa 0 hr, Nrml 100 nM, 10 ng/mL 24 hr, 1 hr Nrml 10 UG1 2.40 HUVEC
Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 48 hr, Nrml 10 UG1
2.39 HUVEC Cells, Unix, HUVEC Cells, t/Dex + TNFa Nrml, 0 hr 100
nM, 10 ng/mL 24 hr, 24 hr Nrml 10 UG1 2.27 HUVEC Cells, Untx, HUVEC
Cells, t/IL10 + TNFa Nrml 10 ng/mL, 10 ng/mL 24 hr, 24 hr, Nrml 10
UG1 2.24 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL Nrml 4 hr,
Nrml 10 UG1 2.18 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
Nrml 1 hr, Nrml 10 UG1 2.17 HUVEC Cells, Untx, HUVEC Cells, t/Dex +
TNFa Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 24 hr Nrml 10 UG1 2.14 HUVEC
Cells, Untx, HUVEC Cells, t/Lipom + TNFa 24 hr 8 mcg/mL, 10 ng/ml 4
hr, 4 hr, 1 hr 10 UG1 2.11 HUVEC Cells, Untx, HUVEC Cells, t/ILlb +
TNFa Nrml 10 ng/mL, 10 ng/mL 24 hr, 24 hr, Nrml 10 UG1 2.04 HUVEC
Cells, Untx, HUVEC Cells, t/IFNg + TNFa Nrml 200 ng/mL, 10 ng/mL 24
hr, 24 hr, Nrml 10 UG1 1.95 HUVEC Cells, Untx, HUVEC Cells, t/Dex +
TNFa Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 1 hr Nrml 10 UG1 1.91 HUVEC
Cells, Untx, HUVEC Cells, t/Lipom + 24 hr asOligo + TNFa 8 mcg/mL,
100 nM, 50 nM, 10 ng/ml 4 hr, 4 hr, 1 hr 10 UG1 1.91 HUVEC Cells,
Untx, HUVEC Cells, t/PD98059 + TNFa Nrml, 24 hr 50 microM, 10 ng/mL
24 hr, 1 hr, Nrml 10 UG1 1.88 HUVEC Cells, Untx, HUVEC Cells, t/Dex
+ TNFa Nrml, 0 hr 100 nM, 10 ng/mL 24 hr, 4 hr Nrml 10 UG1 1.88
HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa Nrml, 0 hr 10 nM, 10
ng/mL 24 hr, 4 hr Nrml 10 UG1 1.86 HUVEC Cells, Untx, HUVEC Cells,
t/IL4 + TNFa 48 hr 10 ng/mL, 10 ng/mL 24 hr, 24 hr 10 UG1 1.82
HUVEC Cells, Untx, HUVEC Cells, t/Lipom + TNFa 24 hr 8 mcg/mL, 10
ng/ml 4 hr, 4 hr, 24 hr 10 UG1 1.82 HUVEC Cells, Untx, HUVEC Cells,
t/IL4 + TNFa 48 hr 10 ng/mL, 10 ng/mL 24 hr, 1 hr 10 UG1 1.76 HUVEC
Cells, Untx, HUVEC Cells, t/IL1b 10 ng/mL Nrml 24 hr, Nrml 10 UG1
1.74 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 24 hr,
Nrml 10 HG3 1.20 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
Nrml 1 hr, Nrml 10 HG3 1.04 HUAEC Cells, Untx, HUAEC Cells, t/TNFa
10 ng/mL Nrml 2 hr, Nrml 12 HG5 1.02 ECV304 Line, t/TNFa ECV304
Line, Untx, Nrml 10 ng/mL 3 d, Nrml 12 HG5 -1.07 HUVEC Cells, Untx
HUVEC Cells, t/IL10 + TNFa 10 ng/mL, 10 ng/mL 24 hr, 4 hr 12 HG5
-1.16 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL Nrml 8 hr,
Nrml 12 HG5 -1.18 HMVEC Cells, Untx, HMVEC Cells, t/TNFa 10 ng/mL
Nrml 24 hr, Nrml 12 HG5 -1.24 HMVEC Cells, Untx, HMVEC Cells,
t/TNFa 10 ng/mL Nrml 8 hr, Nrml 12 HG5 -1.50 HIAEC Cells, Untx,
HIAEC Cells, t/TNFa 10 ng/mL Nrml 8 hr, Nrml 18 HG2 -2.38 HUVEC
Cells, Untx, HUVEC Cells, t/IFNg + TNFa Nrml 10 ng/mL, 10 ng/mL 24
hr, 24 hr, Nrml 32 HG3 -1.13 HUVEC Cells, Untx HUVEC Cells, t/PMA +
TNFa 24 hr, Nrml 10 nM, 10 ng/mL 24 hr, 24 hr, Nrml 33 LG1 -1.17
Tangier Fibroblast Cells, t/LDL Cholesterol, Nrml 34 UG1 -1.04
ECV304 Line, Untx ECV304 Line, t/PMA + Iono 1 microM, 1 meg/ml 4 hr
34 UG1 -1.11 ECV304 Line, Untx ECV304 Line, t/PMA + Iono 1 microM,
1 meg/ml 5 hr
[0221]
Sequence CWU 0
0
* * * * *