U.S. patent application number 09/769970 was filed with the patent office on 2003-09-11 for disease associated protein kinases.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Bandman, Olga, Corley, Neil C., Goli, Surya K., Guegler, Karl J., Hillman, Jennifer L., Lal, Preeti, Shah, Purvi.
Application Number | 20030170219 09/769970 |
Document ID | / |
Family ID | 25373212 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170219 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
September 11, 2003 |
Disease associated protein kinases
Abstract
The invention provides human disease associated protein kinases
and polynucleotides (collectively designated DAPK) which identify
and encode them. The invention also provides expression vectors,
host cells, agonists, antibodies and antagonists. The invention
further provides methods for diagnosing and treating disorders
associated with expression of human disease associated protein
kinases.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Hillman, Jennifer L.; (Mountain View,
CA) ; Corley, Neil C.; (Mountain View, CA) ;
Guegler, Karl J.; (Menlo Park, CA) ; Lal, Preeti;
(Santa Clara, CA) ; Goli, Surya K.; (Sunnyvale,
CA) ; Shah, Purvi; (Sunnyvale, CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
25373212 |
Appl. No.: |
09/769970 |
Filed: |
January 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09769970 |
Jan 24, 2001 |
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09272796 |
Mar 19, 1999 |
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6207148 |
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09272796 |
Mar 19, 1999 |
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08878989 |
Jun 19, 1997 |
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5885803 |
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Current U.S.
Class: |
424/94.1 ;
435/194; 435/325; 435/6.18; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1205 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
424/94.1 ;
435/194; 435/6; 536/23.2; 435/69.1; 435/325 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/02; C12N 009/12 |
Claims
What is claimed is:
1. An isolated polynucleotide, or the complement thereof, encoding
a polypeptide selected from the group consisting of: a) SEQ ID
NOs:2, 4, 5, 6, or 7, and b) a naturally-occurring amino acid
sequence having at least 90% sequence identity over the complete
sequence of SEQ ID NOs:1, 2, 4, or 7, and which retains protein
kinase activity.
2. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 1.
3. A host cell transformed with the recombinant polynucleotide of
claim 2.
4. A method for producing a polypeptide, the method comprising the
steps of: a) culturing the host cell of claim 3 under conditions
suitable for the expression of the polypeptide; and b) recovering
the polypeptide from the host cell culture.
5. A composition comprising the polynucleotide sequence of claim 1
and a pharmaceutically acceptable excipient.
6. An isolated polynucleotide sequence selected from SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID
NO:14, or the complement thereof.
7. A composition comprising the polynucleotide sequence of claim 6
and a pharmaceutically acceptable excipient.
8. A method for detecting a polynucleotide in a sample comprising
the steps of: a) hybridizing the polyucleotide of claim 6 to
nucleic acids of the sample, thereby forming hybridization
complexes; and b) comparing hybridization complex formation with a
standard, wherein the comparison indicates expression of the
polynucleotide in the sample.
9. The method of claim 8 further comprising amplifying the nucleic
acids of the sample prior to hybridization.
10. The method of claim 8 wherein the polynucleotide is attached to
a substrate.
11. A method of using a polynucleotide to screen a plurality of
molecules or compounds for a molecule or compound which
specifically binds the polynucleotide, the method comprising: a)
combining the polynucleotide of claim 6 with a plurality of
molecules or compounds under conditions to allow specific binding;
and b) detecting specific binding, thereby identifying a molecule
or compound which specifically binds the polynucleotide.
12. The method of claim 11 wherein the molecules or compounds are
selected from DNA molecules, RNA molecules, peptide nucleic acids,
artificial chromosome constructions, peptides, transcription
factors, and regulatory molecules.
13. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: a) SEQ ID NOs:1, 2, 4, 5, 6,
or 7, and b) a naturally-occurring amino acid sequence having at
least 90% sequence identity over the complete sequence of SEQ ID
NOs:1,2, 4, or 7, and which retains protein kinase activity.
14. A purified antibody which specifically binds to the polypeptide
of claim 13.
15. The antibody of claim 14, wherein the antibody is: (a) a
chimeric antibody; (b) a single chain antibody; (c) a Fab fragment;
(d) a F(ab').sub.2 fragment; (e) a Fv fragment; or (f) a humanized
antibody.
16. A composition comprising an antibody of claim 14 and a
pharmaceutically acceptable excipient.
17. A method of diagnosing a condition or disease associated with
the expression of DAPK in a subject, comprising administering to
said subject an effective amount of the composition of claim
16.
18. A composition of claim 16, wherein the antibody is labeled.
19. A method of diagnosing a condition or disease associated with
the expression of DAPK in a subject, comprising administering to
said subject an effective amount of the composition of claim
18.
20. A method for detecting a DAPK polypeptide in a sample
comprising the steps of: a) combining the antibody of claim 14 with
a sample under conditions to allow specific binding; and b)
detecting specific binding, wherein specific binding indicates the
presence of the DAPK polypeptide in the sample.
21. A method of using an antibody to purify a DAPK polypeptide from
a sample, the method comprising: a) combining the antibody of claim
14 with a sample under conditions to allow specific binding; and b)
separating the antibody from the protein, thereby obtaining
purified DAPK polypeptide.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/272,796, filed Mar. 19, 1999, which is a
divisional application of U.S. application Ser. No. 08/878,989, now
U.S. Pat. No. 5,885,803.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of human protein kinases which are important in disease
and to the use of these sequences in the diagnosis, prevention, and
treatment of diseases associated with cell proliferation.
BACKGROUND OF THE INVENTION
[0003] Kinases regulate many different cell proliferation,
differentiation, and signaling processes by adding phosphate groups
to proteins. Uncontrolled signaling has been implicated in a
variety of disease conditions including inflammation, cancer,
arteriosclerosis, and psoriasis. Reversible protein phosphorylation
is the main strategy for controlling activities of eukaryotic
cells. It is estimated that more than 1000 of the 10,000 proteins
active in a typical mammalian cell are phosphorylated. The high
energy phosphate which drives activation is generally transferred
from adenosine triphosphate molecules (ATP) to a particular protein
by protein kinases and removed from that protein by protein
phosphatases. Phosphorylation occurs in response to extracellular
signals (hormones, neurotransmitters, growth and differentiation
factors, etc), cell cycle checkpoints, and environmental or
nutritional stresses and is roughly analogous to turning on a
molecular switch. When the switch goes on, the appropriate protein
kinase activates a metabolic enzyme, regulatory protein, receptor,
cytoskeletal protein, ion channel or pump, or transcription
factor.
[0004] The kinases comprise the largest known protein group, a
superfamily of enzymes with widely varied functions and
specificities. They are usually named after their substrate, their
regulatory molecules, or some aspect of a mutant phenotype. With
regard to substrates, the protein kinases may be roughly divided
into two groups; those that phosphorylate tyrosine residues
(protein tyrosine kinases, PTK) and those that phosphorylate serine
or threonine residues (serine/threonine kinases, STK). A few
protein kinases have dual specificity and phosphorylate threonine
and tyrosine residues. Almost all kinases contain a similar 250-300
amino acid catalytic domain. The N-terminal domain, which contains
subdomains I-IV, generally folds into a two-lobed structure which
binds and orients the ATP (or GTP) donor molecule. The larger C
terminal lobe, which contains subdomains VI A-XI, binds the protein
substrate and carries out the transfer of the gamma phosphate from
ATP to the hydroxyl group of a serine, threonine, or tyrosine
residue. Subdomain V spans the two lobes.
[0005] The kinases may be categorized into families by the
different amino acid sequences (generally between 5 and 100
residues) located on either side of, or inserted into loops of, the
kinase domain. These added amino acid sequences allow the
regulation of each kinase as it recognizes and interacts with its
target protein. The primary structure of the kinase domains is
conserved and can be further subdivided into 11 subdomains. Each of
the 11 subdomains contain specific residues and motifs or patterns
of amino acids that are characteristic of that subdomain and are
highly conserved (Hardie, G. and Hanks, S. (1995) The Protein
Kinase Facts Books, Vol I:7-20 Academic Press, San Diego,
Calif.).
[0006] The second messenger dependent protein kinases primarily
mediate the effects of second messengers such as cyclic AMP (cAMP),
cyclic GMP, inositol triphosphate, phosphatidylinositol,
3,4,5-triphosphate, cyclic-ADP-ribose, arachidonic acid,
diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent
protein kinases (PKA) are important members of the STK family.
Cyclic-AMP is an intracellular mediator of hormone action in all
procaryotic and animal cells that have been studied. Such
hormone-induced cellular responses include thyroid hormone
secretion, cortisol secretion, progesterone secretion, glycogen
breakdown, bone resorption, and regulation of heart rate and force
of heart muscle contraction. PKA is found in all animal cells and
is thought to account for the effects of cyclic-AMP in most of
these cells. Altered PKA expression is implicated in a variety of
disorders and diseases including cancer, thyroid disorders,
diabetes, atherosclerosis, and cardiovascular disease (Isselbacher,
K. J. et al. (1994) Harrison's Principles of Internal Medicine,
McGraw-Hill, New York, N.Y., pp. 416-431, 1887).
[0007] Calcium-calmodulin (CaM) dependent protein kinases are also
members of STK family. Calmodulin is a calcium receptor that
mediates many calcium regulated processes by binding to target
proteins in response to the binding of calcium. The principle
target protein in these processes is CaM dependent protein kinases.
CaM-kinases are involved in regulation of smooth muscle contraction
(MLC kinase), glycogen breakdown (phosphorylase kinase), and
neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I
phosphorylates a variety of substrates including the
neurotransmitter related proteins synapsin I and II, the gene
transcription regulator, CREB, and the cystic fibrosis conductance
regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal
14:3679-3686). CaM II kinase also phosphorylates synapsin at
different sites, and controls the synthesis of catecholamines in
the brain through phosphorylation and activation of tyrosine
hydroxylase. Many of the CaM kinases are activated by
phosphorylation in addition to binding to CaM. The kinase may
autophosphorylate itself, or be phosphorylated by another kinase as
part of a "kinase cascade".
[0008] Another ligand-activated protein kinase is 5'-AMP-activated
protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem.
15:8675-8681). Mammaliam AMPK is a regulator of fatty acid and
sterol synthesis through phosphorylation of the enzymes acetyl-CoA
carboxylase and hydroxymethylglutaryl-CoA reductase and mediates
responses of these pathways to cellular stresses such as heat shock
and depletion of glucose and ATP. AMPK is a heterotrimeric complex
comprised of a catalytic alpha subunit and two non-catalytic beta
and gamma subunits that are believed to regulate the activity of
the alpha subunit. Subunits of AMPK have a much wider distribution
in non-lipogenic tissues such as brain, heart, spleen, and lung
than expected. This distribution suggests that its role may extend
beyond regulation of lipid metabolism alone.
[0009] The mitogen-activated protein kinases (MAP) are also members
of the STK family. MAP kinases also regulate intracellular
signaling pathways. They mediate signal transduction from the cell
surface to the nucleus via phosphorylation cascades. Several
subgroups have been identified, and each manifests different
substrate specificities and responds to distinct extracellular
stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature
365:781-783). MAP kinase signaling pathways are present in
mammalian cells as well as in yeast. The extracellular stimuli
which activate mammalian pathways include epidermal growth factor
(EGF), ultraviolet light, hyperosmolar medium, heat shock,
endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines
such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).
[0010] PRK (proliferation-related kinase) is a serun/cytokine
inducible STK that is involved in regulation of the cell cycle and
cell proliferation in human megakaroytic cells (Li, B. et al.
(1996) J. Biol. Chem. 271:19402-19408). PRK is related to the polo
(derived from Drosophila polo gene) family of STKs implicated in
cell division. PRK is downregulated in lung tumor tissue and may be
a proto-oncogene whose deregulated expression in normal tissue
leads to oncogenic transformation. Altered MAP kinase expression is
implicated in a variety of disease conditions including cancer,
inflammation, immune disorders, and disorders affecting growth and
development.
[0011] The cyclin-dependent protein kinases (CDKs) are another
group of STKs that control the progression of cells through the
cell cycle. Cyclins are small regulatory proteins that act by
binding to and activating CDKs which then trigger various phases of
the cell cycle by phosphorylating and activating selected proteins
involved in the mitotic process. CDKs are unique in that they
require multiple inputs to become activated. In addition to the
binding of cyclin, CDK activation requires the phosphorylation of a
specific threonine residue and the dephosphorylation of a specific
tyrosine residue.
[0012] Protein tyrosine kinases, PTKs, specifically phosphorylate
tyrosine residues on their target proteins and may be divided into
transmembrane, receptor PTKs and nontransmembrane, non-receptor
PTKs. Transmembrane protein-tyrosine kinases are receptors for most
growth factors. Binding of growth factor to the receptor activates
the transfer of a phosphate group from ATP to selected tyrosine
side chains of the receptor and other specific proteins. Growth
factors (GF) associated with receptor PTKs include; epidermal GF,
platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and
insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage
colony stimulating factor.
[0013] Non-receptor PTKs lack transmembrane regions and, instead,
form complexes with the intracellular regions of cell surface
receptors. Such receptors that function through non-receptor PTKs
include those for cytokines, hormones (growth hormone and
prolactin) and antigen-specific receptors on T and B
lymphocytes.
[0014] Many of these PTKs were first identified as the products of
mutant oncogenes in cancer cells where their activation was no
longer subject to normal cellular controls. In fact, about one
third of the known oncogenes encode PTKs, and it is well known that
cellular transformation (oncogenesis) is often accompanied by
increased tyrosine phosphorylation activity (Carbonneau H and Tonks
N K (1992) Annu Rev Cell Biol 8:463-493). Regulation of PTK
activity may therefore be an important strategy in controlling some
types of cancer.
[0015] An additional family of protein kinases previously thought
to exist only in procaryotes is the histidine protein kinase family
(HPK). HPKs bear little homology with mammalian STKs or PTKs but
have distinctive sequence motifs of their own (Davie, J. R. et al.
(1995) J. Biol. Chem. 270:19861-19867). A histidine residue in the
N-terminal half of the molecule (region I) is an
autophosphorylation site. Three additional motifs located in the
C-terminal half of the molecule include an invariant asparagine
residue in region II and two glycine-rich loops characteristic of
nucleotide binding domains in regions III and IV. Recently a
branched chain alpha-ketoacid dehydrogenase kinase has been found
with characteristics of HPK in rat (Davie et al., supra).
[0016] The discovery of new human disease associated protein
kinases which are important in disease development, and the
polynucleotides encoding them, satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention and treatment of diseases associated with cell
proliferation, particularly and immune responses and cancers.
SUMMARY OF THE INVENTION
[0017] The invention features substantially purified polypeptides,
human disease associated protein kinases, collectively referred to
as DAPK and individually referred to as DAPK-1, DAPK-2, DAPK-3,
DAPK-4, DAPK-5, DAPK-6, and DAPK-7, having the amino acid sequences
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7,
respectively.
[0018] The invention further provides isolated and substantially
purified polynucleotide sequences encoding DAPK. In a particular
aspect, the polynucleotide is at least one of the nucleotide
sequences selected from the group consisting of SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and
SEQ ID NO:14.
[0019] In addition, the invention provides a polynucleotide
sequence, or fragment thereof, which hybridizes under stringent
conditions to any of the polynucleotide sequences of SEQ ID
NOs:8-14. In another aspect the invention provides compositions
comprising isolated and purified polynucleotide sequences of SEQ ID
NOs:8-14 or fragments thereof.
[0020] The invention further provides a polynucleotide sequence
comprising the complement or fragments thereof of any one of the
polynucleotide sequences encoding DAPK. In another aspect the
invention provides compositions comprising isolated and purified
polynucleotide sequences comprising the complements of SEQ ID
NOs:8-14, or fragments thereof.
[0021] The present invention further provides an expression vector
containing at least a fragment of any one of the polynucleotide
sequences of SEQ ID NOs:8-14. In yet another aspect, the expression
vector containing the polynucleotide sequence is contained within a
host cell.
[0022] The invention also provides a method for producing a
polypeptide or a fragment thereof, the method comprising the steps
of: a) culturing the host cell containing an expression vector
containing at least a fragment of the polynucleotide sequence
encoding an DAPK under conditions suitable for the expression of
the polypeptide; and b) recovering the polypeptide from the host
cell culture.
[0023] The invention also provides a pharmaceutical composition
comprising a substantially purified DAPK in conjunction with a
suitable pharmaceutical carrier.
[0024] The invention also provides a purified antagonist of DAPK.
In one aspect the invention provides a purified antibody which
binds to an DAPK.
[0025] Still further, the invention provides a purified agonist of
DAPK.
[0026] The invention also provides a method for treating or
preventing a cancer comprising administering to a subject in need
of such treatment an effective amount of a pharmaceutical
composition containing DAPK.
[0027] The invention also provides a method for treating or
preventing a cancer comprising administering to a subject in need
of such treatment an effective amount of a pharmaceutical
composition containing DAPK.
[0028] The invention also provides a method for treating or
preventing an immune response associated with the increased
expression or activity of DAPK comprising administering to a
subject in need of such treatment an effective amount of an
antagonist of DAPK.
[0029] The invention also provides a method for stimulating cell
proliferation comprising administering to a cell an effective
amount of DAPK.
[0030] The invention also provides a method for detecting a
polynucleotide which encodes a disease associated protein kinase in
a biological sample comprising the steps of: a) hybridizing a
polynucleotide sequence complementary to a polynucleotide encoding
DAPK to nucleic acid material of a biological sample, thereby
forming a hybridization complex; and b) detecting the hybridization
complex, wherein the presence of the complex correlates with the
presence of a polynucleotide encoding the disease associated
protein kinase in the biological sample.
[0031] The invention also provides a microarray which contains at
least a fragment of at least one of the polynucleotide sequences
encoding DAPK. In a particular aspect, the microarray contains at
least a fragment of at least one of the sequences selected from the
group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
[0032] The invention also provides a method for the simultaneous
detection of the levels of expression of polynucleotides which
encode disease associated protein kinases in a biological sample
comprising the steps of: a) hybridizing said microarray to labeled
complementary nucleotides of a biological sample, comprising at
least a fragment of at least one of the polynucleotides encoding
DAPK, thereby forming hybridization complexes; and b) quantifying
expression, wherein the signal produced by the hybridization
complexes correlates with expression of particular polynucleotides
encoding disease associated protein kinases in the biological
sample. In a preferred embodiment, prior to hybridization, the
nucleic acid material of the biological sample is amplified and
labeled by the polymerase chain reaction.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIGS. 1A, 1B, and 1C show the amino acid sequence alignments
between DAPK-1 (SEQ ID NO:1) and the human proliferation-related
protein kinase, PRK (GI 1488263; SEQ ID NO:15), produced using the
multisequence alignment program of DNASTAR software (DNASTAR Inc,
Madison Wis.).
[0034] FIGS. 2A, and 2B show the amino acid sequence alignments
between DAPK-2 (SEQ ID NO:3) and the human vaccinia virus-related
protein kinase, VRK1 (GI 1827450; SEQ ID NO:16), produced using the
multisequence alignment program of DNASTAR software.
[0035] FIGS. 3A and 3B show the amino acid sequence alignments
between DAPK-3 (SEQ ID NO:3) and the rat MAP-kinase, MEK2 (GI
303804; SEQ ID NO:17), produced using the multisequence alignment
program of DNASTAR software.
[0036] FIGS. 4A and 4B show the amino acid sequence alignments
between DAPK-4 (SEQ ID NO:4) and the human nuclear protein kinase,
Ndr (GI 854170; SEQ ID NO:18), produced using the multisequence
alignment program of DNASTAR software.
[0037] FIGS. 5A and 5B show the amino acid sequence alignments
between DAPK-5 (SEQ ID NO:5) and the human CaM kinase, CaMKI (GI
790790; SEQ ID NO:19), produced using the multisequence alignment
program of DNASTAR software.
[0038] FIGS. 6A and 6B show the amino acid sequence alignments
between DAPK-6 (SEQ ID NO:6) and the rat branched-chain alpha
ketoacid dehydrogenase kinase, BCKDH kinase (GI 924921; SEQ ID
NO:20), produced using the multisequence alignment program of
DNASTAR software.
[0039] FIGS. 7A and 7B show the amino acid sequence alignments
between DAPK-7 (SEQ ID NO:7) and the human 5'-AMP activated protein
kinase gamma subunit, AMPK-gamma (GI 1335856; SEQ ID NO:21),
produced using the multisequence alignment program of DNASTAR
software.
DESCRIPTION OF THE INVENTION
[0040] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be 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.
[0041] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to the "antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0042] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the cell lines, vectors, arrays and methodologies which are
reported in the publications which might be used in connection with
the invention. Nothing herein is to be construed as an admission
that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
[0043] Definitions
[0044] DAPK, as used herein, refers to the amino acid sequences of
substantially purified DAPK obtained from any species, particularly
mammalian, including bovine, ovine, porcine, murine, equine, and
preferably human, from any source whether natural, synthetic,
semi-synthetic, or recombinant.
[0045] The term "agonist", as used herein, refers to a molecule
which, when bound to DAPK, increases or prolongs the duration of
the effect of DAPK. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of DAPK.
[0046] An "allele" or "allelic sequence", as used herein, is an
alternative form of the gene encoding DAPK. Alleles may result from
at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or polypeptides whose structure or function may or
may not be altered. Any given natural or recombinant gene may have
none, one, or many allelic forms. Common mutational changes which
give rise to alleles are generally ascribed to natural deletions,
additions, or substitutions of nucleotides. Each of these types of
changes may occur alone, or in combination with the others, one or
more times in a given sequence.
[0047] "Altered" nucleic acid sequences encoding DAPK as used
herein include those with deletions, insertions, or substitutions
of different nucleotides resulting in a polynucleotide that encodes
the same or a functionally equivalent DAPK. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding DAPK, and improper or unexpected
hybridization to alleles, with a locus other than the normal
chromosomal locus for the polynucleotide sequence encoding DAPK.
The encoded protein may also be "altered" and contain deletions,
insertions, or substitutions of amino acid residues which produce a
silent change and result in a functionally equivalent DAPK.
Deliberate amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues as
long as the biological or immunological activity of DAPK is
retained. For example, negatively charged amino acids may include
aspartic acid and glutamic acid; positively charged amino acids may
include lysine and arginine; and amino acids with uncharged polar
head groups having similar hydrophilicity values may include
leucine, isoleucine, and valine, glycine and alanine, asparagine
and glutamine, serine and threonine, and phenylalanine and
tyrosine.
[0048] "Amino acid sequence" as used herein refers to an
oligopeptide, peptide, polypeptide, or protein sequence, and
fragment thereof, and to naturally occurring or synthetic
molecules. Fragments of DAPK are preferably about 5 to about 15
amino acids in length and retain the biological activity or the
immunological activity of DAPK. Where "amino acid sequence" is
recited herein to refer to an amino acid sequence of a naturally
occurring protein molecule, amino acid sequence, and like terms,
are not meant to limit the amino acid sequence to the complete,
native amino acid sequence associated with the recited protein
molecule.
[0049] "Amplification" as used herein refers to the production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR
Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y.).
[0050] The term "antagonist" as used herein, refers to a molecule
which, when bound to DAPK, decreases the amount or the duration of
the effect of the biological or immunological activity of DAPK.
Antagonists may include proteins, nucleic acids, carbohydrates, or
any other molecules which decrease the effect of DAPK.
[0051] As used herein, the term "antibody" refers to intact
molecules as well as fragments thereof, such as Fab, F(ab').sub.2,
and Fv, which are capable of binding the epitopic determinant.
Antibodies that bind DAPK polypeptides can be prepared using intact
polypeptides or fragments containing small peptides of interest as
the immunizing antigen. The polypeptide or oligopeptide used to
immunize an animal can be derived from the translation of RNA or
synthesized chemically and can be conjugated to a carrier protein,
if desired. Commonly used carriers that are chemically coupled to
peptides include bovine serum albumin and thyroglobulin, keyhole
limpet hemocyanin. The coupled peptide is then used to immunize the
animal (e.g., a mouse, a rat, or a rabbit).
[0052] The term "antigenic determinant", as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or fragment of a protein
is used to immunize a host animal, numerous regions of the protein
may induce the production of antibodies which bind specifically to
a given region or three-dimensional structure on the protein; these
regions or structures are referred to as antigenic determinants. An
antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to elicit the immune response) for binding to an
antibody.
[0053] The term "antisense", as used herein, refers to any
composition containing nucleotide sequences which are complementary
to a specific DNA or RNA sequence. The term "antisense strand" is
used in reference to a nucleic acid strand that is complementary to
the "sense" strand. Antisense molecules include peptide nucleic
acids and may be produced by any method including synthesis or
transcription. Once introduced into a cell, the complementary
nucleotides combine with natural sequences produced by the cell to
form duplexes and block either transcription or translation. The
designation "negative" is sometimes used in reference to the
antisense strand, and "positive" is sometimes used in reference to
the sense strand.
[0054] The term "biologically active", as used herein, refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
DAPK, or any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0055] The terms "complementary" or "complementarity", as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A". Complementarity between two single-stranded molecules may
be "partial", in which only some of the nucleic acids bind, or it
may be complete when total complementarity exists between the
single stranded molecules. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, which depend upon
binding between nucleic acids strands and in the design and use of
PNA molecules.
[0056] A "composition comprising a given polynucleotide sequence"
as used herein refers broadly to any composition containing the
given polynucleotide sequence. The composition may comprise a dry
formulation or an aqueous solution. Compositions comprising
polynucleotide sequences encoding DAPK (SEQ ID NOs:8-14) or
fragments thereof may be employed as hybridization probes. The
probes may be stored in freeze-dried form and may be associated
with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be deployed in an aqueous solution containing salts
(e.g., NaCl), detergents (e.g., SDS) and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.).
[0057] "Consensus", as used herein, refers to a nucleic acid
sequence which has been resequenced to resolve uncalled bases, has
been extended using XL-PCR (Applied Biosystems, Foster City Calif.)
in the 5' and/or the 3' direction and resequenced, or has been
assembled from the overlapping sequences of more than one Incyte
Clone using a computer program for fragment assembly (e.g., GELVIEW
fragment assembly system, GCG, Madison, Wis.). Some sequences have
been both extended and assembled to produce the consensus
sequence.
[0058] The term "correlates with expression of a polynucleotide",
as used herein, indicates that the detection of the presence of a
ribonucleic acid that is similar to a polynucleotide encoding an
DAPK by northern analysis is indicative of the presence of mRNA
encoding DAPK in a sample and thereby correlates with expression of
the transcript from the polynucleotide encoding the protein.
[0059] The term "DAPK" refers to any one or all of the human
polypeptides, DAPK-1, DAPK-2, DAPK-3, DAPK-4, DAPK-5, DAPK-6,
DAPK-7, and DAPK-8.
[0060] A "deletion", as used herein, refers to a change in the
amino acid or nucleotide sequence and results in the absence of one
or more amino acid residues or nucleotides.
[0061] The term "derivative", as used herein, refers to the
chemical modification of a nucleic acid encoding or complementary
to DAPK or the encoded DAPK. Such modifications include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A nucleic acid derivative encodes a polypeptide which retains the
biological or immunological function of the natural molecule. A
derivative polypeptide is one which is modified by glycosylation,
pegylation, or any similar process which retains the biological or
immunological function of the polypeptide from which it was
derived.
[0062] The term "homology", as used herein, refers to a degree of
complementarity. There may be partial homology or complete homology
(i.e., identity). A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous sequence to the target sequence under conditions of low
stringency. This is not to say that conditions of low stringency
are such that non-specific binding is permitted; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be tested by the use of a second target
sequence which lacks even a partial degree of complementarity
(e.g., less than about 30% identity). In the absence of
non-specific binding, the probe will not hybridize to the second
non-complementary target sequence.
[0063] Human artificial chromosomes (HACs) are linear
microchromosomes which may contain DNA sequences of 10K to 10M in
size and contain all of the elements required for stable mitotic
chromosome segregation and maintenance (Harrington, J. J. et al.
(1997) Nat Genet. 15:345-355).
[0064] The term "humanized antibody", as used herein, refers to
antibody molecules in which amino acids have been replaced in the
non-antigen binding regions in order to more closely resemble a
human antibody, while still retaining the original binding
ability.
[0065] The term "hybridization", as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0066] The term "hybridization complex", as used herein, refers to
a complex formed between two nucleic acid sequences by virtue of
the formation of hydrogen bonds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., C.sub.0t or R.sub.0t analysis) or between one
nucleic acid sequence present in solution and another nucleic acid
sequence immobilized on a solid support (e.g., paper, membranes,
filters, chips, pins or glass slides, or any other appropriate
substrate to which cells or their nucleic acids have been
fixed).
[0067] An "insertion" or "addition", as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, as compared to the naturally occurring molecule.
[0068] "Microarray" refers to an array (or arrangement) of distinct
oligonucleotides synthesized on a substrate, such as paper, nylon
or other type of membrane, filter, gel, polymer, chip, glass slide,
or any other suitable support.
[0069] The term "modulate", as used herein, refers to a change in
the activity of DAPK. For example, modulation may cause an increase
or a decrease in protein activity, binding characteristics, or any
other biological, functional or immunological properties of
DAPK.
[0070] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide, or polynucleotide, and fragments
thereof, and to DNA or RNA of genomic or synthetic origin which may
be single- or double-stranded, and represent the sense or antisense
strand. "Fragments" are those nucleic acid sequences which are
greater than 60 nucleotides than in length, and most preferably
includes fragments that are at least 100 nucleotides or at least
1000 nucleotides, and at least 10,000 nucleotides in length.
[0071] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to about 60 nucleotides, preferably
about 15 to 30 nucleotides, and more preferably about 20 to 25
nucleotides, which can be used in PCR amplification or
hybridization assays. As used herein, oligonucleotide is
substantially equivalent to the terms "amplimers", "primers",
"oligomers", and "probes", as commonly defined in the art.
[0072] "Peptide nucleic acid", PNA as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least five nucleotides in length linked to a
peptide backbone of amino acid residues which ends in lysine. The
terminal lysine confers solubility to the composition. PNAs may be
pegylated to extend their lifespan in the cell where they
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation (Nielsen, P. E. et al. (1993) Anticancer
Drug Des. 8:53-63).
[0073] The term "portion", as used herein, with regard to a protein
(as in "a portion of a given protein") refers to fragments of that
protein. The fragments may range in size from five amino acid
residues to the entire amino acid sequence minus one amino acid.
Thus, a protein "comprising at least a portion of the amino acid
sequence of an DAPK encompasses the full-length DAPK and fragments
thereof.
[0074] The term "sample", as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acid
encoding DAPK, or fragments thereof, or DAPK itself may comprise a
bodily fluid, extract from a cell, chromosome, organelle, or
membrane isolated from a cell, a cell, genomic DNA, RNA, or cDNA
(in solution or bound to a solid support, a tissue, a tissue print,
and the like.
[0075] The terms "specific binding" or "specifically binding", as
used herein, refers to that interaction between a protein or
peptide and an agonist, an antibody and an antagonist. The
interaction is dependent upon the presence of a particular
structure (i.e., the antigenic determinant or epitope) of the
protein recognized by the binding molecule. For example, if an
antibody is specific for epitope "A", the presence of a protein
containing epitope A (or free, unlabeled A) in a reaction
containing labeled "A" and the antibody will reduce the amount of
labeled A bound to the antibody.
[0076] The terms "stringent conditions" or "stringency", as used
herein, refer to the conditions for hybridization as defined by the
nucleic acid, salt, and temperature. These conditions are well
known in the art and may be altered in order to identify or detect
identical or related polynucleotide sequences. Numerous equivalent
conditions comprising either low or high stringency depend on
factors such as the length and nature of the sequence (DNA, RNA,
base composition), nature of the target (DNA, RNA, base
composition), milieu (in solution or immobilized on a solid
substrate), concentration of salts and other components (e.g.,
formamide, dextran sulfate and/or polyethylene glycol), and
temperature of the reactions (within a range from about 5.degree.
C. below the melting temperature of the probe to about 20.degree.
C. to 25.degree. C. below the melting temperature). One or more
factors be may be varied to generate conditions of either low or
high stringency different from, but equivalent to, the above listed
conditions.
[0077] The term "substantially purified", as used herein, refers to
nucleic or amino acid sequences that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably 75% free, and most preferably 90% free from other
components with which they are naturally associated.
[0078] A "substitution", as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0079] "Transformation", as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method is selected based
on the type of host cell being transformed and may include, but is
not limited to, viral infection, electroporation, heat shock,
lipofection, and particle bombardment. Such "transformed" cells
include stably transformed cells in which the inserted DNA is
capable of replication either as an autonomously replicating
plasmid or as part of the host chromosome. They also include cells
which transiently express the inserted DNA or RNA for limited
periods of time.
[0080] A "variant" of DAPK, as used herein, refers to an amino acid
sequence that is altered by one or more amino acids. The variant
may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties, e.g., replacement of
leucine with isoleucine. More rarely, a variant may have
"nonconservative" changes, e.g., replacement of a glycine with a
tryptophan. Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
DNASTAR software.
[0081] The Invention
[0082] The invention is based on the discovery of human disease
associated protein kinases (DAPK) and the polynucleotides encoding
DAPK, and the use of these compositions for the diagnosis,
prevention, or treatment of diseases associated with cell
proliferation. Table 1 shows the protein and nucleotide sequence
identification numbers, Incyte Clone number, cDNA library, NCBI
homolog and NCBI sequence identifier for each of the human disease
associated protein kinases disclosed herein.
1TABLE 1 Poly- Poly- peptide nucleotide Inctye Clone Incyte Library
NCBI Homolog Seq 1 Seq 8 39043 HUVENOB01 Human GI 1488263 Seq 2 Seq
9 40194 TBLYNOT01 Human GI 1827450 Seq 3 Seq 10 402339 TMLR3DT01
Rat GI 303804 Seq 4 Seq 11 705365 SYNORAT04 Human GI 854170 Seq 5
Seq 12 827431 PROSNOT06 Human GI 790790 Seq 6 Seq 13 1340712
COLNTUT03 Rat GI 924921 Seq 7 Seq 14 1452972 PENITUT01 Human GI
1335856
[0083] DAPK-1 (SEQ ID NO:1) was first identified in Incyte Clone
39043 from the HUVENOB01 cDNA library using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:8,
was derived from the extended and overlapping nucleic acid
sequences: Incyte Clones 39043/HUVENOB01, 86618/LIVRNOT01,
241996/HIPONOT01, 486079/HNT2RAT01, 1255087/LUNGFET03,
1294238/PGANNOT03, and 2375745/ISLTNOT01.
[0084] Therefore, in one embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:1.
DAPK-1 is 685 amino acids in length and has a potential ATP-binding
sequence at G.sub.89KGGFAKC. As shown in FIG. 1, DAPK-1 has
sequence homology with cytokine-inducible, human
proliferation-related kinase, PRK (GI 1488263). In particular,
DAPK-1 and PRK share 53% homology. DAPK-1 and PRK share the ATP
binding region described above and, in addition, share a presumed
regulatory sequence at K.sub.506WVDYS common to members of the polo
family of protein kinases. DAPK-1 is associated with cDNA libraries
which are immortalized or cancerous and show inflammatory or immune
responses.
[0085] DAPK-2 (SEQ ID NO:2) was first identified in Incyte Clone
40194 from the TBLYNOT01 cDNA library using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:9,
was derived from the extended and overlapping nucleic acid
sequences: Incyte Clones 40194/TBLYNOT01, 278198/TESTNOT03, and
1683885/PROSNOT15.
[0086] Therefore, in one embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:2.
DAPK-2 is 448 amino acids in length and has a potential ATP-binding
sequence at G.sub.36SGGFGLI and an STK specific signature sequence
at Y.sub.162VHGDVKAANLLL. As shown in FIG. 2, DAPK-2 has sequence
homology with the human vaccina virus related kinase, VRK1 (GI
1827450). In particular, DAPK-2 and VRK1 share 65% homology. DAPK-2
and VRK1 share the glycine-rich ATP-binding sequence and the STK
signature sequence described above. DAPK-2 is associated with cDNA
libraries which are immortalized or cancerous and show inflammatory
or immune responses.
[0087] DAPK-3 (SEQ ID NO:3) was first identified in Incyte Clone
402339 from the TMLR3DT01 cDNA library using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:10,
was derived from the extended and overlapping nucleic acid
sequences: Incyte Clones 402339/TMLR3DT01, 495759/HNT2NOT01, and
1931950/COLNNOT16.
[0088] Therefore, in one embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:3.
DAPK-3 is 400 amino acids in length and contains various sequence
motifs characteristic of the catalytic domain of protein kinases.
An ATP-binding sequence is found at G.sub.79AGNGGVV of subdomain I,
and K.sub.101 and E.sub.118 are invariant residues found in
subdomains II and II, respectively. The "catalytic loop" of
subdomain VIB is found in the sequence H.sub.112RDVKPSN, and the
triplet codons D.sub.212FG and A.sub.275PE are characteristic of
subdomains VII and VIII, respectively. As shown in FIG. 3, DAPK-3
has sequence homology with the rat MAP kinase kinase, MEK2 (GI
303804). In particular, DAPK-3 and MEK3 share 94% homology. DAPK-3
is associated with cDNA libraries which are immortalized or
cancerous and show inflammatory or immune responses.
[0089] DAPK-4 (SEQ ID NO:4) was first identified in Incyte Clone
705365 from the SYNORAT04 cDNA library using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:11,
was derived from the extended and overlapping nucleic acid
sequences: Incyte Clones 705365/SYNORAT04, 2529903/GBLANOT02, and
2729238/OVARTUT05.
[0090] Therefore, in one embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:4.
DAPK-4 is 464 amino acids in length and contains various sequence
motifs characteristic the catalytic domain of protein kinases. An
ATP-binding sequence is found at G.sub.97RGAFGEV and the catalytic
loop is found at H.sub.211RDIKPDN. DAPK-4 also contains a nuclear
localization signal at K.sub.266RKAETWKKNR. As shown in FIG. 4,
DAPK-4 has sequence homology with human nuclear protein kinase, Ndr
(GI 854170). In particular, DAPK-4 and Ndr share 87% homology.
DAPK-4 is associated with cDNA libraries which are immortalized or
cancerous and show inflammatory or immune responses.
[0091] DAPK-5 (SEQ ID NO:5) was first identified in Incyte Clone
827431 from the PROSNOT06 cDNA library using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:12,
was derived from the extended and overlapping nucleic acid
sequences: Incyte Clones 755081, 758002 and 760552/BRAITUT02,
827431/PROSNOT06, 1286067/COLNNOT16, and 1503272/BRAITUT07.
[0092] Therefore, in one embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:5.
DAPK-5 is 343 amino acids in length and contains various sequence
motifs characteristic of the catalytic domain of protein kinases.
An ATP-binding sequence is found at G.sub.22SGAFSEV and the
catalytic loop is found at H.sub.134RDLKPEN. The triplet codons
D.sub.157FG and A.sub.180PE characteristic of subdomains VII and
VIII, respectively, are also found. As shown in FIG. 5, DAPK-5 has
sequence homology with the human CaM-kinase, CaMKI (GI 790790). In
particular, DAPK-5 and CaMKI share 64% homology. In addition to the
typical protein kinase motifs mentioned above, DAPK-5 and CaMKI
share T.sub.171 which is a phosphorylation site for CaMKI kinase
and an auto-inhibitory and CaM-binding domain found between
I.sub.1280 and L.sub.313 of DAPK-5. DAPK-5 is associated with cDNA
libraries which are immortalized or cancerous.
[0093] DAPK-6 (SEQ ID NO:6) was first identified in Incyte Clone
1340712 from the COLNTUT03 cDNA library using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:13,
was derived from the extended and overlapping nucleic acid
sequences: Incyte Clones 1340712/COLNTUT03, 1350483/LATRTUT02 and
2631495/COLNTUT15.
[0094] Therefore, in one embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:6.
DAPK-6 is 412 amino acids in length and has characteristics of a
histidine protein kinase (HPK). H.sub.211 in DAPK-6 corresponds to
a potential autophosphorylation site in subdomain I of HPK, and
N.sub.279 is also an invariant residue of subdomain II. The
sequences D.sub.315RGGG and G.sub.365FGFG are characteristic of
subdomain III and IV of HPK. As shown in FIG. 6, DAPK-6 has
sequence homology with a rat branched-chain alpha-ketoacid
dehydrogenase kinase, BCKDH kinase (GI 924921). In particular,
DAPK-6 and BCKDH kinase share 98% homology. BCKDH kinase shares the
characteristic sequences of HPKs described above, but differs by
the presence of a distinctive N-terminal leader sequence in DAPK-6
that may target DAPK-6 to a different subcellular site. DAPK-6 is
associated with cDNA libraries which are immortalized or cancerous
and show inflammatory or immune responses.
[0095] DAPK-7 (SEQ ID NO:7) was first identified in Incyte Clone
1452972 from the PENITUT01 cDNA library using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:14,
was derived from the extended and overlapping nucleic acid
sequences: Incyte Clones 307571/HEARNOT01, 842220/PROSTUT05,
1364737/SCORNON02, 1452972 and 1454802/PENITUT01, and
1479332/CORPNOT02.
[0096] Therefore, in one embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:7.
DAPK-7 is 328 amino acids in length and has potential
cAMP-dependent protein kinase phosphorylation sites at S.sub.72 and
S.sub.217. As shown in FIG. 7, DAPK-7 has sequence homology with
human fetal liver AMPK gamma-subunit (GI 1335856). In particular,
DAPK-7 and AMPK gamma share 73% homology. Several sequences that
are conserved among AMPK gamma isoforms are shared by DAPK-7 and
AMPK gamma. These include L.sub.77TITDFINLHRYYKS, S.sub.217ALPVVDE,
V.sub.228VDIYSKFDVI, and A.sub.286EVHRRLVVV. Sequence differences
between DAPK-7 and other AMPK gamma isoforms, particularly the
distinctive N-terminal portion of DAPK-7, L.sub.2EKLEFEDEAVEDSESG,
may signify different tissue expression and/or regulatory roles for
DAPK-7. DAPK-7 is associated with cDNA libraries which are
immortalized or cancerous and show inflammatory or immune
responses.
[0097] The invention also encompasses DAPK variants which retain
the biological or functional activity of DAPK. A preferred DAPK
variant is one having at least 80%, and more preferably 90%, amino
acid sequence identity to the DAPK amino acid sequence. A most
preferred DAPK variant is one having at least 95% amino acid
sequence identity to an DAPK disclosed herein (SEQ ID NOs:1-7).
[0098] The invention also encompasses polynucleotides which encode
DAPK. Accordingly, any nucleic acid sequence which encodes the
amino acid sequence of DAPK can be used to produce recombinant
molecules which express DAPK. In a particular embodiment, the
invention encompasses a polynucleotide consisting of a nucleic acid
sequence selected from the group consisting of SEQ ID NOs:8-14.
[0099] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
nucleotide sequences encoding DAPK, some bearing minimal homology
to the nucleotide sequences of any known and naturally occurring
gene, may be produced. Thus, the invention contemplates each and
every possible variation of nucleotide sequence that could be made
by selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet
genetic code as applied to the nucleotide sequence of naturally
occurring DAPK, and all such variations are to be considered as
being specifically disclosed.
[0100] Although nucleotide sequences which encode DAPK and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring DAPK under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding DAPK or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding DAPK and its derivatives without altering the
encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0101] The invention also encompasses production of DNA sequences,
or fragments thereof, which encode DAPK and its derivatives,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding DAPK or any fragment
thereof.
[0102] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those shown in SEQ ID NOs:8-14, under
various conditions of stringency as taught in Wahl, G. M. and S. L.
Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R.
(1987; Methods Enzymol. 152:507-511).
[0103] Methods for DNA sequencing which are well known and
generally available in the art and may be used to practice any of
the embodiments of the invention. These methods employ enzymes such
as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA
polymerase and thermostable T7 DNA polymerase (Amersham Pharmacia
Biotech (APB), Piscataway N.J.), or combinations of polymerases and
proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg Md.).
Preferably, sequence preparation is automated with machines such as
the MICROLAB 2200 system (Hamilton, Reno Nev.) and the DNA ENGINE
thermal cycler (MJ Research, Watertown Mass.). Machines commonly
used for sequencing include the ABI PRISM 3700, 377 or 373 DNA
sequencing systems (Applied Biosystems), the MEGABACE 1000 DNA
sequencing system (APB), and the like.
[0104] The nucleic acid sequences encoding DAPK may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences such as
promoters and regulatory elements. For example, one method which
may be employed, "restriction-site" PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus (Sarkar, G.
(1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA
is first amplified in the presence of primer to a linker sequence
and a primer specific to the known region. The amplified sequences
are then subjected to a second round of PCR with the same linker
primer and another specific primer internal to the first one.
Products of each round of PCR are transcribed with an appropriate
RNA polymerase and sequenced using reverse transcriptase.
[0105] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region (Triglia, T. et al.
(1988) Nucleic Acids Res. 16:8186). The primers may be designed
using commercially available software such as OLIGO 4.06 Primer
Analysis software (National Biosciences Inc., Plymouth, Minn.), or
another appropriate program, to be 22-30 nucleotides in length, to
have a GC content of 50% or more, and to anneal to the target
sequence at temperatures about 68.degree.-72.degree. C. The method
uses several restriction enzymes to generate a suitable fragment in
the known region of a gene. The fragment is then circularized by
intramolecular ligation and used as a PCR template.
[0106] Another method which may be used is capture PCR which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA (Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119). In this method,
multiple restriction enzyme digestions and ligations may also be
used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR.
[0107] Another method which may be used to retrieve unknown
sequences is that of Parker, J. D. et al. (1991; Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries to walk genomic DNA (Clontech, Palo Alto,
Calif.). This process avoids the need to screen libraries and is
useful in finding intron/exon junctions. When screening for
full-length cDNAs, it is preferable to use libraries that have been
size-selected to include larger cDNAs. Also, random-primed
libraries are preferable, in that they will contain more sequences
which contain the 5' regions of genes. Use of a randomly primed
library may be especially preferable for situations in which an
oligo d(T) library does not yield a full-length cDNA. Genomic
libraries may be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0108] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and detection of the emitted
wavelengths by a charge coupled devise camera. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g. GENOTYPER and SEQUENCE NAVIGATOR, Applied
Biosystems) and the entire process from loading of samples to
computer analysis and electronic data display may be computer
controlled. Capillary electrophoresis is especially preferable for
the sequencing of small pieces of DNA which might be present in
limited amounts in a particular sample.
[0109] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode DAPK may be used in
recombinant DNA molecules to direct expression of DAPK, fragments
or functional equivalents thereof, in appropriate host cells. Due
to the inherent degeneracy of the genetic code, other DNA sequences
which encode substantially the same or a functionally equivalent
amino acid sequence may be produced, and these sequences may be
used to clone and express DAPK.
[0110] As will be understood by those of skill in the art, it may
be advantageous to produce DAPK-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0111] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter DAPK encoding sequences for a variety of reasons, including
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0112] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding DAPK may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of DAPK activity, it may
be useful to encode a chimeric DAPK protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the DAPK
encoding sequence and the heterologous protein sequence, so that
DAPK may be cleaved and purified away from the heterologous
moiety.
[0113] In another embodiment, sequences encoding DAPK may be
synthesized, in whole or in part, using chemical methods well known
in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Symp.
Ser. 7:215-223, Horn, T. et al. (1980) Nucl. Acids. Symp. Ser.
7:225-232). Alternatively, the protein itself may be produced using
chemical methods to synthesize the amino acid sequence of DAPK, or
a fragment thereof. For example, peptide synthesis can be performed
using various solid-phase techniques (Roberge, J. Y. et al. (1995)
Science 269:202-204) and automated synthesis may be achieved, for
example, using the ABI 431A peptide synthesizer (Applied
Biosystems).
[0114] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins, Structures and Molecular Principles,
W H Freeman and Co., New York, N.Y.). The composition of the
synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure; Creighton,
supra). Additionally, the amino acid sequence of DAPK, or any part
thereof, may be altered during direct synthesis and/or combined
using chemical methods with sequences from other proteins, or any
part thereof, to produce a variant polypeptide.
[0115] In order to express a biologically active DAPK, the
nucleotide sequences encoding DAPK or functional equivalents, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0116] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding DAPK and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y.
[0117] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding DAPK. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0118] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1
plasmid (Life Technologies) and the like may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO; and storage protein genes) or from
plant viruses (e.g., viral promoters or leader sequences) may be
cloned into the vector. In mammalian cell systems, promoters from
mammalian genes or from mammalian viruses are preferable. If it is
necessary to generate a cell line that contains multiple copies of
the sequence encoding DAPK, vectors based on SV40 or EBV may be
used with an appropriate selectable marker.
[0119] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for DAPK. For example,
when large quantities of DAPK are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, the multifunctional E. coli
cloning and expression vectors such as BLUESCRIPT (Stratagene), in
which the sequence encoding DAPK may be ligated into the vector in
frame with sequences for the amino-terminal Met and the subsequent
7 residues of .beta.-galactosidase so that a hybrid protein is
produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509); and the like. PGEX vectors (Promega,
Madison, Wis.) may also be used to express foreign polypeptides as
fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems may be designed to include heparin, thrombin, or factor XA
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0120] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol.
153:516-544.
[0121] In cases where plant expression vectors are used, the
expression of sequences encoding DAPK may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196.
[0122] An insect system may also be used to express DAPK. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding DAPK may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of DAPK will
render the polyhedrin gene inactive and produce recombinant virus
lacking coat protein. The recombinant viruses may then be used to
infect, for example, S. frugiperda cells or Trichoplusia larvae in
which DAPK may be expressed (Engelhard, E. K. et al. (1994) Proc.
Nat. Acad. Sci. 91:3224-3227).
[0123] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding DAPK may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing DAPK in
infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl.
Acad. Sci. 81:3655-3659). In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0124] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of 6 to 10M are constructed and delivered via
conventional delivery methods (liposomes, polycationic amino
polymers, or vesicles) for therapeutic purposes.
[0125] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding DAPK. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding DAPK, its initiation codon, and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0126] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (ATCC; Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0127] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express DAPK may be transformed using expression
vectors which may contain viral origins of replication and/or
endogenous expression elements and a selectable marker gene on the
same or on a separate vector. Following the introduction of the
vector, cells may be allowed to grow for 1-2 days in an enriched
media before they are switched to selective media. The purpose of
the selectable marker is to confer resistance to selection, and its
presence allows growth and recovery of cells which successfully
express the introduced sequences. Resistant clones of stably
transformed cells may be proliferated using tissue culture
techniques appropriate to the cell type.
[0128] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1980) Cell 22:817-23) genes which can be employed in tk.sup.-
or aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic
or herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-51). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta. glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin,
being widely used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131).
[0129] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. For example, if the
sequence encoding DAPK is inserted within a marker gene sequence,
transformed cells containing sequences encoding DAPK can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding DAPK
under the control of a single promoter. Expression of the marker
gene in response to induction or selection usually indicates
expression of the tandem gene as well.
[0130] Alternatively, host cells which contain the nucleic acid
sequence encoding DAPK and express DAPK may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein. The
presence of polynucleotide sequences encoding DAPK can be detected
by DNA-DNA or DNA-RNA hybridization or amplification using probes
or fragments or fragments of polynucleotides encoding DAPK. Nucleic
acid amplification based assays involve the use of oligonucleotides
or oligomers based on the sequences encoding DAPK to detect
transformants containing DNA or RNA encoding DAPK.
[0131] A variety of protocols for detecting and measuring the
expression of DAPK, using either polyclonal or monoclonal
antibodies specific for the protein are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on DAPK is preferred, but
a competitive binding assay may be employed. These and other assays
are described, among other places, in Hampton, R. et al. (1990;
Serological Methods, a Laboratory Manual, APS Press, St Paul,
Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
158:1211-1216).
[0132] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding DAPK include oligolabeling, nick
translation, end-labeling or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding DAPK, or any
fragments thereof may be cloned into a vector for the production of
an mRNA probe. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a
variety of commercially available kits (Pharmacia & Upjohn,
(Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical
Corp.(Cleveland, Ohio). Suitable reporter molecules or labels,
which may be used for ease of detection, include radionuclides,
enzymes, fluorescent, chemiluminescent, or chromogenic agents as
well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
[0133] Host cells transformed with nucleotide sequences encoding
DAPK may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode DAPK may be designed to
contain signal sequences which direct secretion of DAPK through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding DAPK to nucleotide sequence
encoding a polypeptide domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAG
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.) between the purification domain and DAPK may be used to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing DAPK and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography as described in Porath, J. et al. (1992, Prot. Exp.
Purif. 3: 263-281) while the enterokinase cleavage site provides a
means for purifying DAPK from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
[0134] In addition to recombinant production, fragments of DAPK may
be produced by direct peptide synthesis using solid-phase
techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154).
Protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
Applied Biosystems 431A peptide synthesizer (Applied Biosystems).
Various fragments of DAPK may be chemically synthesized separately
and combined using chemical methods to produce the full length
molecule.
[0135] Therapeutics
[0136] Chemical and structural homology exits among the human
protein kinases of the invention. The expression of DAPK is closely
associated with cell proliferation. Therefore, in cancers or immune
disorders where DAPK is being expressed, or is promoting cell
proliferation; it is desirable to decrease the expression of DAPK.
In cancers where expression of DAPK is decreased, it is desirable
to provide the protein or increase the expression of DAPK.
[0137] In one embodiment, DAPK or a fragment or derivative thereof
may be administered to a subject to prevent or treat cancer which
is associated with decreased expression of DAPK. Such cancers
include, but are not limited to, adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma and
cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus.
[0138] In another embodiment, an agonist which is specific for DAPK
may be administered to a subject to prevent or treat cancer
including, but not limited to, those cancers listed above. In
another further embodiment, a vector capable of expressing DAPK, or
a fragment or a derivative thereof, may be administered to a
subject to prevent or treat cancer including, but not limited to,
those cancers listed above.
[0139] In a further embodiment, antagonists which decrease the
expression and activity of DAPK may be administered to a subject to
prevent or treat cancer which is associated with increased
expression of DAPK. Such cancers include, but are not limited to,
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and
teratocarcinoma and cancers of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect,
antibodies which specifically bind DAPK may be used directly as an
antagonist or indirectly as a targeting or delivery mechanism for
bringing a pharmaceutical agent to cells or tissue which express
DAPK.
[0140] In another embodiment, a vector expressing the complement of
the polynucleotide encoding DAPK may be administered to a subject
to treat or prevent cancer including, but not limited to, those
cancers listed above.
[0141] In one embodiment, an antagonist of DAPK may be administered
to a subject to prevent or treat an immune response. Such responses
may be associated with AIDS, Addison's disease, adult respiratory
distress syndrome, allergies, anemia, asthma, atherosclerosis,
bronchitis, cholecystitis, Crohn's disease, ulcerative colitis,
atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
atrophic gastritis, glomerulonephritis, gout, Graves' disease,
hypereosinophilia, irritable bowel syndrome, lupus erythematosus,
multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, and autoimmune thyroiditis; complications of cancer,
hemodialysis, extracorporeal circulation; viral, bacterial, fungal,
parasitic, protozoal, and helminthic infections and trauma. In one
aspect, antibodies which specifically bind DAPK may be used
directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue
which express DAPK.
[0142] In another embodiment, a vector expressing the complement of
the polynucleotide encoding DAPK may be administered to a subject
to treat or prevent an immune response including, but not limited
to, those listed above.
[0143] In a further embodiment, DAPK or a fragment or derivative
thereof may be added to cells to stimulate cell proliferation. In
particular, DAPK may be added to a cell in culture or cells in vivo
using delivery mechanisms such as liposomes, viral based vectors,
or electroinjection for the purpose of promoting cell proliferation
and tissue or organ regeneration. Specifically, DAPK may be added
to a cell, cell line, tissue or organ culture in vitro or ex vivo
to stimulate cell proliferation for use in heterologous or
autologous transplantation. In some cases, the cell will have been
preselected for its ability to fight an infection or a cancer or to
correct a genetic defect in a disease such as sickle cell anemia,
.beta. thalassemia, cystic fibrosis, or Huntington's chorea.
[0144] In another embodiment, an agonist which is specific for DAPK
may be administered to a cell to stimulate cell proliferation, as
described above.
[0145] In another embodiment, a vector capable of expressing DAPK,
or a fragment or a derivative thereof, may be administered to a
cell to stimulate cell proliferation, as described above.
[0146] In other embodiments, any of the therapeutic proteins,
antagonists, antibodies, agonists, complementary sequences or
vectors of the invention may be administered 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 effect the treatment or prevention of the
various disorders described above. 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.
[0147] Antagonists or inhibitors of DAPK may be produced using
methods which are generally known in the art. In particular,
purified DAPK may be used to produce antibodies or to screen
libraries of pharmaceutical agents to identify those which
specifically bind DAPK.
[0148] Antibodies to DAPK may be generated using methods that are
well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, single chain, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies, (i.e., those which inhibit dimer
formation) are especially preferred for therapeutic use.
[0149] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with DAPK or any fragment or oligopeptide thereof which
has immunogenic properties. Depending on the host species, various
adjuvants may be used to increase immunological response. Such
adjuvants include, but are not limited to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable.
[0150] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to DAPK have an amino acid
sequence consisting of at least five amino acids and more
preferably at least 10 amino acids. It is also preferable that they
are identical to a portion of the amino acid sequence of the
natural protein, and they may contain the entire amino acid
sequence of a small, naturally occurring molecule. Short stretches
of DAPK amino acids may be fused with those of another protein such
as keyhole limpet hemocyanin and antibody produced against the
chimeric molecule.
[0151] Monoclonal antibodies to DAPK may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.
80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120).
[0152] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison, S. L. et
al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et
al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature
314:452-454). Alternatively, techniques described for the
production of single chain antibodies may be adapted, using methods
known in the art, to produce DAPK-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic
composition, may be generated by chain shuffling from random
combinatorial immunoglobulin libraries (Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:11120-3).
[0153] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991)
Nature 349:293-299).
[0154] Antibody fragments which contain specific binding sites for
DAPK may also be generated. For example, such fragments include,
but are not limited to, the F(ab')2 fragments which can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 254:1275-1281).
[0155] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between DAPK and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering DAPK epitopes
is preferred, but a competitive binding assay may also be employed
(Maddox, supra).
[0156] In another embodiment of the invention, the polynucleotides
encoding DAPK, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding DAPK may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding DAPK. Thus, complementary molecules or
fragments may be used to modulate DAPK activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments, can be designed from various locations along the coding
or control regions of sequences encoding DAPK.
[0157] Expression vectors derived from retro viruses, adenovirus,
herpes or vaccinia viruses, or from various bacterial plasmids may
be used for delivery of nucleotide sequences to the targeted organ,
tissue or cell population. Methods which are well known to those
skilled in the art can be used to construct vectors which will
express nucleic acid sequence which is complementary to the
polynucleotides of the gene encoding DAPK. These techniques are
described both in Sambrook et al. (supra) and in Ausubel et al.
(supra).
[0158] Genes encoding DAPK can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide or fragment thereof which encodes DAPK. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector and even longer if appropriate replication elements are part
of the vector system.
[0159] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5' or regulatory
regions of the gene encoding DAPK (signal sequence, promoters,
enhancers, and introns). Oligonucleotides derived from the
transcription initiation site, e.g., between positions -10 and +10
from the start site, are preferred. Similarly, inhibition can be
achieved using "triple helix" base-pairing methodology. Triple
helix pairing is useful because it causes inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I.
Carr, Molecular and Immunologic Approaches, Futura Publishing Co.,
Mt. Kisco, N.Y.). The complementary sequence or antisense molecule
may also be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0160] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Examples which may be used include engineered hammerhead
motif ribozyme molecules that can specifically and efficiently
catalyze endonucleolytic cleavage of sequences encoding DAPK.
[0161] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0162] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding DAPK. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA constitutively or inducibly can
be introduced into cell lines, cells, or tissues. RNA molecules may
be modified to increase intracellular stability and half-life.
Possible modifications include, but are not limited to, the
addition of flanking sequences at the 5' and/or 3' ends of the
molecule or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the backbone of the molecule.
This concept is inherent in the production of PNAs and can be
extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0163] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections or polycationic amino polymers (Goldman, C.
K. et al. (1997) Nature Biotechnology 15:462-466; incorporated
herein by reference) may be achieved using methods which are well
known in the art.
[0164] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0165] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of DAPK, antibodies to DAPK, mimetics, agonists,
antagonists, or inhibitors of DAPK. The compositions may be
administered alone or in combination with at least one other agent,
such as stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0166] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0167] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising 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 may be found in the latest edition
of Remington's Pharmaceutical Sciences(Maack Publishing Co.,
Easton, Pa.).
[0168] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0169] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0170] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0171] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0172] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0173] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art. The
pharmaceutical compositions of the present invention may be
manufactured in a manner that is known in the art, e.g., by means
of conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, or lyophilizing
processes.
[0174] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents than are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder which
may contain any or all of the following: 1-50 mM histidine, 0.1%-2%
sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is
combined with buffer prior to use.
[0175] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of DAPK, such
labeling would include amount, frequency, and method of
administration.
[0176] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0177] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually mice, rabbits, dogs,
or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0178] A therapeutically effective dose refers to that amount of
active ingredient, for example DAPK or fragments thereof,
antibodies of DAPK, agonists, antagonists or inhibitors of DAPK,
which ameliorates the symptoms or condition. Therapeutic efficacy
and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., ED50
(the dose therapeutically effective in 50% of the population) and
LD50 (the dose lethal to 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index, and
it can be expressed as the ratio, LD50/ED50. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies is
used in formulating a range of dosage for human use. The dosage
contained in such compositions is preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0179] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active moiety or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0180] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0181] Diagnostics
[0182] In another embodiment, antibodies which specifically bind
DAPK may be used for the diagnosis of conditions or diseases
characterized by expression of DAPK, or in assays to monitor
patients being treated with DAPK, agonists, antagonists or
inhibitors. The antibodies useful for diagnostic purposes may be
prepared in the same manner as those described above for
therapeutics. Diagnostic assays for DAPK include methods which
utilize the antibody and a label to detect DAPK in human body
fluids or extracts of cells or tissues. The antibodies may be used
with or without modification, and may be labeled by joining them,
either covalently or non-covalently, with a reporter molecule. A
wide variety of reporter molecules which are known in the art may
be used, several of which are described above.
[0183] A variety of protocols including ELISA, RIA, and FACS for
measuring DAPK are known in the art and provide a basis for
diagnosing altered or abnormal levels of DAPK expression. Normal or
standard values for DAPK expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to DAPK under conditions suitable
for complex formation. The amount of standard complex formation may
be quantified by various methods, but preferably by photometric
means. Quantities of DAPK expressed in subject samples, control and
diseased, from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes
the parameters for diagnosing disease.
[0184] In another embodiment of the invention, the polynucleotides
encoding DAPK may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of DAPK may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
DAPK, and to monitor regulation of DAPK levels during therapeutic
intervention.
[0185] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding DAPK or closely related molecules, may be used
to identify nucleic acid sequences which encode DAPK. The
specificity of the probe, whether it is made from a highly specific
region, e.g., 10 unique nucleotides in the 5' regulatory region, or
a less specific region, e.g., especially in the 3' coding region,
and the stringency of the hybridization or amplification (maximal,
high, intermediate, or low) will determine whether the probe
identifies only naturally occurring sequences encoding DAPK,
alleles, or related sequences.
[0186] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides from any of the DAPK encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
derived from the nucleotide sequence of SEQ ID Nos:8-14 or from
genomic sequence including promoter, enhancer elements, and introns
of the naturally occurring DAPK.
[0187] Means for producing specific hybridization probes for DNAs
encoding DAPK include the cloning of nucleic acid sequences
encoding DAPK or DAPK derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, radionuclides
such as 32P or 35S, or enzymatic labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
[0188] Polynucleotide sequences encoding DAPK may be used for the
diagnosis of conditions, disorders, or diseases which are
associated with either increased or decreased expression of DAPK.
Examples of such conditions or diseases include adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma,
and cancers of the adrenal gland, bladder, bone, brain, breast,
cervix, gall bladder, ganglia, gastrointestinal tract, heart,
kidney, liver, lung, bone marrow, muscle, ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus; and immune disorders such as
AIDS, Addison's disease, adult respiratory distress syndrome,
allergies, anemia, asthma, atherosclerosis, bronchitis,
cholecystitus, Crohn's disease, ulcerative colitis, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic
gastritis, glomerulonephritis, gout, Graves' disease,
hypereosinophilia, irritable bowel syndrome, lupus erythematosus,
multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, and thyroiditis. The polynucleotide sequences encoding
DAPK may be used in Southern or northern analysis, dot blot, or
other membrane-based technologies; in PCR technologies; or in
dipstick, pin, ELISA assays or microarrays utilizing fluids or
tissues from patient biopsies to detect altered DAPK expression.
Such qualitative or quantitative methods are well known in the
art.
[0189] In a particular aspect, the nucleotide sequences encoding
DAPK may be useful in assays that detect activation or induction of
various cancers, particularly those mentioned above. The nucleotide
sequences encoding DAPK may be labeled by standard methods, and
added to a fluid or tissue sample from a patient under conditions
suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is
quantitated and compared with a standard value. If the amount of
signal in the biopsied or extracted sample is significantly altered
from that of a comparable control sample, the nucleotide sequences
have hybridized with nucleotide sequences in the sample, and the
presence of altered levels of nucleotide sequences encoding DAPK in
the sample indicates the presence of the associated disease. Such
assays may also be used to evaluate the efficacy of a particular
therapeutic treatment regimen in animal studies, in clinical
trials, or in monitoring the treatment of an individual
patient.
[0190] In order to provide a basis for the diagnosis of disease
associated with expression of DAPK, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
which encodes DAPK, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with those from
an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for disease. Deviation between
standard and subject values is used to establish the presence of
disease.
[0191] Once disease is established and a treatment protocol is
initiated, hybridization assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that which is observed in the normal patient. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0192] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0193] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding DAPK may involve the use of PCR. Such
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably consist of two
nucleotide sequences, one with sense orientation (5'->3') and
another with antisense (3'<-5'), employed under optimized
conditions for identification of a specific gene or condition. The
same two oligomers, nested sets of oligomers, or even a degenerate
pool of oligomers may be employed under less stringent conditions
for detection and/or quantitation of closely related DNA or RNA
sequences.
[0194] Methods which may also be used to quantitate the expression
of DAPK include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and standard curves onto
which the experimental results are interpolated (Melby, P. C. et
al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al.
(1993) Anal. Biochem. 212:229-236). The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
[0195] In further embodiments, oligonucleotides derived from any of
the polynucleotide sequences described herein may be used as
targets in a microarray. The microarray can be used to monitor the
expression level of large numbers of genes simultaneously (to
produce a transcript image), and to identify genetic variants,
mutations and polymorphisms. This information may be used to
determine gene function, understanding the genetic basis of
disease, diagnosing disease, and in developing and in monitoring
the activities of therapeutic agents.
[0196] In one embodiment, the microarray is prepared and used
according to the methods described in PCT application WO95/11995
(Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14:
1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93:
10614-10619), all of which are incorporated herein in their
entirety by reference.
[0197] The microarray is preferably composed of a large number of
unique, single-stranded nucleic acid sequences, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides are preferably about 6-60
nucleotides in length, more preferably 15-30 nucleotides in length,
and most preferably about 20-25 nucleotides in length. For a
certain type of microarray, it may be preferable to use
oligonucleotides which are only 7-10 nucleotides in length. The
microarray may contain oligonucleotides which cover the known 5',
or 3', sequence, or contain sequential oligonucleotides which cover
the full length sequence; or unique oligonucleotides selected from
particular areas along the length of the sequence. Polynucleotides
used in the microarray may be oligonucleotides that are specific to
a gene or genes of interest in which at least a fragment of the
sequence is known or that are specific to one or more unidentified
cDNAs which are common to a particular cell type, developmental or
disease state. In certain situations it may be appropriate to use
pairs of oligonucleotides on a microarray. The "pairs" will be
identical, except for one nucleotide which preferably is located in
the center of the sequence. The second oligonucleotide in the pair
(mismatched by one) serves as a control. The number of
oligonucleotide pairs may range from 2 to one million.
[0198] In order to produce oligonucleotides to a known sequence for
a microarray, the gene of interest is examined using a computer
algorithm which starts at the 5' or more preferably at the 3' end
of the nucleotide sequence. The algorithm identifies oligomers of
defined length that are unique to the gene, have a GC content
within a range suitable for hybridization, and lack predicted
secondary structure that may interfere with hybridization. The
oligomers are synthesized at designated areas on a substrate using
a light-directed chemical process. The substrate may be paper,
nylon or other type of membrane, filter, chip, glass slide or any
other suitable solid support.
[0199] In another aspect, the oligonucleotides may be synthesized
on the surface of the substrate by using a chemical coupling
procedure and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array may be produced
by hand or using available devices (slot blot or dot blot
apparatus), materials and machines (including robotic instruments)
and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or
any other multiple from 2 to one million which lends itself to the
efficient use of commercially available instrumentation.
[0200] In order to conduct sample analysis using the microarrays,
the RNA or DNA from a biological sample is made into hybridization
probes. The mRNA is isolated, and cDNA is produced and used as a
template to make antisense RNA (aRNA). The aRNA is amplified in the
presence of fluorescent nucleotides, and labeled probes are
incubated with the microarray so that the probe sequences hybridize
to complementary oligonucleotides of the microarray. Incubation
conditions are adjusted so that hybridization occurs with precise
complementary matches or with various degrees of less
complementarity. After removal of nonhybridized probes, a scanner
is used to determine the levels and patterns of fluorescence. The
scanned images are examined to determine degree of complementarity
and the relative abundance of each oligonucleotide sequence on the
microarray. The biological samples may be obtained from any bodily
fluids (such as blood, urine, saliva, phlegm, gastric juices,
etc.), cultured cells, biopsies, or other tissue preparations. A
detection system may be used to measure the absence, presence, and
amount of hybridization for all of the distinct sequences
simultaneously. This data may be used for large scale correlation
studies or functional analysis of the sequences, mutations,
variants, or polymorphisms among samples (Heller, R. A. et al.,
(1997) Proc. Natl. Acad. Sci. 94:2150-2155).
[0201] In another embodiment of the invention, the nucleic acid
sequences which encode DAPK may also be used to generate
hybridization probes which are useful for mapping the naturally
occurring genomic sequence. The sequences may be mapped to a
particular chromosome, to a specific region of a chromosome or to
artificial chromosome constructions, such as human artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial
artificial chromosomes (BACs), bacterial P1 constructions or single
chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood
Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet.
7:149-154.
[0202] Fluorescent in situ hybridization (FISH as described in
Verma et al. (1988) Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York, N.Y.) may be correlated with
other physical chromosome mapping techniques and genetic map data.
Examples of genetic map data can be found in various scientific
journals or at Online Mendelian Inheritance in Man (OMIM).
Correlation between the location of the gene encoding DAPK on a
physical chromosomal map and a specific disease, or predisposition
to a specific disease, may help delimit the region of DNA
associated with that genetic disease. The nucleotide sequences of
the subject invention may be used to detect differences in gene
sequences between normal, carrier, or affected individuals.
[0203] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms, or parts
thereof, by physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, for example, AT to 11q22-23 (Gatti, R. A. et al.
(1988) Nature 336:577-580), any sequences mapping to that area may
represent associated or regulatory genes for further investigation.
The nucleotide sequence of the subject invention may also be used
to detect differences in the chromosomal location due to
translocation, inversion, etc. among normal, carrier, or affected
individuals.
[0204] In another embodiment of the invention, DAPK, its catalytic
or immunogenic fragments or oligopeptides thereof, can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes, between DAPK and the agent being tested, may be
measured.
[0205] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564. In this method, as applied to
DAPK large numbers of different small test compounds are
synthesized on a solid substrate, such as plastic pins or some
other surface. The test compounds are reacted with DAPK, or
fragments thereof, and washed. Bound DAPK is then detected by
methods well known in the art. Purified DAPK can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0206] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding DAPK specifically compete with a test compound for binding
DAPK. In this manner, the antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with DAPK.
[0207] In additional embodiments, the nucleotide sequences which
encode DAPK may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0208] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0209] For purposes of example, the preparation and sequencing of
the TMLR3DT01 cDNA library, from which Incyte Clone 402339 was
isolated, is described. Preparation and sequencing of cDNAs in
libraries in the LIFESEQ database have varied over time, and the
gradual changes involved use of particular kits, plasmids, and
machinery available at the particular time the library was made and
analyzed.
[0210] I TMLR3DT01 cDNA Library Construction
[0211] The TMLR3DT01 cDNA library was constructed from normal
peripheral blood T-lymphocytes obtained from two unrelated
Caucasian males aged 25 and 29 years. This library represents a
mixture of allogeneically stimulated human T cell populations
obtained from Ficoll/Hypaque purified buffy coats. The cells from
the two different donors (not typed for HLA alleles) were incubated
at a density of 1.times.10.sup.6/ml, cultured for 96 hours in DME
containing 10% human serum, washed in PBS, scraped and lyzed
immediately in buffer containing guanidinium isothiocyanate. The
lysate was extracted twice with a mixture of phenol and chloroform,
pH 8.0 and centrifuged over a CsCl cushion using an SW28 rotor in a
L8-70M ultracentrifuge (Beckman Instruments, Fullerton Calif.). The
RNA was precipitated using 0.3 M sodium acetate and 2.5 volumes of
ethanol, resuspended in water and DNase treated for 15 min at
37.degree. C. The total RNA was isolated using the OLIGOTEX kit
(QIAGEN Inc, Chatsworth Calif.). B lymphocytes were not removed,
and some contaminating macrophages may also have been present.
[0212] Stratagene (La Jolla Calif.) used the total RNA to construct
a custom cDNA library. First strand cDNA synthesis was accomplished
using an oligo d(T) primer/linker which also contained an XhoI
restriction site. Second strand synthesis was performed using a
combination of DNA polymerase I, E. coli ligase and RNase H,
followed by the addition of an EcoRI adaptor to the blunt ended
cDNA. The EcoRI adapted, double-stranded cDNA was then digested
with XhoI restriction enzyme and fractionated on Sephacryl S400 to
obtain sequences which exceeded 800 bp in size. The size-selected
cDNAs were inserted into the LAMBDAZAP vector system (Stratagene);
and the vector which contains the PBLUESCRIPT phagemid (Stratagene)
was transformed into cells of E. coli, strain XL1-BLUEMRF
(Stratagene).
[0213] The phagemid forms of individual cDNA clones were obtained
by the in vivo excision process. Enzymes from both PBLUESCRIPT and
a co-transformed f1 helper phage nicked the DNA, initiated new DNA
synthesis, and created the smaller, single-stranded, circular
phagemid molecules which contained the cDNA insert. The phagemid
DNA was released, purified, and used to reinfect fresh host cells
(SOLR, Stratagene). Presence of the phagemid which carries the gene
for .beta.-lactamase allowed transformed bacteria to grow on medium
containing ampicillin.
[0214] II Isolation and Sequencing of cDNA Clones
[0215] Plasmid DNA was released from the cells and purified using
the MINIPREP Kit (Edge Biosystems, Gaithersburg Md.). This kit
consists of a 96 well block with reagents for 960 purifications.
The recommended protocol was employed except for the following
changes: 1) the 96 wells were each filled with only 1 ml of sterile
TERRIFIC BROTH (BD Biosciences, Sparks Md.) with carbenicillin at
25 mg/L and glycerol at 0.4%; 2) the bacteria were cultured for 24
hours after the wells were inoculated and then lysed with 60 .mu.l
of lysis buffer; 3) a centrifugation step employing the BECKMAN
GS-6R centrifuge (Beckman Instruments) at 2900 rpm for 5 min was
performed before the contents of the block were added to the
primary filter plate; and 4) the optional step of adding
isopropanol to TRIS buffer was not routinely performed. After the
last step in the protocol, samples were transferred to a 96-well
block for storage.
[0216] The cDNAs were prepared for sequencing using the MICROLAB
2200 system (Hamilton) in combination with the DNA ENGINE thermal
cyclers (MJ Research). The cDNAs were sequenced by the method of
Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM
377 sequencing system (Applied Biosystems) and the reading frame
was determined.
[0217] III Homology Searching of cDNA Clones and their Deduced
Proteins
[0218] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST, which stands for
Basic Local Alignment Search Tool (Altschul, S. F. (1993) J. Mol.
Evol 36:290-300; Altschul, et al. (1990) J. Mol. Biol.
215:403-410).
[0219] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms such as the one described in Smith, T. et
al. (1992, Protein Engineering 5:35-51), incorporated herein by
reference, could have been used when dealing with primary sequence
patterns and secondary structure gap penalties. The sequences
disclosed in this application have lengths of at least 49
nucleotides, and no more than 12% uncalled bases (where N is
recorded rather than A, C, G, or T).
[0220] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-14 for peptides.
[0221] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam); and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp) for homology. The relevant database for a
particular match were reported as GIxxx.+-.p (where xxx is pri,
rod, etc., and if present, p=peptide).
[0222] IV Northern Analysis
[0223] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound
(Sambrook et al., supra).
[0224] Analogous computer techniques use BLAST to search for
identical or related molecules in nucleotide databases such as
GenBank or the LIFESEQ database (Incyte Genomics, Palo Alto
Calif.). This analysis is much faster than multiple, membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or homologous.
[0225] The basis of the search is the product score which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0226] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1-2% error; and at 70, the match will be exact.
Homologous molecules are usually identified by selecting those
which show product scores between 15 and 40, although lower scores
may identify related molecules.
[0227] The results of northern analysis are reported as a list of
libraries in which the transcript encoding DAPK occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0228] V Extension of DAPK Encoding Polynucleotides
[0229] The nucleic acid sequence of an Incyte Clone disclosed in
the Sequence Listing was used to design oligonucleotide primers for
extending a partial nucleotide sequence to full length. One primer
was synthesized to initiate extension in the antisense direction,
and the other was synthesized to extend sequence in the sense
direction. Primers were used to facilitate the extension of the
known sequence "outward" generating amplicons containing new,
unknown nucleotide sequence for the region of interest. The initial
primers were designed from the cDNA using OLIGO 4.06 primer
analysis software (National Biosciences), or another appropriate
program, to be about 22 to about 30 nucleotides in length, to have
a GC content of 50% or more, and to anneal to the target sequence
at temperatures of about 68.degree. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0230] Selected human cDNA libraries (Life Technologies) were used
to extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0231] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Applied Biosystems) and thoroughly
mixing the enzyme and reaction mix. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR was performed using the DNA ENGINE thermal cycler
(MJ Research) and the following parameters:
2 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat step 4-6 for 15 additional
cycles Step 8 94.degree. C. for 15 sec Step 9 65.degree. C. for 1
min Step 10 68.degree. C. for 7:15 min Step 11 Repeat step 8-10 for
12 cycles Step 12 72.degree. C. for 8 min Step 13 4.degree. C. (and
holding)
[0232] A 5-10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
mini-gel to determine which reactions were successful in extending
the sequence. Bands thought to contain the largest products were
excised from the gel, purified using QIAQUICK (QIAGEN Inc.,
Chatsworth, Calif.), and trimmed of overhangs using Klenow enzyme
to facilitate religation and cloning.
[0233] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2-3 hours or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook et al., supra). After
incubation for one hour at 37.degree. C., the E. coli mixture was
plated on Luria Bertani (LB)-agar (Sambrook et al., supra)
containing 2.times.Carb. The following day, several colonies were
randomly picked from each plate and cultured in 150 .mu.l of liquid
LB/2.times.Carb medium placed in an individual well of an
appropriate, commercially-available, sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and after dilution
1:10 with water, 5 .mu.l of each sample was transferred into a PCR
array.
[0234] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
3 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0235] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0236] In like manner, the nucleotide sequence of SEQ ID NO:8-14
are used to obtain 5' regulatory sequences using the procedure
above, oligonucleotides designed for 5' extension, and an
appropriate genomic library.
[0237] VI Labeling and Use of Individual Hybridization Probes
[0238] Hybridization probes derived from SEQ ID NOs:8-14 are
employed to screen cDNAs, genomic DNAs, or mRNAs. Although the
labeling of oligonucleotides, consisting of about 20 base-pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 (National
Biosciences), labeled by combining 50 pmol of each oligomer and 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (APB) and T4
polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeled
oligonucleotides are substantially purified with SEPHADEX G-25
superfine resin column (APB). A aliquot containing 10.sup.7 counts
per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of human genomic DNA digested with one of
the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,
or Pvu II; DuPont NEN).
[0239] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (NYTRAN PLUS,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times.saline sodium
citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are
visualized using autoradiography or an alternative imaging means
and compared.
[0240] VII Microarrays
[0241] To produce oligonucleotides for a microarray, SEQ ID
Nos:8-14 were examined using a computer algorithm which starts at
the 3' end of the nucleotide sequence. The algorithm identified
oligomers of defined length that are unique to the gene, have a GC
content within a range suitable for hybridization, and lack
predicted secondary structure that would interfere with
hybridization. The algorithm identified approximately 20
sequence-specific oligonucleotides of 20 nucleotides in length
(20-mers). A matched set of oligonucleotides was created in which
one nucleotide in the center of each sequence was altered. This
process was repeated for each gene in the microarray, and double
sets of twenty 20 mers were synthesized and arranged on the surface
of the silicon chip using a light-directed chemical process (Chee,
M. et al., PCT/WO95/11995, incorporated herein by reference).
[0242] In the alternative, a chemical coupling procedure and an ink
jet device were used to synthesize oligomers on the surface of a
substrate (Baldeschweiler, J. D. et al., PCT/WO95/25116,
incorporated herein by reference). In another alternative, a
"gridded" array analogous to a dot (or slot) blot was used to
arrange and link cDNA fragments or oligonucleotides to the surface
of a substrate using a vacuum system, thermal, UV, mechanical or
chemical bonding procedures. A typical array may be produced by
hand or using available materials and machines and contain grids of
8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots. After
hybridization, the microarray was washed to remove nonhybridized
probes, and a scanner was used to determine the levels and patterns
of fluorescence. The scanned images were examined to determine
degree of complementarity and the relative abundance/expression
level of each oligonucleotide sequence in the micro-array.
[0243] VIII Complementary Polynucleotides
[0244] Sequence complementary to the sequence encoding DAPK, or any
part thereof, is used to detect, decrease or inhibit expression of
naturally occurring DAPK. Although use of oligonucleotides
comprising from about 15 to about 30 base-pairs is described,
essentially the same procedure is used with smaller or larger
sequence fragments. Appropriate oligonucleotides are designed using
OLIGO 4.06 primer analysis software and the coding sequence of
DAPK, SEQ ID NOs:8-14. To inhibit transcription, a complementary
oligonucleotide is designed from the most unique 5' sequence and
used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent
ribosomal binding to the transcript encoding DAPK.
[0245] IX Expression of DAPK
[0246] Expression of DAPK is accomplished by subcloning the cDNAs
into appropriate vectors and transforming the vectors into host
cells. In this case, the cloning vector is also used to express
DAPK in E. coli. Upstream of the cloning site, this vector contains
a promoter for .beta.-galactosidase, followed by sequence
containing the amino-terminal Met, and the subsequent seven
residues of .beta.-galactosidase. Immediately following these eight
residues is a bacteriophage promoter useful for transcription and a
linker containing a number of unique restriction sites.
[0247] Induction of an isolated, transformed bacterial strain with
IPTG using standard methods produces a fusion protein which
consists of the first eight residues of .beta.-galactosidase, about
5 to 15 residues of linker, and the full length protein. The signal
residues direct the secretion of DAPK into the bacterial growth
media which can be used directly in the following assay for
activity.
[0248] X Demonstration of DAPK Activity
[0249] DAPK activity may be measured by phosphorylation of a
protein substrate using gamma-labeled .sup.32P-ATP and quantitation
of the incorporated radioactivity using a gamma radioisotope
counter. DAPK is incubated with the protein substrate,
.sup.32P-ATP, and a kinase buffer. The .sup.32P incorporated into
the substrate is then separated from free .sup.32P-ATP by
electrophoresis and the incorporated .sup.32P is counted. The
amount of .sup.32P recovered is proportional to the activity of
DAPK in the assay. A determination of the specific amino acid
residues phosphorylated is made by phosphoamino acid analysis of
the hydrolyzed protein.
[0250] XI Production of DAPK Specific Antibodies
[0251] DAPK that is substantially purified using PAGE
electrophoresis (Sambrook, supra), or other purification
techniques, is used to immunize rabbits and to produce antibodies
using standard protocols. The amino acid sequence deduced from SEQ
ID NOs:8-14 is analyzed using DNASTAR software (DNASTAR Inc) to
determine regions of high immunogenicity and a corresponding
oligopeptide is synthesized and used to raise antibodies by means
known to those of skill in the art. Selection of appropriate
epitopes, such as those near the C-terminus or in hydrophilic
regions, is described by Ausubel et al. (supra), and others.
[0252] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems peptide synthesizer Model
431A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin
(KLH, Sigma, St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,
supra). Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. The resulting antisera are tested for
antipeptide activity, for example, by binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio iodinated, goat anti-rabbit
IgG.
[0253] XII Purification of Naturally Occurring DAPK Using Specific
Antibodies
[0254] Naturally occurring or recombinant DAPK is substantially
purified by immunoaffinity chromatography using antibodies specific
for DAPK. An immunoaffinity column is constructed by covalently
coupling DAPK antibody to an activated chromatographic resin, such
as CNBr-activated Sepharose (Pharmacia & Upjohn). After the
coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0255] Media containing DAPK is passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of DAPK (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/protein binding (eg, a buffer of
pH 2-3 or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and DAPK is collected.
[0256] XIII Identification of Molecules which Interact with
DAPK
[0257] DAPK or biologically active fragments thereof are labeled
with .sup.125I Bolton-Hunter reagent (Bolton et al. (1973) Biochem.
J. 133: 529). Candidate molecules previously arrayed in the wells
of a multi-well plate are incubated with the labeled DAPK, washed
and any wells with labeled DAPK complex are assayed. Data obtained
using different concentrations of DAPK are used to calculate values
for the number, affinity, and association of DAPK with the
candidate molecules.
[0258] All publications and patents mentioned in the above
specification are herein incorporated 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 which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
* * * * *