U.S. patent application number 10/121925 was filed with the patent office on 2003-06-05 for nucleic acid molecules encoding human kinase and phosphatase homologues and uses therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Robison, Keith E..
Application Number | 20030104505 10/121925 |
Document ID | / |
Family ID | 23528952 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104505 |
Kind Code |
A1 |
Robison, Keith E. |
June 5, 2003 |
Nucleic acid molecules encoding human kinase and phosphatase
homologues and uses therefor
Abstract
The invention provides isolated nucleic acids molecules,
designated Kinase and Phosphatase nucleic acid molecules, which
encode novel protein kinase and protein phosphatase polypeptides.
The invention also provides antisense nucleic acid molecules,
recombinant expression vectors containing Kinase and Phosphatase
nucleic acid molecules, host cells into which the expression
vectors have been introduced, and nonhuman transgenic animals in
which a Kinase and Phosphatase gene has been introduced or
disrupted. The invention still further provides isolated Kinase and
Phosphatase proteins, fusion proteins, antigenic peptides and
anti-Kinase and Phosphatase antibodies. Diagnostic, screening, and
therapeutic methods utilizing compositions of the invention are
also provided.
Inventors: |
Robison, Keith E.;
(Wilmington, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
23528952 |
Appl. No.: |
10/121925 |
Filed: |
April 12, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10121925 |
Apr 12, 2002 |
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09948802 |
Sep 7, 2001 |
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6465232 |
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09948802 |
Sep 7, 2001 |
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09387212 |
Aug 31, 1999 |
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6309849 |
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Current U.S.
Class: |
435/7.92 ;
435/6.18; 435/7.31 |
Current CPC
Class: |
G01N 2333/9121 20130101;
G01N 2500/00 20130101; G01N 33/573 20130101; C12N 9/12 20130101;
C07K 2319/00 20130101; C12N 9/16 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/7.92 ;
435/7.31; 435/6 |
International
Class: |
G01N 033/53; G01N
033/569; G01N 033/537; G01N 033/543; C12Q 001/68 |
Claims
What is claimed:
1. A method for detecting the presence of a Kinase or Phosphatase
encoded by the nucleotide sequence shown in SEQ ID NOs: 1-14, in a
sample comprising: a) contacting the sample with a compound which
selectively binds to the Kinase or Phosphatase; and b) determining
whether the compound binds to the Kinase or Phosphatase in the
sample to thereby detect the presence of a Kinase or Phosphatase in
the sample.
2. The method of claim 1, wherein the compound which binds to the
Kinase or Phosphatase is an antibody.
3. A method for detecting the presence of a Kinase or Phosphatase
nucleic acid molecule shown in SEQ ID NOs: 1-14 in a sample
comprising: a) contacting the sample with a nucleic acid probe or
primer which selectively hybridizes to the nucleic acid molecule;
and b) determining whether the nucleic acid probe or primer binds
to a nucleic acid molecule in the sample to thereby detect the
presence of a Kinase or Phosphatase nucleic acid molecule in the
sample.
4. The method of claim 3, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
5. A method for identifying a compound which binds to a Kinase or
Phosphatase encoded by the nucleotide sequence shown in SEQ ID NOs:
1-14 comprising: a) contacting the Kinase or Phosphatase with a
test compound under conditions suitable for binding; and b)
detecting binding of the test compound to the Kinase or
Phosphatase.
6. The method of claim 5, wherein said detection is by direct
binding.
7. The method of claim 6, wherein said direct binding is determined
by an immunoprecipitation.
8. The method of claim 6, wherein said direct binding is determined
by a yeast two-hybrid assay.
9. A method for modulating the activity of a Kinase or Phosphatase
encoded by the nucleotide sequence shown in SEQ ID NOs: 1-14
comprising contacting the Kinase or Phosphatase with a compound
which binds to the Kinase or Phosphatase in a sufficient
concentration to modulate the activity of the Kinase or
Phosphatase.
10. A method for identifying a compound which modulates the
activity of a Kinase or Phosphatase encoded by the nucleotide
sequence shown in SEQ ID NOs: 1-14 comprising: a) contacting a
Kinase or Phosphatase with a test compound; and b) determining the
effect of the test compound on the activity of the Kinase or
Phosphatase to thereby identify a compound which modulates the
activity of the Kinase or Phosphatase.
Description
BACKGROUND OF THE INVENTION
[0001] Phosphate tightly associated with a molecule, e.g., a
protein, has been known since the late nineteenth century. Since
then, a variety of covalent linkages of phosphate to proteins have
been found. The most common involve esterification of phosphate to
serine, threonine, and tyrosine with smaller amounts being linked
to lysine, arginine, histidine, aspartic acid, glutamic acid, and
cysteine. The occurrence of phosphorylated molecules, e.g.,
proteins implies the existence of one or more kinases, e.g.,
protein kinases, capable of phosphorylating various molecules,
e.g., amino acid residues on proteins, and also of phosphatases,
e.g., protein phosphatases, capable of hydrolyzing various
phosphorylated molecules, e.g., phosphorylated amino acid residues
on proteins.
[0002] Protein kinases and phosphatases play critical roles in the
regulation of biochemical and morphological changes associated with
cellular growth and division (D'Urso, G. et al. (1990) Science 250:
786-791; Birchmeier. C. et al. (1993) Bioassays 15: 185-189). They
serve as growth factor receptors and signal transducers and have
been implicated in cellular transformation and malignancy (Hunter,
T. et al. (1992) Cell 70: 375-387; Posada, J. et al. (1992) Mol.
Biol. Cell 3: 583-592; Hunter, T. et al. (1994) Cell 79: 573-582).
For example, protein kinases have been shown to participate in the
transmission of signals from growth-factor receptors (Sturgill, T.
W. et al. (1988) Nature 344: 715-718; Gomez, N. et al. (1991)
Nature 353: 170-173), control of entry of cells into mitosis
(Nurse, P. (1990) Nature 344: 503-508; Maller, J. L. (1991) Curr.
Opin. Cell Biol. 3: 269-275) and regulation of actin bundling
(Husain-Chishti, A. et al. (1988) Nature 334: 718-721).
[0003] Protein kinases and phosphatases can be divided into
different groups based on either amino acid sequence similarity or
specificity for either serine/threonine or tyrosine residues. A
small number of dual-specificity kinases and phosphatases have also
been described. Within the broad classification, kinases and
phosphatases can be further sub-divided into families whose members
share a higher degree of catalytic domain amino acid sequence
identity and also have similar biochemical properties. Most protein
kinase and phosphatase family members also share structural
features outside the kinase and phosphatase domain, respectively,
that reflect their particular cellular roles. These include
regulatory domains that control kinase or phosphatase activity or
interaction with other proteins (Hanks, S. K. et al. (1988) Science
241: 42-52).
SUMMARY OF THE INVENTION
[0004] The present invention is based, at least in part, on the
discovery of novel nucleic acid molecules and polypeptides encoded
by such nucleic acid molecules, referred to herein as "Kinases" and
"Phosphatases". The Kinase and Phosphatase nucleic acid and
polypeptide molecules of the present invention are useful as
modulating agents in regulating a variety of cellular processes.
Accordingly, in one aspect, this invention provides isolated
nucleic acid molecules encoding Kinase and Phosphatase
polypeptides, as well as nucleic acid fragments suitable as primers
or hybridization probes for the detection of Kinase- and
Phosphatase-encoding nucleic acids.
[0005] In one embodiment, a Kinase and a Phosphatase nucleic acid
molecule of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98% or more homologous to a nucleotide sequence (e.g., to
the entire length of the nucleotide sequence) including SEQ ID NO:
1-14, or a complement thereof.
[0006] In a preferred embodiment, the isolated nucleic acid
molecule includes the nucleotide sequence shown SEQ ID NO: 1-14, or
a complement thereof. In another preferred embodiment, an isolated
nucleic acid molecule of the invention encodes the amino acid
sequence of a human Kinase or Phosphatase polypeptide.
[0007] Another embodiment of the invention features nucleic acid
molecules, preferably Kinase and Phosphatase nucleic acid
molecules, which specifically detect Kinase and Phosphatase nucleic
acid molecules relative to nucleic acid molecules encoding
non-Kinase and non-Phosphatase polypeptides. For example, in one
embodiment, such a nucleic acid molecule is at least 20, 30, 40,
50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, or 800 nucleotides in length and hybridizes under
stringent conditions to a nucleic acid molecule comprising the
nucleotide sequence shown in SEQ ID NO: 1-14, or a complement
thereof.
[0008] In other preferred embodiments, the nucleic acid molecule
encodes a naturally occurring allelic variant of a human Kinase or
Phosphatase polypeptide, wherein the nucleic acid molecule
hybridizes to a nucleic acid molecule which includes SEQ ID NO: 1
-14 under stringent conditions.
[0009] Another embodiment of the invention provides an isolated
nucleic acid molecule which is antisense to a Kinase or a
Phosphatase nucleic acid molecule, e.g., the coding strand of a
Kinase or a Phosphatase nucleic acid molecule.
[0010] Another aspect of the invention provides a vector comprising
a Kinase or a Phosphatase nucleic acid molecule. In certain
embodiments, the vector is a recombinant expression vector. In
another embodiment, the invention provides a host cell containing a
vector of the invention. The invention also provides a method for
producing a polypeptide, preferably a Kinase or a Phosphatase
polypeptide, by culturing in a suitable medium, a host cell, e.g.,
a mammalian host cell such as a non-human mammalian cell, of the
invention containing a recombinant expression vector, such that the
polypeptide is produced.
[0011] Another aspect of this invention features isolated or
recombinant Kinase polypeptides and proteins. In one embodiment,
the isolated polypeptide, preferably a Kinase polypeptide, is a
eukaryotic protein kinase. Another aspect of this invention
features isolated or recombinant Phosphatase polypeptides and
proteins.
[0012] In a further embodiment, the isolated polypeptide,
preferably a Kinase or a Phosphatase polypeptide, plays a role in
signaling pathways associated with cellular growth, e.g., signaling
pathways associated with cell cycle regulation. In another
embodiment, the isolated polypeptide, preferably a Kinase or a
Phosphatase polypeptide is encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO: 1-14.
[0013] Another embodiment of the invention features an isolated
polypeptide, preferably a Kinase and a Phosphatase polypeptide,
which is encoded by a nucleic acid molecule having a nucleotide
sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98% or more homologous to a nucleotide sequence (e.g., to
the entire length of the nucleotide sequence) including SEQ ID NO:
1-14 or a complement thereof.
[0014] This invention further features an isolated polypeptide,
preferably a Kinase and a Phosphatase polypeptide, which is encoded
by a nucleic acid molecule having a nucleotide sequence which
hybridizes under stringent hybridization conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:
1-14, or a complement thereof.
[0015] In another aspect, the invention pertains to any individual
Kinase or Phosphatase nucleic acid molecule from the
above-identified group (SEQ ID NO: 1-14), as well as any subgroups
from within the above-identified group. Furthermore, the subgroups
can preferably consist of at least 1, 5, 10, or more members of the
group identified above. For example, the group consisting of the
Kinase and Phosphatase nucleic acid molecules of SEQ ID NO: 1-14
can be divided into one or more subgroups as follows: SEQ ID NO:
1-4, SEQ ID NO: 4-8, SEQ ID NO: 8-12, and SEQ ID NO: 12-14, or any
combinations thereof.
[0016] The polypeptides of the present invention can be operatively
linked to a non-Kinase and a non-Phosphatase polypeptide (e.g.,
heterologous amino acid sequences) to form fusion proteins. The
invention further features antibodies, such as monoclonal or
polyclonal antibodies, that specifically bind polypeptides of the
invention, preferably Kinase and Phosphatase polypeptides. In
addition, the Kinase and Phosphatase polypeptides, e.g.,
biologically active polypeptides, can be incorporated into
pharmaceutical compositions, which optionally include
pharmaceutically acceptable carriers.
[0017] In another aspect, the present invention provides a method
for detecting the presence of a Kinase and a Phosphatase nucleic
acid molecule, polypeptide or polypeptide in a biological sample by
contacting the biological sample with an agent capable of detecting
a Kinase and Phosphatase nucleic acid molecule, polypeptide or
polypeptide such that the presence of a Kinase and a Phosphatase
nucleic acid molecule, polypeptide or polypeptide is detected in
the biological sample.
[0018] In another aspect, the present invention provides a method
for detecting the presence of Kinase and Phosphatase activity in a
biological sample by contacting the biological sample with an agent
capable of detecting an indicator of Kinase and Phosphatase
activity such that the presence of Kinase and Phosphatase activity
is detected in the biological sample.
[0019] In another aspect, the invention provides a method for
modulating Kinase and Phosphatase activity comprising contacting a
cell capable of expressing Kinase and Phosphatase with an agent
that modulates Kinase and Phosphatase activity such that Kinase and
Phosphatase activity in the cell is modulated. In one embodiment,
the agent inhibits Kinase and Phosphatase activity. In another
embodiment, the agent stimulates Kinase and Phosphatase activity.
In one embodiment, the agent is an antibody that specifically binds
to a Kinase and Phosphatase polypeptide. In another embodiment, the
agent modulates expression of Kinase and Phosphatase by modulating
transcription of a Kinase and Phosphatase gene or translation of a
Kinase and Phosphatase mRNA. In yet another embodiment, the agent
is a nucleic acid molecule having a nucleotide sequence that is
antisense to the coding strand of a Kinase or Phosphatase mRNA or a
Kinase or Phosphatase gene.
[0020] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
Kinase or Phosphatase polypeptide or nucleic acid expression or
activity by administering an agent which is a Kinase or a
Phosphatase modulator to the subject. In one embodiment, the Kinase
and Phosphatase modulator is a Kinase and a Phosphatase
polypeptide, respectively. In another embodiment the Kinase and
Phosphatase modulator is a Kinase and Phosphatase nucleic acid
molecule, respectively. In yet another embodiment, the Kinase and
Phosphatase modulator is a peptide, peptidomimetic, or other small
molecule. In a preferred embodiment, the disorder characterized by
aberrant Kinase or Phosphatase polypeptide or nucleic acid
expression is a cellular growth related disorder, e.g., a
proliferative disorder such as cancer.
[0021] The present invention also provides a diagnostic assay for
identifying the presence or absence of a genetic alteration
characterized by at least one of (i) aberrant modification or
mutation of a gene encoding a Kinase or a Phosphatase polypeptide;
(ii) mis-regulation of the gene; and (iii) aberrant
post-translational modification of a Kinase or a Phosphatase
polypeptide, wherein a wild-type form of the gene encodes a
polypeptide with a Kinase or a Phosphatase activity.
[0022] In another aspect the invention provides a method for
identifying a compound that binds to or modulates the activity of a
Kinase or a Phosphatase polypeptide. The method includes providing
an indicator composition comprising a Kinase or a Phosphatase
polypeptide having Kinase or Phosphatase activity, respectively,
contacting the indicator composition with a test compound, and
determining the effect of the test compound on Kinase or
Phosphatase activity in the indicator composition to identify a
compound that modulates the activity of a Kinase or a Phosphatase
polypeptide.
[0023] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 depicts a human cDNA sequence designated 14803C1 (SEQ
ID NO: 1).
[0025] FIG. 2 depicts a human CDNA sequence designated 16328S1 (SEQ
ID NO: 2).
[0026] FIG. 3 depicts a human cDNA sequence designated 16328S2 (SEQ
ID NO: 3).
[0027] FIG. 4 depicts a human CDNA sequence designated 16358C1 (SEQ
ID NO: 4).
[0028] FIG. 5 depicts a human cDNA sequence designated 16676S1 (SEQ
ID NO: 5).
[0029] FIG. 6 depicts a human CDNA sequence designated 16692C1 (SEQ
ID NO: 6).
[0030] FIG. 7 depicts a human cDNA sequence designated 16692C2 (SEQ
ID NO: 7).
[0031] FIG. 8 depicts a human cDNA sequence designated 23552C2 (SEQ
ID NO: 8).
[0032] FIG. 9 depicts a human cDNA sequence designated 23552C2 (SEQ
ID NO: 9).
[0033] FIG. 10 depicts a human cDNA sequence designated 32641S1
(SEQ ID NO: 10).
[0034] FIG. 11 depicts a human cDNA sequence designated 42960C1
(SEQ ID NO: 11).
[0035] FIG. 12 depicts a human cDNA sequence designated 43043C1
(SEQ ID NO: 12).
[0036] FIG. 13 depicts a human cDNA sequence designated 42957C1
(SEQ ID NO: 13).
[0037] FIG. 14 depicts a human cDNA sequence designated 42958S1
(SEQ ID NO: 14).
1 Detailed Description of the Invention I. Isolated Nucleic Acid
Molecules -10- II. Isolated Kinase and Phosphatase Proteins and
Anti-Kinase -19- and Anti-Phosphatase Antibodies III. Recombinant
Expression Vectors and Host Cells -29- IV. Pharmaceutical
Compositions -37- V. Uses and Methods of the Invention -40- A.
Screening Assays -41- B. Detection Assays -47- 1. Chromosome
Mapping -48- 2. Tissue Typing -50- 3. Use of Partial Kinase and
Phosphatase Sequences -51- in Forensic Biology C. Predictive
Medicine -51- 1. Diagnostic Assays -52- 2. Prognostic Assays -54-
3. Monitoring of Effects During Clinical Trials -59- D. Methods of
Treatment -60- 1. Prophylactic Methods -60- 2. Therapeutic Methods
-61- 3. Pharmacogenomics -62-
[0038] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as "Kinase" and
"Phosphatase" nucleic acid and polypeptide molecules, which play a
role in or function in signaling pathways associated with cellular
growth and/or cellular metabolic pathways. These growth and
metabolic pathways are described in Lodish H. et al. Molecular Cell
Biology, (Scientific American Books Inc., New York, N.Y., 1995) and
Stryer L., Biochemistry, (W. H. Freeman, New York), the contents of
which are incorporated herein by reference. In one embodiment, the
Kinase and Phosphatase molecules modulate the activity of one or
more proteins involved in cellular growth or differentiation, e.g.,
cardiac, epithelial, or neuronal cell growth or differentiation. In
another embodiment, the Kinase and Phosphatase molecules of the
present invention are capable of modulating the phosphorylation
state of a Kinase or Phosphatase molecule or the phosphorylation
state of one or more proteins involved in cellular growth or
differentiation, e.g., cardiac, epithelial, or neuronal cell growth
or differentiation, as described in, for example, Lodish H. et al.
Molecular Cell Biology, (Scientific American Books Inc., New York,
N.Y., 1995) and Stryer L., Biochemistry, (W. H. Freeman, New York),
the contents of which are incorporated herein by reference. In
addition, Kinases and Phosphatases of the present invention are
targets of drugs described in Goodman and Gilman, The
Pharmacological Basis of Therapeutics (9.sup.th Edition) (Hartman
& Limbard Editors, 1996), the contents of which are
incorporated herein by reference.
[0039] As used herein, the term "Kinase" includes a protein,
polypeptide, or other non-proteinaceous molecule which is capable
of modulating its own phosphorylation state or the phosphorylation
state of a different protein, polypeptide, or other
non-proteinaceous molecule. Kinases can have a specificity for
(i.e., a specificity to phosphorylate) serine/threonine residues,
tyrosine residues, or both serine/threonine and tyrosine residues,
e.g., the dual specificity kinases. As referred to herein, Kinases
such as protein Kinases, preferably include a catalytic domain of
about 200-400 amino acid residues in length, preferably about
200-300 amino acid residues in length, or more preferably about
250-300 amino acid residues in length, which includes preferably
5-20, more preferably 5-15, or preferably 11 highly conserved
motifs or subdomains separated by sequences of amino acids with
reduced or minimal conservation. Specificity of a Kinase for
phosphorylation of either tyrosine or serine/threonine can be
predicted by the sequence of two of the subdomains (VIb and VIII)
in which different residues are conserved in each class (as
described in, for example, Hanks et al. (1988) Science 241:42-52)
the contents of which are incorporated herein by reference). These
subdomains are also described in further detail herein.
[0040] As used herein, the term "Phosphatase" includes a protein or
polypeptide, e.g., an enzyme, or another non-proteinaceous molecule
which is capable of facilitating, e.g., catalyzing, the removal of
a phosphate group from, for example, a protein or polypeptide which
is phosphorylated. Phosphatases can have a specificity for (i.e. a
specificity to dephosphorylate) serine/threonine residues, tyrosine
residues, or both serine/threonine and tyrosine residues. As
referred to herein, a Phosphatase such as a protein Phosphatase,
can preferably include a catalytic domain of at least about 200-400
amino acid residues in length, preferably about 200-300 amino acid
residues in length, and more preferably about 250-300 amino acid
residues in length, which includes preferably 2-20, more preferably
2-15, or preferably 2-8 highly conserved motifs or subdomains
separated by sequences of amino acids with reduced or minimal
conservation. Phosphatases can be either soluble or membrane bound
(see e.g., Brautigan et al. (1992) Biochem. biophys. Acta,
1114:63-77; Charbonneau et al. (1992) Ann. Rev. Cell Biol., 8:
463-493; Fisher et al. (1991) Science, 253:401-406; and Hunter et
al. (1989) Cell, 58:1013-1016). Membrane bound Phosphatases
typically contain receptor-like extracellular regions connected to
the intracellular (catalytic) domains by a short transmembrane
segment (Streuli and Saito, (1993) Adv. Prot. Phosphatases
7:67-94). The non-transmembrane (cytoplasmic) Phosphatases
typically include at least one catalytic domain (Koch et al.,
(1991) Science 252:668-674).
[0041] Kinases and Phosphatases play a role in signaling pathways
associated with cellular growth. For example, protein Kinases and
protein Phosphatases are involved in the regulation of signal
transmission from cellular receptors, e.g., growth-factor
receptors; entry of cells into mitosis; and the regulation of
cytoskeleton function, e.g., actin bundling. Thus, the Kinase and
Phosphatase molecules of the present invention may be involved in:
1) the regulation of transmission of signals from cellular
receptors, e.g., growth factor receptors; 2) the modulation of the
entry of cells into mitosis; 3) the modulation of cellular
differentiation; 4) the modulation of cell death; and 5) the
regulation of cytoskeleton function, e.g., actin bundling.
[0042] Inhibition or over stimulation of the activity of Kinases
and Phosphatases involved in signaling pathways associated with
cellular growth can lead to perturbed cellular growth, which can in
turn lead to cellular growth related disorders. As used herein, a
"cellular growth related disorder" includes a disorder, disease, or
condition characterized by a deregulation, e.g., an upregulation or
a downregulation, of cellular growth. Cellular growth deregulation
may be due to a deregulation of cellular proliferation, cell cycle
progression, cellular differentiation and/or cellular hypertrophy.
Examples of cellular growth related disorders include
cardiovascular disorders such as heart failure, hypertension,
atrial fibrillation, dilated cardiomyopathy, idiopathic
cardiomyopathy, or angina; proliferative disorders or
differentiative disorders such as cancer, e.g., melanoma, prostate
cancer, cervical cancer, breast cancer, colon cancer, or
sarcoma.
[0043] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as Kinase and
Phosphatase protein and nucleic acid molecules, which comprise a
family of molecules having certain conserved structural and
functional features. The term "family" when referring to the
protein and nucleic acid molecules of the invention is intended to
mean two or more proteins or nucleic acid molecules having a common
structural domain or motif and having sufficient amino acid or
nucleotide sequence homology as defined herein. Such family members
can be naturally or non-naturally occurring and can be from either
the same or different species. For example, a family can contain a
first protein of human origin, as well as other, distinct proteins
of human origin or alternatively, can contain homologues of
non-human origin. Members of a family may also have common
functional characteristics.
[0044] One embodiment of the invention features Kinase and
Phosphatase nucleic acid molecules, preferably human Kinase and
Phosphatase molecules, which were identified based on a consensus
motif or protein domain characteristic of a Kinase or Phosphatase
family of proteins. (Such families are described below). The Kinase
and Phosphatase nucleic acid and polypeptide molecules of the
invention are described in further detail in the following
subsections.
[0045] A. The Eucaryotic Protein Kinase Nucleic Acid and
Polypeptide Molecules
[0046] In one embodiment, the isolated nucleic acid molecules of
the present invention encode eukaryotic protein kinase
polypeptides. Eukaryotic protein kinases (described in, for
example, Hanks S. K. et al. (1995) FASEB J. 9:576-596) are enzymes
that belong to an extensive family of proteins which share a
conserved catalytic core common to both serine/threonine and
tyrosine protein kinases. There are a number of conserved regions
in the catalytic domain of protein kinases. One of this regions,
located in the N-terminal extremity of the catalytic domain, is a
glycine-rich stretch of residues in the vicinity of a lysine
residue, which has been shown to be involved in ATP binding.
Another region, located in the central part of the catalytic
domain, contains a conserved aspartic acid residue which is
important for the catalytic activity of the enzyme (Knighton D. R.
et al. (1991) Science 253:407-414). Two signature patterns have
been described for this region: one specific for serine/ threonine
kinases and one for tyrosine kinases.
[0047] Eukaryotic protein kinase polypeptides of the present
invention preferably include one of the following consensus
sequences:
2 (SEQ ID NO:15) [LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}-
-[LIVCAT]- {PD}-x [GSTACLIVMFY]-x(5,18)-[LIVMFYWC-
STAR]-[AIVP]-[LIVMF AGCKR]-K [K binds ATP]
[LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x(2)-N-[LIVMFYCT](3) (SEQ ID NO:
16) [D is an active site residue]
[LIVMFYC]-x-[HY]-x-D-[LIVMFY]-[RSTAC]-x(2)-N-[LIVMFYC](3) (SEQ ID
NO: 17) [D is an active site residue]
[0048] SEQ ID NOs: 1-8, 10-12, 13, and 14, shown in FIGS. 1-8,
10-12, 13, and 14, respectively, are part of cDNAs that encode
eukaryotic protein kinase polypeptides.
[0049] B. The Adenylate Kinase Nucleic Acid and Polypeptide
Molecules
[0050] In one embodiment, the isolated nucleic acid molecules of
the present invention encode adenylate kinase polypeptides.
Adenylate kinase (AK) (described in Schulz G. E. (1987) Cold Spring
Harbor Symp. Quant. Biol. 52:429-439) is a monomeric enzyme that
catalyzes the reversible transfer of MgATP to AMP
(MgATP+AMP=MgADP+ADP).
[0051] In mammals there are three different isozymes AK1 (or
myokinase) which is cytosolic; AK2, which is located in the outer
compartment of mitochondria; and AK3 (or GTP:AMP
phosphotransferase), which is located in the mitochondrial matrix
and which uses MgGTP instead of MgATP.
[0052] Several regions of AK family enzymes are well conserved,
including the ATP-binding domains. This region includes an aspartic
acid residue that is part of the catalytic cleft of the enzyme and
is involved in a salt bridge.
[0053] It also includes an arginine residue whose modification
leads to inactivation of the enzyme. Adenylate kinase polypeptides
of the present invention preferably include the following consensus
sequence:
[LIVMFYW](3)-D-G-[FYI]-P-R-x(3)-[NQ](SEQ ID NO: 18)
[0054] SEQ ID NO: 9 shown in FIG. 9 is part of a cDNA that encodes
an adenylate kinase polypeptide.
[0055] Various aspects of the invention are described in further
detail in the following subsections:
[0056] I. Isolated Nucleic Acid Molecules
[0057] One aspect of the invention pertains to isolated nucleic
acid molecules that encode Kinase and Phosphatase proteins or
biologically active portions thereof, as well as nucleic acid
fragments sufficient for use as hybridization probes to identify
Kinase and Phosphatase-encoding nucleic acids (e.g., Kinase and
Phosphatase mRNA) and fragments for use as PCR primers for the
amplification or mutation of Kinase and Phosphatase nucleic acid
molecules. As used herein, the term "nucleic acid molecule" is
intended to include DNA molecules (e.g., cDNA or genomic DNA) and
RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0058] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid. For example, with regards
to genomic DNA, the term "isolated" includes nucleic acid molecules
which are separated from the chromosome with which the genomic DNA
is naturally associated. Preferably, an "isolated" nucleic acid is
free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
For example, in various embodiments, the isolated Kinase and
Phosphatase nucleic acid molecule can contain less than about 5 kb,
4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences
which naturally flank the nucleic acid molecule in genomic DNA of
the cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0059] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:
1-14, or a portion thereof, can be isolated using standard
molecular biology techniques and the sequence information provided
herein. For example, using all or portion of the nucleic acid
sequence of SEQ ID NO: 1-14, as a hybridization probe, Kinase and
Phosphatase nucleic acid molecules can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0060] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO: 1-14 can be isolated by the polymerase chain
reaction (PCR) using synthetic oligonucleotide primers designed
based upon the sequence of SEQ ID NO: 1-14, respectively.
[0061] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to Kinase and
Phosphatase nucleotide sequences can be prepared by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
[0062] In a preferred embodiment, an isolated nucleic acid molecule
of the invention comprises the nucleotide sequence shown in SEQ ID
NO: 1-14.
[0063] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
is a complement of the nucleotide sequence shown in SEQ ID NO:
1-14, or a portion of any of these nucleotide sequences. A nucleic
acid molecule which is complementary to the nucleotide sequence
shown in SEQ ID NO: 1-14, is one which is sufficiently
complementary to the nucleotide sequence shown in SEQ ID NO: 1-14,
respectively, such that it can hybridize to the nucleotide sequence
shown in SEQ ID NO: 1-14, respectively, thereby forming a stable
duplex.
[0064] In still another preferred embodiment, an isolated nucleic
acid molecule of the present invention comprises a nucleotide
sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 98% or more homologous to the nucleotide sequence
(e.g., to the entire length of the nucleotide sequence) shown in
SEQ ID NO: 1-14, or a portion of any of these nucleotide
sequences.
[0065] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the nucleic acid sequence of SEQ ID NO:
1-14, for example a fragment which can be used as a probe or primer
or a fragment encoding a biologically active portion of a Kinase
and Phosphatase protein. The nucleotide sequence determined from
the cloning of the Kinase and Phosphatase gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning other Kinase and Phosphatase family members, as well
as Kinase and Phosphatase homologues from other species. The
probe/primer typically comprises substantially purified
oligonucleotide The oligonucleotide typically comprises a region of
nucleotide sequence that hybridizes under stringent conditions to
at least about 12 or 15, preferably about 20 or 25, more preferably
about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides
of a sense sequence of SEQ ID NO: 1- 14, or of a naturally
occurring allelic variant or mutant of SEQ ID NO: 1-14. In an
exemplary embodiment, a nucleic acid molecule of the present
invention comprises a nucleotide sequence which is at least 100,
150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or
800 nucleotides in length and hybridizes under stringent
hybridization conditions to a nucleic acid molecule of SEQ ID NO:
1-14.
[0066] Probes based on the Kinase and Phosphatase nucleotide
sequences can be used to detect transcripts or genomic sequences
encoding the same or homologous proteins. In preferred embodiments,
the probe further comprises a label group attached thereto, e.g.,
the label group can be a radioisotope, a fluorescent compound, an
enzyme, or an enzyme co-factor. Such probes can be used as a part
of a diagnostic test kit for identifying cells or tissues which
misexpress a Kinase and Phosphatase protein, such as by measuring a
level of a Kinase and Phosphatase-encoding nucleic acid in a sample
of cells from a subject e.g., detecting Kinase and Phosphatase mRNA
levels or determining whether a genomic Kinase and Phosphatase gene
has been mutated or deleted.
[0067] A nucleic acid fragment encoding a "biologically active
portion of a Kinase and Phosphatase protein" can be prepared by
isolating a portion of the nucleotide sequence of SEQ ID NO: 1-14,
which encodes a polypeptide having a Kinase and Phosphatase
biological activity (the biological activities of the Kinase and
Phosphatase proteins are described herein), expressing the encoded
portion of the Kinase and Phosphatase protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the Kinase and Phosphatase protein.
[0068] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO: 1-14,
due to the degeneracy of the genetic code and, thus, encode the
same Kinase and Phosphatase proteins as those encoded by the
nucleotide sequence shown in SEQ ID NO: 1-14. In another
embodiment, an isolated nucleic acid molecule of the invention has
a nucleotide sequence encoding a Kinase or Phosphatase protein.
[0069] In addition to the Kinase and Phosphatase nucleotide
sequences shown in SEQ ID NO: 1-14, it will be appreciated by those
skilled in the art that DNA sequence polymorphisms that lead to
changes in the amino acid sequences of the Kinase and Phosphatase
proteins may exist within a population (e.g., the human
population). Such genetic polymorphism in the Kinase and
Phosphatase genes may exist among individuals within a population
due to natural allelic variation. As used herein, the terms "gene"
and "recombinant gene" refer to nucleic acid molecules which
include an open reading frame encoding an Kinase and Phosphatase
protein, preferably a mammalian Kinase and Phosphatase protein, and
can further include non-coding regulatory sequences, and introns.
Such natural allelic variations include both functional and
non-functional Kinase and Phosphatase proteins and can typically
result in 1-5% variance in the nucleotide sequence of a Kinase and
Phosphatase gene. Any and all such nucleotide variations and
resulting amino acid polymorphisms in Kinase and Phosphatase genes
that are the result of natural allelic variation and that do not
alter the functional activity of a Kinase and Phosphatase protein
are intended to be within the scope of the invention.
[0070] Moreover, nucleic acid molecules encoding other Kinase and
Phosphatase family members and, thus, which have a nucleotide
sequence which differs from the Kinase and Phosphatase sequences of
SEQ ID NO: 1-14 are intended to be within the scope of the
invention. For example, another Kinase and Phosphatase cDNA can be
identified based on the nucleotide sequence of human Kinase and
Phosphatase. Moreover, nucleic acid molecules encoding Kinase and
Phosphatase proteins from different species, and thus which have a
nucleotide sequence which differs from the Kinase and Phosphatase
sequences of SEQ ID NO: 1-14 are intended to be within the scope of
the invention. For example, a mouse Kinase and Phosphatase cDNA can
be identified based on the nucleotide sequence of a human Kinase
and Phosphatase.
[0071] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the Kinase and Phosphatase cDNAs of the
invention can be isolated based on their homology to the Kinase and
Phosphatase nucleic acids disclosed herein using the cDNAs
disclosed herein, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions.
[0072] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 15, 20, 25, 30 or more
nucleotides in length and hybridizes under stringent conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO: 1-14. In other embodiment, the nucleic acid is at least 30,
50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600
nucleotides in length. As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
30%, 40%, 50%, or 60% homologous to each other typically remain
hybridized to each other. Preferably, the conditions are such that
sequences at least about 70%, more preferably at least about 80%,
even more preferably at least about 85% or 90% homologous to each
other typically remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found
in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. Preferably, an isolated nucleic acid molecule of
the invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO: 1-14 corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein).
[0073] In addition to naturally-occurring allelic variants of the
Kinase and Phosphatase sequences that may exist in the population,
the skilled artisan will further appreciate that changes can be
introduced by mutation into the nucleotide sequences of SEQ ID NO:
1-14, thereby leading to changes in the amino acid sequence of the
encoded Kinase and Phosphatase proteins, without altering the
functional ability of the Kinase and Phosphatase proteins. For
example, nucleotide substitutions leading to amino acid
substitutions at "non-essential" amino acid residues can be made in
the sequence of a Kinase or Phosphatase protein. A "non-essential"
amino acid residue is a residue that can be altered from the
wild-type sequence of Kinase and Phosphatase without altering the
biological activity, whereas an "essential" amino acid residue is
required for biological activity: For example, amino acid residues
that are conserved among the Kinase and Phosphatase proteins of the
present invention, are predicted to be particularly unamenable to
alteration. Furthermore, additional amino acid residues that are
conserved between the Kinase and Phosphatase proteins of the
present invention and other Kinase and Phosphatase family members
are not likely to be amenable to alteration.
[0074] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding Kinase and Phosphatase proteins
that contain changes in amino acid residues that are not essential
for activity.
[0075] An isolated nucleic acid molecule encoding a Kinase and
Phosphatase protein homologous to the Kinase and Phosphatase
proteins of the present invention can be created by introducing one
or more nucleotide substitutions, additions or deletions into the
nucleotide sequence of SEQ ID NO: 1-14, such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into SEQ ID NO: 1-14
by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis. Preferably, conservative amino acid
substitutions are made at one or more predicted non-essential amino
acid residues. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a Kinase and
Phosphatase protein is preferably replaced with another amino acid
residue from the same side chain family. Alternatively, in another
embodiment, mutations can be introduced randomly along all or part
of a Kinase and Phosphatase coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for Kinase
and Phosphatase biological activity to identify mutants that retain
activity. Following mutagenesis of SEQ ID NO: 1-14, the encoded
protein can be expressed recombinantly and the activity of the
protein can be determined.
[0076] In a preferred embodiment, a mutant Kinase and Phosphatase
protein can be assayed for the ability to: 1) regulate trasmission
of signals from cellular receptors, e.g., growth factor receptors;
2) control entry of cells, e.g., epithelial cells, into mitosis; 3)
modulate cellular differentiation; 4) modulate cell death; or 5)
regulate cytoskeleton function, e.g., actin bundling.
[0077] In addition to the nucleic acid molecules encoding Kinase
and Phosphatase proteins described above, another aspect of the
invention pertains to isolated nucleic acid molecules which are
antisense thereto. An "antisense" nucleic acid comprises a
nucleotide sequence which is complementary to a "sense" nucleic
acid encoding a protein, e.g., complementary to the coding strand
of a double-stranded cDNA molecule or complementary to an mRNA
sequence. Accordingly, an antisense nucleic acid can hydrogen bond
to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire Kinase and Phosphatase coding strand, or
only to a portion thereof. In one embodiment, an antisense nucleic
acid molecule is antisense to a "coding region" of the coding
strand of a nucleotide sequence encoding Kinase and Phosphatase.
The term "coding region" refers to the region of the nucleotide
sequence comprising codons which are translated into amino acid
residues. In another embodiment, the antisense nucleic acid
molecule is antisense to a "noncoding region" of the coding strand
of a nucleotide sequence encoding Kinase and Phosphatase. The term
"noncoding region" refers to 5' and 3' sequences which flank the
coding region that are not translated into amino acids (i.e., also
referred to as 5' and 3' untranslated regions).
[0078] Given the coding strand sequences encoding Kinase and
Phosphatase disclosed herein, antisense nucleic acids of the
invention can be designed according to the rules of Watson and
Crick base pairing. The antisense nucleic acid molecule can be
complementary to the entire coding region of Kinase and Phosphatase
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of Kinase and
Phosphatase mRNA. For example, the antisense oligonucleotide can be
complementary to the region surrounding the translation start site
of Kinase and Phosphatase mRNA. An antisense oligonucleotide can
be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50
nucleotides in length. An antisense nucleic acid of the invention
can be constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomet- hyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1
-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0079] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a Kinase and Phosphatase protein to thereby inhibit
expression of the protein, e.g., by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the
case of an antisense nucleic acid molecule which binds to DNA
duplexes, through specific interactions in the major groove of the
double helix. An example of a route of administration of antisense
nucleic acid molecules of the invention include direct injection at
a tissue site. Alternatively, antisense nucleic acid molecules can
be modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[0080] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0081] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave Kinase and Phosphatase MRNA
transcripts to thereby inhibit translation of Kinase and
Phosphatase mRNA. A ribozyme having specificity for a Kinase and
Phosphatase-encoding nucleic acid can be designed based upon the
nucleotide sequence of a Kinase and Phosphatase cDNA disclosed
herein (i.e., SEQ ID NO: 1-14). For example, a derivative of a
Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide
sequence of the active site is complementary to the nucleotide
sequence to be cleaved in a Kinase and Phosphatase-encoding mRNA.
See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.
U.S. Pat. No. 5,116,742. Alternatively, Kinase and Phosphatase mRNA
can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.
[0082] Alternatively, Kinase and Phosphatase gene expression can be
inhibited by targeting nucleotide sequences complementary to the
regulatory region of the Kinase and Phosphatase (e.g., the Kinase
and Phosphatase promoter and/or enhancers) to form triple helical
structures that prevent transcription of the Kinase and Phosphatase
gene in target cells. See generally, Helene, C. (1991) Anticancer
Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad.
Sci. 660:27-36; and Maher, L. J. (1992) Bioassays
14(12):807-15.
[0083] In yet another embodiment, the Kinase and Phosphatase
nucleic acid molecules of the present invention can be modified at
the base moiety, sugar moiety or phosphate backbone to improve,
e.g., the stability, hybridization, or solubility of the molecule.
For example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):
5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g.; DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl.
Acad. Sci. 93: 14670-675.
[0084] PNAs of Kinase and Phosphatase nucleic acid molecules can be
used in therapeutic and diagnostic applications. For example, PNAs
can be used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of Kinase and Phosphatase nucleic acid molecules can also be used
in the analysis of single base pair mutations in a gene, (e.g., by
PNA-directed PCR clamping); as `artificial restriction enzymes`
when used in combination with other enzymes, (e.g., S1 nucleases
(Hyrup B. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. (1996) supra;.
Perry-O'Keefe supra).
[0085] In another embodiment, PNAs of Kinase and Phosphatase can be
modified, (e.g., to enhance their stability or cellular uptake), by
attaching lipophilic or other helper groups to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques of drug delivery known in the art. For example, PNA-DNA
chimeras of Kinase and Phosphatase nucleic acid molecules can be
generated which may combine the advantageous properties of PNA and
DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H
and DNA polymerases), to interact with the DNA portion while the
PNA portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis
of PNA-DNA chimeras can be performed as described in Hyrup B.
(1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res.
24(17): 335763. For example, a DNA chain can be synthesized on a
solid support using standard phosphoramidite coupling chemistry and
modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used as a between the PNA and the 5' end of DNA (Mag, M. et al.
(1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment (Finn P. J. et al. (1996)
supra). Alternatively, chimeric molecules can be synthesized with a
5' DNA segment and a 3' PNA segment (Peterser, K. H. et al. (1975)
Bioorganic Med. Chem. Lett. 5: 1119-11124).
[0086] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. W088/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.
(1988) Bio-Techniques 6:958-976) or intercalating agents. (See,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the
oligonucleotide may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0087] II. Isolated Kinase and Phosphatase Proteins and Anti-Kinase
and Anti-Phosphatase Antibodies
[0088] One aspect of the invention pertains to isolated Kinase and
Phosphatase proteins, and biologically active portions thereof, as
well as polypeptide fragments suitable for use as immunogens to
raise anti-Kinase and Phosphatase antibodies. In one embodiment,
native Kinase and Phosphatase proteins can be isolated from cells
or tissue sources by an appropriate purification scheme using
standard protein purification techniques. In another embodiment,
Kinase and Phosphatase proteins are produced by recombinant DNA
techniques. Alternative to recombinant expression, a Kinase and
Phosphatase protein or polypeptide can be synthesized chemically
using standard peptide synthesis techniques.
[0089] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the Kinase and Phosphatase protein is derived, or substantially
free from chemical precursors or other chemicals when chemically
synthesized. The language "substantially free of cellular material"
includes preparations of Kinase and Phosphatase protein in which
the protein is separated from cellular components of the cells from
which it is isolated or recombinantly produced. In one embodiment,
the language "substantially free of cellular material" includes
preparations of Kinase and Phosphatase protein having less than
about 30% (by dry weight) of non-Kinase and Phosphatase protein
(also referred to herein as a "contaminating protein"), more
preferably less than about 20% of non-Kinase and Phosphatase
protein, still more preferably less than about 10% of non-Kinase
and Phosphatase protein, and most preferably less than about 5%
non-Kinase and Phosphatase protein. When the Kinase and Phosphatase
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, more
preferably less than about 10%, and most preferably less than about
5% of the volume of the protein preparation.
[0090] The language "substantially free of chemical precursors or
other chemicals" includes preparations of Kinase and Phosphatase
protein in which the protein is separated from chemical precursors
or other chemicals which are involved in the synthesis of the
protein. In one embodiment, the language "substantially free of
chemical precursors or other chemicals" includes preparations of
Kinase and Phosphatase protein having less than about 30% (by dry
weight) of chemical precursors or non-Kinase and Phosphatase
chemicals, more preferably less than about 20% chemical precursors
or non-Kinase and Phosphatase chemicals, still more preferably less
than about 10% chemical precursors or non-Kinase and Phosphatase
chemicals, and most preferably less than about 5% chemical
precursors or non-Kinase and Phosphatase chemicals.
[0091] Biologically active portions of a Kinase and Phosphatase
protein include peptides comprising amino acid sequences
sufficiently homologous to or derived from the amino acid sequence
of the Kinase and Phosphatase protein, which include less amino
acids than the full length Kinase and Phosphatase proteins, and
exhibit at least one activity of a Kinase and Phosphatase protein.
Typically, biologically active portions comprise a domain or motif
with at least one activity of the Kinase and Phosphatase protein. A
biologically active portion of a Kinase and Phosphatase protein can
be a polypeptide which is, for example, at least 10, 25, 50, 100 or
more amino acids in length.
[0092] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence. The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position (as used herein amino acid or nucleic acid "identity" is
equivalent to amino acid or nucleic acid "homology"). The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences, taking into account
the number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences.
[0093] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blosum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1,2,3,4,5,or6.
[0094] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to Kinase and Phosphatase
nucleic acid molecules of the invention. BLAST protein searches can
be performed with the XBLAST program, score=50, wordlength=3 to
obtain amino acid sequences homologous to Kinase and Phosphatase
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) can be used.
See http://www.ncbi.nlm.nih.gov.
[0095] The invention also provides Kinase and Phosphatase chimeric
or fusion proteins. As used herein, a Kinase and Phosphatase
"chimeric protein" or "fusion protein" comprises a Kinase and
Phosphatase polypeptide operatively linked to a non-Kinase and
Phosphatase polypeptide. An "Kinase and Phosphatase polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to Kinase and Phosphatase, whereas a "non-Kinase and Phosphatase
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
the Kinase and Phosphatase protein, e.g., a protein which is
different from the Kinase and Phosphatase protein and which is
derived from the same or a different organism. Within a Kinase and
Phosphatase fusion protein the Kinase and Phosphatase polypeptide
can correspond to all or a portion of a Kinase and Phosphatase
protein. In a preferred embodiment, a Kinase and Phosphatase fusion
protein comprises at least one biologically active portion of a
Kinase and Phosphatase protein. In another preferred embodiment, a
Kinase and Phosphatase fusion protein comprises at least two
biologically active portions of a Kinase and Phosphatase protein.
Within the fusion protein, the term "operatively linked" is
intended to indicate that the Kinase and Phosphatase polypeptide
and the non-Kinase and Phosphatase polypeptide are fused in-frame
to each other. The non-Kinase and Phosphatase polypeptide can be
fused to the N-terminus or C-terminus of the Kinase and Phosphatase
polypeptide.
[0096] For example, in one embodiment, the fusion protein is a
GST-Kinase and Phosphatase fusion protein in which the Kinase and
Phosphatase sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of
recombinant Kinase and Phosphatase.
[0097] In another embodiment, the fusion protein is a Kinase and
Phosphatase protein containing a heterologous signal sequence at
its N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of Kinase and Phosphatase can be
increased through use of a heterologous signal sequence.
[0098] The Kinase and Phosphatase fusion proteins of the invention
can be incorporated into pharmaceutical compositions and
administered to a subject in vivo. The Kinase and Phosphatase
fusion proteins can be used to affect the bioavailability of a
Kinase and Phosphatase substrate. Use of Kinase and Phosphatase
fusion proteins may be useful therapeutically for the treatment of
cellular growth related disorders, e.g., cancer. Moreover, the
Kinase and Phosphatase-fusion proteins of the invention can be used
as immunogens to produce anti-Kinase and Phosphatase antibodies in
a subject, to purify Kinase and Phosphatase ligands and in
screening assays to identify molecules which inhibit the
interaction of Kinase and Phosphatase with a Kinase and Phosphatase
substrate.
[0099] Preferably, a Kinase and Phosphatase chimeric or fusion
protein of the invention is produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance
with conventional techniques, for example by employing blunt-ended
or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A Kinase and Phosphatase-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the Kinase and Phosphatase
protein.
[0100] The present invention also pertains to variants of the
Kinase and Phosphatase proteins which function as either Kinase and
Phosphatase agonists (mimetics) or as Kinase and Phosphatase
antagonists. Variants of the Kinase and Phosphatase proteins can be
generated by mutagenesis, e.g., discrete point mutation or
truncation of a Kinase and Phosphatase protein. An agonist of the
Kinase and Phosphatase proteins can retain substantially the same,
or a subset, of the biological activities of the naturally
occurring form of a Kinase and Phosphatase protein. An antagonist
of a Kinase and Phosphatase protein can inhibit one or more of the
activities of the naturally occurring form of the Kinase and
Phosphatase protein by, for example, competitively modulating a
cellular activity of a Kinase and Phosphatase protein. Thus,
specific biological effects can be elicited by treatment with a
variant of limited function. In one embodiment, treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein has fewer side
effects in a subject relative to treatment with the naturally
occurring form of the Kinase and Phosphatase protein.
[0101] In one embodiment, variants of a Kinase and Phosphatase
protein which function as either Kinase and Phosphatase agonists
(mimetics) or as Kinase and Phosphatase antagonists can be
identified by screening combinatorial libraries of mutants, e.g.,
truncation mutants, of a Kinase and Phosphatase protein for Kinase
and Phosphatase protein agonist or antagonist activity. In one
embodiment, a variegated library of Kinase and Phosphatase variants
is generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of Kinase and Phosphatase variants can be produced by, for example,
enzymatically ligating a mixture of synthetic oligonucleotides into
gene sequences such that a degenerate set of potential Kinase and
Phosphatase sequences is expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage
display) containing the set of Kinase and Phosphatase sequences
therein. There are a variety of methods which can be used to
produce libraries of potential Kinase and Phosphatase variants from
a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential Kinase and Phosphatase
sequences. Methods for synthesizing degenerate oligonucleotides are
known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al.
(1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.
11:477.
[0102] In addition, libraries of fragments of a Kinase and
Phosphatase protein coding sequence can be used to generate a
variegated population of Kinase and Phosphatase fragments for
screening and subsequent selection of variants of a Kinase and
Phosphatase protein. In one embodiment, a library of coding
sequence fragments can be generated by treating a double stranded
PCR fragment of a Kinase and Phosphatase coding sequence with a
nuclease under conditions wherein nicking occurs only about once
per molecule, denaturing the double stranded DNA, renaturing the
DNA to form double stranded DNA which can include sense/antisense
pairs from different nicked products, removing single stranded
portions from reformed duplexes by treatment with S1 nuclease, and
ligating the resulting fragment library into an expression vector.
By this method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the Kinase and Phosphatase protein.
[0103] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of Kinase and Phosphatase proteins. The most widely
used techniques, which are amenable to high through-put analysis,
for screening large gene libraries typically include cloning the
gene library into replicable expression vectors, transforming
appropriate cells with the resulting library of vectors, and
expressing the combinatorial genes under conditions in which
detection of a desired activity facilitates isolation of the vector
encoding the gene whose product was detected. Recrusive ensemble
mutagenesis (REM), a new technique which enhances the frequency of
functional mutants in the libraries, can be used in combination
with the screening assays to identify Kinase and Phosphatase
variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA
89:7811-7815; Delgrave et al. (1993) Protein Engineering
6(3):327-33 1).
[0104] In one embodiment, cell based assays can be exploited to
analyze a variegated Kinase and Phosphatase library. For example, a
library of expression vectors can be transfected into a cell line
which ordinarily synthesizes and secretes Kinase and Phosphatase.
The transfected cells are then cultured such that Kinase and
Phosphatase and a particular mutant Kinase and Phosphatase are
secreted and the effect of expression of the mutant on Kinase and
Phosphatase activity in cell supernatants can be detected, e.g., by
any of a number of enzymatic assays. Plasmid DNA can then be
recovered from the cells which score for inhibition, or
alternatively, potentiation of Kinase and Phosphatase activity, and
the individual clones further characterized.
[0105] An isolated Kinase and Phosphatase protein, or a portion or
fragment thereof, can be used as an immunogen to generate
antibodies that bind Kinase and Phosphatase using standard
techniques for polyclonal and monoclonal antibody preparation. A
full-length Kinase and Phosphatase protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of Kinase and Phosphatase for use as immunogens. The antigenic
peptide of Kinase and Phosphatase comprises at least 8 amino acid
residues and encompasses an epitope of Kinase and Phosphatase such
that an antibody raised against the peptide forms a specific immune
complex with Kinase and Phosphatase. Preferably, the antigenic
peptide comprises at least 10 amino acid residues, more preferably
at least 15 amino acid residues, even more preferably at least 20
amino acid residues, and most preferably at least 30 amino acid
residues.
[0106] Preferred epitopes encompassed by the antigenic peptide are
regions of Kinase and Phosphatase that are located on the surface
of the protein, e.g., hydrophilic regions.
[0107] A Kinase and Phosphatase immunogen typically is used to
prepare antibodies by immunizing a suitable subject, (e.g., rabbit,
goat, mouse or other mammal) with the immunogen. An appropriate
immunogenic preparation can contain, for example, recombinantly
expressed Kinase and Phosphatase protein or a chemically
synthesized Kinase and Phosphatase polypeptide. The preparation can
further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic Kinase and
Phosphatase preparation induces a polyclonal anti-Kinase and
Phosphatase antibody response.
[0108] Accordingly, another aspect of the invention pertains to
anti-Kinase and Phosphatase antibodies. The term "antibody" as used
herein refers to immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antigen binding site which specifically binds
(immunoreacts with) an antigen, such as Kinase and Phosphatase.
Examples of immunologically active portions of immunoglobulin
molecules include F(ab) and F(ab').sub.2 fragments which can be
generated by treating the antibody with an enzyme such as pepsin.
The invention provides polyclonal and monoclonal antibodies that
bind Kinase and Phosphatase. The term "monoclonal antibody" or
"monoclonal antibody composition", as used herein, refers to a
population of antibody molecules that contain only one species of
an antigen binding site capable of immunoreacting with a particular
epitope of Kinase and Phosphatase. A monoclonal antibody
composition thus typically displays a single binding affinity for a
particular Kinase and Phosphatase protein with which it
immunoreacts.
[0109] Polyclonal anti-Kinase and Phosphatase antibodies can be
prepared as described above by immunizing a suitable subject with a
Kinase and Phosphatase immunogen. The anti-Kinase and Phosphatase
antibody titer in the immunized subject can be monitored over time
by standard techniques, such as with an enzyme linked immunosorbent
assay (ELISA) using immobilized Kinase and Phosphatase. If desired,
the antibody molecules directed against Kinase and Phosphatase can
be isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography
to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the anti-Kinase and Phosphatase antibody
titers are highest, antibody-producing cells can be obtained from
the subject and used to prepare monoclonal antibodies by standard
techniques, such as the hybridoma technique originally described by
Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et
al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol.
Chem .255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76.2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological
Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A.
Lerner (1981) Yale J. Biol. Med, 54:387-402; M. L. Gefter et al.
(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell
line (typically a myeloma) is fused to lymphocytes (typically
splenocytes) from a mammal immunized with a Kinase and Phosphatase
immunogen as described above, and the culture supernatants of the
resulting hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds Kinase and
Phosphatase.
[0110] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-Kinase and Phosphatase monoclonal
antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052;
Gefter et al. Somatic Cell Genet., cited supra; Lemer, Yale J.
Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited
supra). Moreover, the ordinarily skilled worker will appreciate
that there are many variations of such methods which also would be
useful. Typically, the immortal cell line (e.g., a myeloma cell
line) is derived from the same mammalian species as the
lymphocytes. For example, murine hybridomas can be made by fusing
lymphocytes from a mouse immunized with an immunogenic preparation
of the present invention with an immortalized mouse cell line.
Preferred immortal cell lines are mouse myeloma cell lines that are
sensitive to culture medium containing hypoxanthine, aminopterin
and thymidine ("HAT medium"). Any of a number of myeloma cell lines
can be used as a fusion partner according to standard techniques,
e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma
lines. These myeloma lines are available from ATCC. Typically,
HAT-sensitive mouse myeloma cells are fused to mouse splenocytes
using polyethylene glycol ("PEG"). Hybridoma cells resulting from
the fusion are then selected using HAT medium, which kills unfused
and unproductively fused myeloma cells (unfused splenocytes die
after several days because they are not transformed). Hybridoma
cells producing a monoclonal antibody of the invention are detected
by screening the hybridoma culture supernatants for antibodies that
bind Kinase and Phosphatase, e.g., using a standard ELISA
assay.
[0111] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-Kinase and Phosphatase antibody can
be identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with Kinase and Phosphatase to thereby isolate immunoglobulin
library members that bind Kinase and Phosphatase. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfZAP.TM. Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods
and reagents particularly amenable for use in generating and
screening antibody display library can be found in, for example,
Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al. PCT
International Publication No. WO 91/17271; Winter et al. PCT
International Publication WO 92/20791; Markland et al. PCT
International Publication No. WO 92/15679; Breitling et al. PCT
International Publication WO 93/01288; McCafferty et al. PCT
International Publication No. WO 92/01047; Garrard et al. PCT
International Publication No. WO 92/09690; Ladner et al. PCT
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J.
Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad
et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991)
Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad.
Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990)
348:552-554.
[0112] Additionally, recombinant anti-Kinase and Phosphatase
antibodies, such as chimeric and humanized monoclonal antibodies,
comprising both human and non-human portions, which can be made
using standard recombinant DNA techniques, are within the scope of
the invention. Such chimeric and humanized monoclonal antibodies
can be produced by recombinant DNA techniques known in the art, for
example using methods described in Robinson et al. International
Application No. PCT/US86/02269; Akira, et al. European Patent
Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison et al. European Patent Application 173,494;
Neuberger et al. PCT International Publication No. WO 86/01533;
Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European
Patent Application 125,023; Better et al. (1988) Science
240:1041-1043; Liuet al. (1987) Proc. Natl. Acad Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et
al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:40534060.
[0113] An anti-Kinase and Phosphatase antibody (e.g., monoclonal
antibody) can be used to isolate Kinase and Phosphatase by standard
techniques, such as affinity chromatography or immunoprecipitation.
An anti-Kinase and Phosphatase antibody can facilitate the
purification of natural Kinase and Phosphatase from cells and of
recombinantly produced Kinase and Phosphatase expressed in host
cells. Moreover, an anti-Kinase and Phosphatase antibody can be
used to detect Kinase and Phosphatase protein (e.g., in a cellular
lysate or cell supernatant) in order to evaluate the abundance and
pattern of expression of the Kinase and Phosphatase protein.
Anti-Kinase and Phosphatase antibodies can be used diagnostically
to monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling (i.e.,
physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, -galactosidase, or acetylcholinesterase; examples of
suitable prosthetic group complexes include streptavidinibiotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0114] III. Recombinant Expression Vectors and Host Cells
[0115] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
Kinase and Phosphatase protein (or a portion thereof). As used
herein, the term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other-vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are
referred to herein as "expression vectors". In general, expression
vectors of utility in recombinant DNA techniques are often in the
form of plasmids. In the present specification, "plasmid" and
"vector" can be used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors, such as viral
vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0116] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nuclootide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to includes promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, and the
like. The expression vectors of the invention can be introduced
into host cells to thereby produce proteins or peptides, including
fusion proteins or peptides, encoded by nucleic acids as described
herein (e.g., Kinase and Phosphatase proteins, mutant forms of
Kinase and Phosphatase proteins, fusion proteins, and the
like).
[0117] The recombinant expression vectors of the invention can be
designed for expression of Kinase and Phosphatase proteins in
prokaryotic or eukaryotic cells. For example, Kinase and
Phosphatase proteins can be expressed in bacterial cells such as E.
coli, insect cells (using baculovirus expression vectors) yeast
cells or mammalian cells. Suitable host cells are discussed further
in Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0118] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[0119] Purified fusion proteins can be utilized in Kinase and
Phosphatase activity assays, (e.g., direct assays or competitive
assays described in detail below), or to generate antibodies
specific for Kinase and Phosphatase proteins, for example. In a
preferred embodiment, a Kinase and Phosphatase fusion protein
expressed in a retroviral expression vector of the present
invention can be utilized to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six (6) weeks).
[0120] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11 d vector relies on
transcription from a T7 gnl10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gnl gene under the transcriptional control
of the lacUV 5 promoter.
[0121] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially utilized in E.
coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0122] In another embodiment, the Kinase and Phosphatase expression
vector is a yeast expression vector. Examples of vectors for
expression in yeast S. cerivisae include pYepSec1 (Baldari, et al.,
(1987) Embo J. 6:229-234), pMFa (Kuijan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[0123] Alternatively, Kinase and Phosphatase proteins can be
expressed in insect cells using baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL
series (Lucklow and Summers (1989) Virology 170:31-39).
[0124] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd,
ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0125] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0126] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to Kinase and Phosphatase
mRNA. Regulatory sequences operatively linked to a nucleic acid
cloned in the antisense orientation can be chosen which direct the
continuous expression of the antisense RNA molecule in a variety of
cell types, for instance viral promoters and/or enhancers, or
regulatory sequences can be chosen which direct constitutive,
tissue specific or cell type specific expression of antisense RNA.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus in which antisense nucleic
acids are produced under the control of a high efficiency
regulatory region, the activity of which can be determined by the
cell type into which the vector is introduced. For a discussion of
the regulation of gene expression using antisense genes see
Weintraub, H. et al., Antisense RNA as a molecular tool for genetic
analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986.
[0127] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0128] A host cell can be any prokaryotic or eukaryotic cell. For
example, a Kinase and Phosphatase protein can be expressed in
bacterial cells such as E. coli, insect cells, yeast or mammalian
cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other suitable host cells are known to those skilled in the
art.
[0129] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0130] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding a Kinase and Phosphatase protein or can be introduced
on a separate vector. Cells stably transfected with the introduced
nucleic acid can be identified by drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0131] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) a Kinase and Phosphatase protein. Accordingly, the
invention further provides methods for producing a Kinase and
Phosphatase protein using the host cells of the invention. In one
embodiment, the method comprises culturing the host cell of
invention (into which a recombinant expression vector encoding a
Kinase and Phosphatase protein has been introduced) in a suitable
medium such that a Kinase and Phosphatase protein is produced. In
another embodiment, the method further comprises isolating a Kinase
and Phosphatase protein from the medium or the host cell.
[0132] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which Kinase and Phosphatase-coding sequences have
been introduced. Such host cells can then be used to create
non-human transgenic animals in which exogenous Kinase and
Phosphatase sequences have been introduced into their genome or
homologous recombinant animals in which endogenous Kinase and
Phosphatase sequences have been altered. Such animals are useful
for studying the function and/or activity of a Kinase and
Phosphatase and for identifying and/or evaluating modulators of
Kinase and Phosphatase activity. As used herein, a "transgenic
animal" is a non-human animal, preferably a mammal, more preferably
a rodent such as a rat or mouse, in which one or more of the cells
of the animal includes a transgene. Other examples of transgenic
animals include non-human primates, sheep, dogs, cows, goats,
chickens, amphibians, and the like. A transgene is exogenous DNA
which is integrated into the genome of a cell from which a
transgenic animal develops and which remains in the genome of the
mature animal, thereby directing the expression of an encoded gene
product in one or more cell types or tissues of the transgenic
animal. As used herein, a "homologous recombinant animal" is a
non-human animal, preferably a mammal, more preferably a mouse, in
which an endogenous Kinase and Phosphatase gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0133] A transgenic animal of the invention can be created by
introducing a Kinase and Phosphatase-encoding nucleic acid into the
male pronuclei of a fertilized oocyte, e.g., by microinjection,
retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. The Kinase and Phosphatase
cDNA sequence of SEQ ID NO: 1-14 can be introduced as a transgene
into the genome of a non-human animal. Alternatively, a nonhuman
homologue of a human Kinase and Phosphatase gene, such as a mouse
or rat Kinase and Phosphatase gene, can be used as a transgene.
Alternatively, a Kinase and Phosphatase gene homologue, such as
another Kinase and Phosphatase family member, can be isolated based
on hybridization to the Kinase and Phosphatase cDNA sequences of
SEQ ID NO: 1-14 (described further in subsection I above) and used
as a transgene. Intronic sequences and polyadenylation signals can
also be included in the transgene to increase the efficiency of
expression of the transgene. A tissue-specific regulatory
sequence(s) can be operably linked to a Kinase and Phosphatase
transgene to direct expression of a Kinase and Phosphatase protein
to particular cells. Methods for generating transgenic animals via
embryo manipulation and microinjection, particularly animals such
as mice, have become conventional in the art and are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder
et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of a Kinase and
Phosphatase transgene in its genome and/or expression of Kinase and
Phosphatase mRNA in tissues or cells of the animals. A transgenic
founder animal can then be used to breed additional animals
carrying the transgene. Moreover, transgenic animals carrying a
transgene encoding a Kinase and Phosphatase protein can further be
bred to other transgenic animals carrying other transgenes.
[0134] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a Kinase and
Phosphatase gene into which a deletion, addition or substitution
has been introduced to thereby alter, e.g., functionally disrupt,
the Kinase and Phosphatase gene. The Kinase and Phosphatase gene
can be a human gene (e.g., the SEQ ID NO: 1-14), but more
preferably, is a non-human homologue of a human Kinase and
Phosphatase gene (e.g., a cDNA isolated by stringent hybridization
with the nucleotide sequence of SEQ ID NO: 1-14). For example, a
mouse Kinase and Phosphatase gene can be used to construct a
homologous recombination vector suitable for altering an endogenous
Kinase and Phosphatase gene in the mouse genome. In a preferred
embodiment, the vector is designed such that, upon homologous
recombination, the endogenous Kinase and Phosphatase gene is
functionally disrupted (i.e., no longer encodes a functional
protein; also referred to as a "knock out" vector). Alternatively,
the vector can be designed such that, upon homologous
recombination, the endogenous Kinase and Phosphatase gene is
mutated or otherwise altered but still encodes a functional protein
(e.g., the upstream regulatory region can be altered to thereby
alter the expression of the endogenous Kinase and Phosphatase
protein). In the homologous recombination vector, the altered
portion of the Kinase and Phosphatase gene is flanked at its 5' and
3' ends by additional nucleic acid sequence of the Kinase and
Phosphatase gene to allow for homologous recombination to occur
between the exogenous Kinase and Phosphatase gene carried by the
vector and an endogenous Kinase and Phosphatase gene in an
embryonic stem cell. The additional flanking Kinase and Phosphatase
nucleic acid sequence is of sufficient length for successful
homologous recombination with the endogenous gene. Typically,
several kilobases of flanking DNA (both at the 5' and 3' ends) are
included in the vector (see e.g., Thomas, K. R. and Capecchi, M. R.
(1987) Cell 51:503 for a description of homologous recombination
vectors). The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced Kinase
and Phosphatase gene has homologously recombined with the
endogenous Kinase and Phosphatase gene are selected (see, e.g., Li,
E. et al. (1992) Cell 69:915). The selected cells are then injected
into a blastocyst of an animal (e.g., a mouse) to form aggregation
chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,
Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
into a suitable pseudopregnant female foster animal and the embryo
brought to term. Progeny harboring the homologously recombined DNA
in their germ cells can be used to breed animals in which all cells
of the animal contain the homologously recombined DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination vectors and homologous recombinant animals are
described further in Bradley, A. (1991) Current Opinion in
Biotechnology 2:823-829 and in PCT International Publication Nos.:
WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.;
WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.
[0135] In another embodiment, transgenic non-humans animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al. (1991) Science 251:1351 -1355. If a
cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[0136] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
recontructed oocyte is then cultured such that it develops to
morula or blastocyte and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[0137] IV. Pharmaceutical Compositions
[0138] The Kinase and Phosphatase nucleic acid molecules, Kinase
and Phosphatase proteins, and anti-Kinase and anti-Phosphatase
antibodies (also referred to herein as "active compounds") of the
invention can be incorporated into pharmaceutical compositions
suitable for administration. Such compositions typically comprise
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0139] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0140] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0141] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a Kinase and Phosphatase
protein or anti-Kinase and Phosphatase antibody) in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0142] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0143] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0144] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0145] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0146] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0147] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0148] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 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. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0149] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0150] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments. In a preferred example, a subject is
treated with antibody, protein, or polypeptide in the range of
between about 0.1 to 20 mg/kg body weight, one time per week for
between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. It will also be appreciated that the
effective dosage of antibody, protein, or polypeptide used for
treatment may increase or decrease over the course of a particular
treatment. Changes in dosage may result and become apparent from
the results of diagnostic assays as described herein.
[0151] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e,. including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0152] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the ken of the
ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention.
[0153] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein.
[0154] When one or more of these small molecules is to be
administered to an animal (e.g., a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0155] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0156] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0157] V. Uses and Methods of the Invention
[0158] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic). The isolated nucleic acid molecules
of the invention can be used, for example, to express Kinase and
Phosphatase protein (e.g., via a recombinant expression vector in a
host cell in gene therapy applications), to detect Kinase and
Phosphatase mRNA (e.g., in a biological sample) or a genetic
alteration in a Kinase and Phosphatase gene, and to modulate Kinase
and Phosphatase activity, as described further below. The Kinase
and Phosphatase proteins can be used to treat disorders
characterized by insufficient or excessive production of a Kinase
and Phosphatase substrate or production of Kinase and Phosphatase
inhibitors. In addition, the Kinase and Phosphatase proteins can be
used to screen for naturally occurring Kinase and Phosphatase
substrates, to screen for drugs or compounds which modulate Kinase
and Phosphatase activity, as well as to treat disorders
characterized by insufficient or excessive production of Kinase and
Phosphatase protein or production of Kinase and Phosphatase protein
forms which have decreased or aberrant activity compared to Kinase
and Phosphatase wild type protein. Moreover, the anti-Kinase and
Phosphatase antibodies of the invention can be used to detect and
isolate Kinase and Phosphatase proteins, regulate the
bioavailability of Kinase and Phosphatase proteins, and modulate
Kinase and Phosphatase activity.
[0159] A. Screening Assays:
[0160] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) which bind to Kinase and Phosphatase
proteins, have a stimulatory or inhibitory effect on, for example,
Kinase and Phosphatase expression or Kinase and Phosphatase
activity, or have a stimulatory or inhibitory effect on, for
example, the expression or activity of a Kinase and Phosphatase
substrate.
[0161] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
Kinase and Phosphatase protein or polypeptide or biologically
active portion thereof In another embodiment, the invention
provides assays for screening candidate or test compounds which
bind to or modulate the activity of a Kinase and Phosphatase
protein or polypeptide or biologically active portion thereof,
e.g., modulate the ability of Kinase and Phosphatase to interact
with its cognate ligand. The test compounds of the present
invention can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam,
K. S. (1997) Anticancer Drug Des. 12:145).
[0162] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233.
[0163] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner USP 5,223,409), spores (Ladner U.S. Pat. No.
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.).
[0164] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a Kinase and Phosphatase
target molecule (e.g., a Kinase and Phosphatase phosphorylation
substrate) with a test compound and determining the ability of the
test compound to modulate (e.g. stimulate or inhibit) the activity
of the Kinase and Phosphatase target molecule. Determining the
ability of the test compound to modulate the activity of a Kinase
and Phosphatase target molecule can be accomplished, for example,
by determining the ability of the Kinase and Phosphatase protein to
bind to or interact with the Kinase and Phosphatase target
molecule, or by determining the ability of the Kinase and
Phosphatase protein to phosphorylate the Kinase and Phosphatase
target molecule.
[0165] The ability of the Kinase and Phosphatase protein to
phosphorylate a Kinase and Phosphatase target molecule can be
determined by, for example, an in vitro kinase assay. Briefly, a
Kinase and Phosphatase target molecule, e.g., an immunoprecipitated
Kinase and Phosphatase target molecule from a cell line expressing
such a molecule, can be incubated with the Kinase and Phosphatase
protein and radioactive ATP, e.g., [.gamma.-.sup.32P] ATP, in a
buffer containing MgCl.sub.2 and MnCl.sub.2, e.g., 10 mM MgCl.sub.2
and 5 mM MnCl.sub.2. Following the incubation, the
immunoprecipitated Kinase and Phosphatase target molecule can be
separated by SDS-polyacrylamide gel electrophoresis under reducing
conditions, transferred to a membrane, e.g., a PVDF membrane, and
autoradiographed. The appearance of detectable bands on the
autoradiograph indicates that the Kinase and Phosphatase substrate
has been phosphorylated. Phosphoaminoacid analysis of the
phosphorylated substrate can also be performed in order to
determine which residues on the Kinase and Phosphatase substrate
are phosphorylated. Briefly, the radiophosphorylated protein band
can be excised from the SDS gel and subjected to partial acid
hydrolysis. The products can then be separated by one-dimensional
electrophoresis and analyzed on, for example, a phosphoimager and
compared to ninhydrin-stained phosphoaminoacid standards.
[0166] Determining the ability of the Kinase and Phosphatase
protein to bind to or interact with a Kinase and Phosphatase target
molecule can be accomplished by determining direct binding.
Determining the ability of the Kinase and Phosphatase protein to
bind to or interact with a Kinase and Phosphatase target molecule
can be accomplished, for example, by coupling the Kinase and
Phosphatase protein with a radioisotope or enzymatic label such
that binding of the Kinase and Phosphatase protein to a Kinase and
Phosphatase target molecule can be determined by detecting the
labeled Kinase and Phosphatase protein in a complex. For example,
Kinase and Phosphatase molecules, e.g., Kinase and Phosphatase
proteins, can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemmission or by scintillation
counting. Alternatively, Kinase and Phosphatase molecules can be
enzymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[0167] It is also within the scope of this invention to determine
the ability of a compound to modulate the interaction between
Kinase and Phosphatase and its target molecule, without the
labeling of any of the interactants. For example, a
microphysiometer can be used to detect the interaction of Kinase
and Phosphatase with its target molecule without the labeling of
either Kinase and Phosphatase or the target molecule. McConnell, H.
M. et al. (1992) Science 257:1906-1912. As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between compound and receptor.
[0168] In a preferred embodiment, determining the ability of the
Kinase and Phosphatase protein to bind to or interact with a Kinase
and Phosphatase target molecule can be accomplished by determining
the activity of the target molecule. For example, the activity of
the target molecule can be determined by detecting induction of a
cellular second messenger of the target (e.g., intracellular
Ca.sup.2+, diacylglycerol, IP.sub.3, etc.), detecting
catalytic/enzymatic activity of the target an appropriate
substrate, detecting the induction of a reporter gene (comprising a
target-responsive regulatory element operatively linked to a
nucleic acid encoding a detectable marker, e.g., chloramphenicol
acetyl transferase), or detecting a target-regulated cellular
response.
[0169] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a Kinase and Phosphatase protein or
biologically active portion thereof is contacted with a test
compound and the ability of the test compound to bind to the Kinase
and Phosphatase protein or biologically active portion thereof is
determined. Binding of the test compound to the Kinase and
Phosphatase protein can be determined either directly or indirectly
as described above. In a preferred embodiment, the assay includes
contacting the Kinase and Phosphatase protein or biologically
active portion thereof with a known compound which binds Kinase and
Phosphatase to form an assay mixture, contacting the assay mixture
with a test compound, and determining the ability of the test
compound to interact with a Kinase and Phosphatase protein, wherein
determining the ability of the test compound to interact with a
Kinase and Phosphatase protein comprises determining the ability of
the test compound to preferentially bind to Kinase and Phosphatase
or biologically active portion thereof as compared to the known
compound.
[0170] In another embodiment, the assay is a cell-free assay in
which a Kinase and Phosphatase protein or biologically active
portion thereof is contacted with a test compound and the ability
of the test compound to modulate (e.g., stimulate or inhibit) the
activity of the Kinase and Phosphatase protein or biologically
active portion thereof is determined. Determining the ability of
the test compound to modulate the activity of a Kinase and
Phosphatase protein can be accomplished, for example, by
determining the ability of the Kinase and Phosphatase protein to
bind to a Kinase and Phosphatase target molecule by one of the
methods described above for determining direct binding. Determining
the ability of the Kinase and Phosphatase protein to bind to a
Kinase and Phosphatase target molecule can also be accomplished
using a technology such as real-time Biomolecular Interaction
Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem.
63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705. As used herein, "BIA" is a technology for studying
biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the optical phenomenon of
surface plasmon resonance (SPR) can be used as an indication of
real-time reactions between biological molecules.
[0171] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of a Kinase and Phosphatase
protein can be accomplished by determining the ability of the
Kinase and Phosphatase protein to further modulate the activity of
a Kinase and Phosphatase target molecule (e.g., a Kinase and
Phosphatase mediated signal transduction pathway component). For
example, the activity of the effector molecule on an appropriate
target can be determined, or the binding of the effector to an
appropriate target can be determined as previously described.
[0172] In yet another embodiment, the cell-free assay involves
contacting a Kinase and Phosphatase protein or biologically active
portion thereof with a known compound which binds the Kinase and
Phosphatase protein to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the
test compound to interact with the Kinase and Phosphatase protein,
wherein determining the ability of the test compound to interact
with the Kinase and Phosphatase protein comprises determining the
ability of the Kinase and Phosphatase protein to preferentially
bind to or modulate the activity of a Kinase and Phosphatase target
molecule.
[0173] The cell-free assays of the present invention are amenable
to use of both soluble and/or membrane-bound forms of proteins
(e.g., Kinase and Phosphatase proteins or biologically active
portions thereof, or receptors to which Kinase and Phosphatase
binds). In the case of cell-free assays in which a membrane-bound
form a protein is used (e.g., a cell surface Kinase and Phosphatase
receptor) it may be desirable to utilize a solubilizing agent such
that the membrane-bound form of the protein is maintained in
solution. Examples of such solubilizing agents include non-ionic
detergents such as n-octylglucoside, n-dodecylglucoside,
n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM.X-100, Triton.RTM.X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
3-[(3-cholamidopropyl)dimethylamminio])1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0174] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
Kinase and Phosphatase or its target molecule to facilitate
separation of complexed from uncomplexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to a Kinase and Phosphatase protein, or
interaction of a Kinase and Phosphatase protein with a target
molecule in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/Kinase and Phosphatase fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or Kinase and Phosphatase protein, and
the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtitre plate wells are
washed to remove any unbound components, the matrix immobilized in
the case of beads, complex determined either directly or
indirectly, for example, as described above. Alternatively, the
complexes can be dissociated from the matrix, and the level of
Kinase and Phosphatase binding or activity determined using
standard techniques.
[0175] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a Kinase and Phosphatase protein or a Kinase and Phosphatase
target molecule can be immobilized utilizing conjugation of biotin
and streptavidin. Biotinylated Kinase and Phosphatase protein or
target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with Kinase
and Phosphatase protein or target molecules but which do not
interfere with binding of the Kinase and Phosphatase protein to its
target molecule can be derivatized to the wells of the plate, and
unbound target or Kinase and Phosphatase protein trapped in the
wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the Kinase and Phosphatase protein
or target molecule, as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the Kinase and
Phosphatase protein or target molecule.
[0176] In another embodiment, modulators of Kinase and Phosphatase
expression are identified in a method wherein a cell is contacted
with a candidate compound and the expression of Kinase and
Phosphatase mRNA or protein in the cell is determined. The level of
expression of Kinase and Phosphatase mRNA or protein in the
presence of the candidate compound is compared to the level of
expression of Kinase and Phosphatase mRNA or protein in the absence
of the candidate compound. The candidate compound can then be
identified as a modulator of Kinase and Phosphatase expression
based on this comparison. For example, when expression of Kinase
and Phosphatase mRNA or protein is greater (statistically
significantly greater) in the presence of the candidate compound
than in its absence, the candidate compound is identified as a
stimulator of Kinase and Phosphatase mRNA or protein expression.
Alternatively, when expression of Kinase and Phosphatase mRNA or
protein is less (statistically significantly less) in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of Kinase and Phosphatase
mRNA or protein expression. The level of Kinase and Phosphatase
mRNA or protein expression in the cells can be determined by
methods described herein for detecting Kinase and Phosphatase MRNA
or protein.
[0177] In yet another aspect of the invention, the Kinase and
Phosphatase proteins can be used as "bait proteins" in a two-hybrid
assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317;
Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol.
Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques
14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent
WO94/10300), to identify other proteins, which bind to or interact
with Kinase and Phosphatase ("Kinase and Phosphatase-binding
proteins" or "Kinase and Phosphatase-bp") and are involved in
Kinase and Phosphatase activity. Such Kinase and
Phosphatase-binding proteins are also likely to be involved in the
propagation of signals by the Kinase and Phosphatase proteins or
Kinase and Phosphatase targets as, for example, downstream elements
of a Kinase and Phosphatase-mediated signaling pathway.
Alternatively, such Kinase and Phosphatase-binding proteins are
likely to be Kinase and Phosphatase inhibitors.
[0178] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a Kinase and
Phosphatase protein is fused to a gene encoding the DNA binding
domain of a known transcription factor (e.g., GAL-4). In the other
construct, a DNA sequence, from a library of DNA sequences, that
encodes an unidentified protein ("prey" or "sample") is fused to a
gene that codes for the activation domain of the known
transcription factor. If the "bait" and the "prey" proteins are
able to interact, in vivo, forming a Kinase and
Phosphatase-dependent complex, the DNA-binding and activation
domains of the transcription factor are brought into close
proximity. This proximity allows transcription of a reporter gene
(e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the Kinase and Phosphatase protein.
[0179] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a Kinase and
Phosphatase modulating agent, an antisense Kinase and Phosphatase
nucleic acid molecule, a Kinase and Phosphatase-specific antibody,
or a Kinase and Phosphatase-binding partner) can be used in an
animal model to determine the efficacy, toxicity, or side effects
of treatment with such an agent. Alternatively, an agent identified
as described herein can be used in an animal model to determine the
mechanism of action of such an agent Furthermore, this invention
pertains to uses of novel agents identified by the above-described
screening assays for treatments as described herein.
[0180] B. Detection Assays
[0181] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of
a biological sample. These applications are described in the
subsections below.
[0182] 1. Chromosome Mapping
[0183] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the Kinase and
Phosphatase nucleotide sequences, described herein, can be used to
map the location of the Kinase and Phosphatase genes on a
chromosome. The mapping of the Kinase and Phosphatase sequences to
chromosomes is an important first step in correlating these
sequences with genes associated with disease.
[0184] Briefly, Kinase and Phosphatase genes can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp in
length) from the Kinase and Phosphatase nucleotide sequences.
Computer analysis of the Kinase and Phosphatase sequences can be
used to predict primers that do not span more than one exon in the
genomic DNA, thus complicating the amplification process. These
primers can then be used for PCR screening of somatic cell hybrids
containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the Kinase and
Phosphatase sequences will yield an amplified fragment.
[0185] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio P. et al. (1983)
Science 220:919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[0186] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the Kinase and Phosphatase nucleotide sequences to
design oligonucleotide primers, sublocalization can be achieved
with panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a 9o, 1p, or 1v
sequence to its chromosome include in situ hybridization (described
in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),
pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries.
[0187] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical such as colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual of Basic
Techniques (Pergamon Press, New York 1988).
[0188] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0189] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[0190] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the Kinase and Phosphatase gene, can be determined. If a mutation
is observed in some or all of the affected individuals but not in
any unaffected individuals, then the mutation is likely to be the
causative agent of the particular disease. Comparison of affected
and unaffected individuals generally involves first looking for
structural alterations in the chromosomes, such as deletions or
translocations that are visible from chromosome spreads or
detectable using PCR based on that DNA sequence. Ultimately,
complete sequencing of genes from several individuals can be
performed to confirm the presence of a mutation and to distinguish
mutations from polymorphisms.
[0191] 2. Tissue Typing
[0192] The Kinase and Phosphatase sequences of the present
invention can also be used to identify individuals from minute
biological samples. The United States military, for example, is
considering the use of restriction fragment length polymorphism
(RFLP) for identification of its personnel. In this technique, an
individual's genomic DNA is digested with one or more restriction
enzymes, and probed on a Southern blot to yield unique bands for
identification. This method does not suffer from the current
limitations of "Dog Tags" which can be lost, switched, or stolen,
making positive identification difficult. The sequences of the
present invention are useful as additional DNA markers for RFLP
(described in U.S. Pat. No. 5,272,057).
[0193] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the Kinase and Phosphatase nucleotide
sequences described herein can be used to prepare two PCR primers
from the 5' and 3' ends of the sequences. These primers can then be
used to amplify an individual's DNA and subsequently sequence
it.
[0194] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The Kinase and
Phosphatase nucleotide sequences of the invention uniquely
represent portions of the human genome. Allelic variation occurs to
some degree in the coding regions of these sequences, and to a
greater degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a frequency
of about once per each 500 bases. Each of the sequences described
herein can, to some degree, be used as a standard against which DNA
from an individual can be compared for identification purposes.
Because greater numbers of polymorphisms occur in the noncoding
regions, fewer sequences are necessary to differentiate
individuals. The noncoding sequences of SEQ ID NO: 1-161, can
comfortably provide positive individual identification with a panel
of perhaps 10 to 1,000 primers which each yield a noncoding
amplified sequence of 100 bases. If predicted coding sequences are
used, a more appropriate number of primers for positive individual
identification would be 500-2,000.
[0195] If a panel of reagents from Kinase and Phosphatase
nucleotide sequences described herein is used to generate a unique
identification database for an individual, those same reagents can
later be used to identify tissue from that individual. Using the
unique identification database, positive identification of the
individual, living or dead, can be made from extremely small tissue
samples.
[0196] 3. Use of Partial Kinase and Phosphatase Sequences in
Forensic Biology
[0197] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0198] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions are particularly appropriate for this use as
greater numbers of polymorphisms occur in the noncoding regions,
making it easier to differentiate individuals using this technique.
Examples of polynucleotide reagents include the Kinase and
Phosphatase nucleotide sequences or portions thereof having a
length of at least 20 bases, preferably at least 30 bases.
[0199] The Kinase and Phosphatase nucleotide sequences described
herein can further be used to provide polynucleotide reagents,
e.g., labeled or labelable probes which can be used in, for
example, an in situ hybridization technique, to identify a specific
tissue, e.g., brain tissue. This can be very useful in cases where
a forensic pathologist is presented with a tissue of unknown
origin. Panels of such Kinase and Phosphatase probes can be used to
identify tissue by species and/or by organ type.
[0200] In a similar fashion, these reagents, e.g., Kinase and
Phosphatase primers or probes can be used to screen tissue culture
for contamination (i.e. screen for the presence of a mixture of
different types of cells in a culture).
[0201] C. Predictive Medicine:
[0202] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining Kinase and Phosphatase protein
and/or nucleic acid expression as well as Kinase and Phosphatase
activity, in the context of a biological sample (e.g., blood,
serum, cells, tissue) to thereby determine whether an individual is
afflicted with a disease or disorder, or is at risk of developing a
disorder, associated with aberrant Kinase and Phosphatase
expression or activity. The invention also provides for prognostic
(or predictive) assays for determining whether an individual is at
risk of developing a disorder associated with Kinase and
Phosphatase protein, nucleic acid expression or activity. For
example, mutations in a Kinase and Phosphatase gene can be assayed
in a biological sample. Such assays can be used for prognostic or
predictive purpose to thereby phophylactically treat an individual
prior to the onset of a disorder characterized by or associated
with Kinase and Phosphatase protein, nucleic acid expression or
activity.
[0203] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on the expression or
activity of Kinase and Phosphatase in clinical trials.
[0204] These and other agents are described in further detail in
the following sections.
[0205] 1. Diagnostic Assays
[0206] An exemplary method for detecting the presence or absence of
Kinase and Phosphatase protein or nucleic acid in a biological
sample involves obtaining a biological sample from a test subject
and contacting the biological sample with a compound or an agent
capable of detecting Kinase and Phosphatase protein or nucleic acid
(e.g., mRNA, genomic DNA) that encodes Kinase and Phosphatase
protein such that the presence of Kinase and Phosphatase protein or
nucleic acid is detected in the biological sample. A preferred
agent for detecting Kinase and Phosphatase mRNA or genomic DNA is a
labeled nucleic acid probe capable of hybridizing to Kinase and
Phosphatase mRNA or genomic DNA. The nucleic acid probe can be, for
example, a human Kinase and Phosphatase nucleic acid, such as the
nucleic acid of SEQ ID NO: 1-14, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and sufficient to specifically hybridize under stringent
conditions to Kinase and Phosphatase mRNA or genomic DNA. Other
suitable probes for use in the diagnostic assays of the invention
are described herein.
[0207] A preferred agent for detecting Kinase and Phosphatase
protein is an antibody capable of binding to Kinase and Phosphatase
protein, preferably an antibody with a detectable label. Antibodies
can be polyclonal, or more preferably, monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be
used. The term "labeled", with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or antibody by
coupling (i.e., physically linking) a detectable substance to the
probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect Kinase and
Phosphatase mRNA, protein, or genomic DNA in a biological sample in
vitro as well as in vivo. For example, in vitro techniques for
detection of Kinase and Phosphatase mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of Kinase and Phosphatase protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. In vitro techniques for detection of Kinase
and Phosphatase genomic DNA include Southern hybridizations.
Furthermore, in vivo techniques for detection of Kinase and
Phosphatase protein include introducing into a subject a labeled
anti-Kinase and Phosphatase antibody. For example, the antibody can
be labeled with a radioactive marker whose presence and location in
a subject can be detected by standard imaging techniques.
[0208] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a serum sample isolated by conventional means from a
subject.
[0209] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting Kinase
and Phosphatase protein, mRNA, or genomic DNA, such that the
presence of Kinase and Phosphatase protein, mRNA or genomic DNA is
detected in the biological sample, and comparing the presence of
Kinase and Phosphatase protein, mRNA orgenomic DNA in the control
sample with the presence of Kinase and Phosphatase protein, mRNA or
genomic DNA in the test sample.
[0210] The invention also encompasses kits for detecting the
presence of Kinase and Phosphatase in a biological sample. For
example, the kit can comprise a labeled compound or agent capable
of detecting Kinase and Phosphatase protein or mRNA in a biological
sample; means for determining the amount of Kinase and Phosphatase
in the sample; and means for comparing the amount of Kinase and
Phosphatase in the sample with a standard. The compound or agent
can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect Kinase and
Phosphatase protein or nucleic acid.
[0211] 2. Prognostic Assays
[0212] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
disease or disorder associated with aberrant Kinase and Phosphatase
expression or activity. For example, the assays described herein,
such as the preceding diagnostic assays or the following assays,
can be utilized to identify a subject having or at risk of
developing a disorder associated with Kinase and Phosphatase
protein, nucleic acid expression or activity. Thus, the present
invention provides a method for identifying a disease or disorder
associated with aberrant Kinase and Phosphatase expression or
activity in which a test sample is obtained from a subject and
Kinase and Phosphatase protein or nucleic acid (e.g., mRNA, genomic
DNA) is detected, wherein the presence of Kinase and Phosphatase
protein or nucleic acid is diagnostic for a subject having or at
risk of developing a disease or disorder associated with aberrant
Kinase and Phosphatase expression or activity. As used herein, a
"test sample" refers to a biological sample obtained from a subject
of interest. For example, a test sample can be a biological fluid
(e.g., serum), cell sample, or tissue.
[0213] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant Kinase and Phosphatase
expression or activity. Thus, the present invention provides
methods for determining whether a subject can be effectively
treated with an agent for a disorder associated with aberrant
Kinase and Phosphatase expression or activity in which a test
sample is obtained and Kinase and Phosphatase protein or nucleic
acid expression or activity is detected (e.g., wherein the
abundance of Kinase and Phosphatase protein or nucleic acid
expression or activity is diagnostic for a subject that can be
administered the agent to treat a disorder associated with aberrant
Kinase and Phosphatase expression or activity).
[0214] The methods of the invention can also be used to detect
genetic alterations in a Kinase and Phosphatase gene, thereby
determining if a subject with the altered gene is at risk for a
disorder associated with the Kinase and Phosphatase gene. In
preferred embodiments, the methods include detecting, in a sample
of cells from the subject, the presence or absence of a genetic
alteration characterized by at least one of an alteration affecting
the integrity of a gene encoding a Kinase and Phosphatase-protein,
or the mis-expression of the Kinase and Phosphatase gene. For
example, such genetic alterations can be detected by ascertaining
the existence of at least one of 1) a deletion of one or more
nucleotides from a Kinase and Phosphatase gene; 2) an addition of
one or more nucleotides to a Kinase and Phosphatase gene; 3) a
substitution of one or more nucleotides of a Kinase and Phosphatase
gene, 4) a chromosomal rearrangement of a Kinase and Phosphatase
gene; 5) an alteration in the level of a messenger RNA transcript
of a Kinase and Phosphatase gene, 6) aberrant modification of a
Kinase and Phosphatase gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of a Kinase and Phosphatase
gene, 8) a non-wild type level of a Kinase and Phosphatase-protein,
9) allelic loss of a Kinase and Phosphatase gene, and 10)
inappropriate post-translational modification of a Kinase and
Phosphatase-protein. As described herein, there are a large number
of assay techniques known in the art which can be used for
detecting alterations in a Kinase and Phosphatase gene. A preferred
biological sample is a tissue or serum sample isolated by
conventional means from a subject, e.g., a cardiac tissue
sample.
[0215] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364),
the latter of which can be particularly useful for detecting point
mutations in the Kinase and Phosphatase-gene (see Abravaya et al.
(1995) Nucleic Acids Res .23:675-682). This method can include the
steps of collecting a sample of cells from a patient, isolating
nucleic acid (e.g., genomic, mRNA or both) from the cells of the
sample, contacting the nucleic acid sample with one or more primers
which specifically hybridize to a Kinase and Phosphatase gene under
conditions such that hybridization and amplification of the Kinase
and Phosphatase-gene (if present) occurs, and detecting the
presence or absence of an amplification product, or detecting the
size of the amplification product and comparing the length to a
control sample. It is anticipated that PCR and/or LCR may be
desirable to use as a preliminary amplification step in conjunction
with any of the techniques used for detecting mutations described
herein.
[0216] Alternative amplification methods include: self sustained
sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988)
Bio-Technology 6:1197), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers.
[0217] In an alternative embodiment, mutations in a Kinase and
Phosphatase gene from a sample cell can be identified by
alterations in restriction enzyme cleavage patterns. For example,
sample and control DNA is isolated, amplified (optionally),
digested with one or more restriction endonucleases, and fragment
length sizes are determined by gel electrophoresis and compared.
Differences in fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use of
sequence specific ribozymes (see, for example, U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0218] In other embodiments, genetic mutations in Kinase and
Phosphatase can be identified by hybridizing a sample and control
nucleic acids, e.g., DNA or RNA, to high density arrays containing
hundreds or thousands of oligonucleotides probes (Cronin, M. T. et
al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996)
Nature Medicine 2: 753-759). For example, genetic mutations in
Kinase and Phosphatase can be identified in two dimensional arrays
containing light-generated DNA probes as described in Cronin, M. T.
et al. supra. Briefly, a first hybridization array of probes can be
used to scan through long stretches of DNA in a sample and control
to identify base changes between the sequences by making linear
arrays of sequential ovelapping probes. This step allows the
identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0219] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
Kinase and Phosphatase gene and detect mutations by comparing the
sequence of the sample Kinase and Phosphatase with the
corresponding wild-type (control) sequence. Examples of sequencing
reactions include those based on techniques developed by Maxam and
Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger
((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also
contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Biotechniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT International Publication No. WO
94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and
Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
[0220] Other methods for detecting mutations in the Kinase and
Phosphatase gene include methods in which protection from cleavage
agents is used to detect mismatched bases in RNA/RNA or RNA/DNA
heteroduplexes (Myers et al. (1985) Science 230:1242). In general,
the art technique of "mismatch cleavage" starts by providing
heteroduplexes formed by hybridizing (labeled) RNA or DNA
containing the wild-type Kinase and Phosphatase sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The
double-stranded duplexes are treated with an agent which cleaves
single-stranded regions of the duplex such as which will exist due
to basepair mismatches between the control and sample strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA
hybrids treated with S1 nuclease to enzymatically digesting the
mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA
duplexes can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions. After
digestion of the mismatched regions, the resulting material is then
separated by size on denaturing polyacrylamide gels to determine
the site of mutation. See, for example, Cotton et al. (1988) Proc.
Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol.
217:286-295. In a preferred embodiment, the control DNA or RNA can
be labeled for detection.
[0221] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in Kinase
and Phosphatase cDNAs obtained from samples of cells. For example,
the mutY enzyme of E. coli cleaves A at G/A mismatches and the
thymidine DNA glycosylase from HeLa cells cleaves T at G/T
mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
According to an exemplary embodiment, a probe based on a Kinase and
Phosphatase sequence, e.g., a wild-type Kinase and Phosphatase
sequence, is hybridized to a cDNA or other DNA product from a test
cell(s). The duplex is treated with a DNA mismatch repair enzyme,
and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[0222] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in Kinase and
Phosphatase genes. For example, single strand conformation
polymorphism (SSCP) may be used to detect differences in
electrophoretic mobility between mutant and wild type nucleic acids
(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also
Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal
Tech Appl 9:73-79). Single-stranded DNA fragments of sample and
control Kinase and Phosphatase nucleic acids will be denatured and
allowed to renature. The secondary structure of single-stranded
nucleic acids varies according to sequence, the resulting
alteration in electrophoretic mobility enables the detection of
even a single base change. The DNA fragments may be labeled or
detected with labeled probes. The sensitivity of the assay may be
enhanced by using RNA (rather than DNA), in which the secondary
structure is more sensitive to a change in sequence. In a preferred
embodiment, the subject method utilizes heteroduplex analysis to
separate double stranded heteroduplex molecules on the basis of
changes in electrophoretic mobility (Keen et al. (1991) Trends
Genet 7:5).
[0223] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR- In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[0224] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[0225] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner et al. (1993) Tibtech
11:238). In addition it may be desirable to introduce a novel
restriction site in the region of the mutation to create
cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes
6:1). It is anticipated that in certain embodiments amplification
may also be performed using Taq ligase for amplification (Barany
(1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation
will occur only if there is a perfect match at the 3' end of the 5'
sequence making it possible to detect the presence of a known
mutation at a specific site by looking for the presence or absence
of amplification.
[0226] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a Kinase and Phosphatase gene.
[0227] Furthermore, any cell type or tissue in which Kinase and
Phosphatase is expressed may be utilized in the prognostic assays
described herein.
[0228] 3. Monitoring of Effects During Clinical Trials
[0229] Monitoring the influence of agents (e.g., drugs or
compounds) on the expression or activity of a Kinase and
Phosphatase protein can be applied not only in basic drug
screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as
described herein to increase Kinase and Phosphatase gene
expression, protein levels, or upregulate Kinase and Phosphatase
activity, can be monitored in clinical trials of subjects
exhibiting decreased Kinase and Phosphatase gene expression,
protein levels, or downregulated Kinase and Phosphatase activity.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease Kinase and Phosphatase gene expression,
protein levels, or downregulate Kinase and Phosphatase activity,
can be monitored in clinical trials of subjects exhibiting
increased Kinase and Phosphatase gene expression, protein levels,
or upregulated Kinase and Phosphatase activity. In such clinical
trials, the expression or activity of a Kinase and Phosphatase
gene, and preferably, other genes that have been implicated in a
disorder can be used as a "read out" or markers of the phenotype of
a particular cell.
[0230] For example, and not by way of limitation, genes, including
Kinase and Phosphatase, that are modulated in cells by treatment
with an agent (e.g., compound, drug or small molecule) which
modulates Kinase and Phosphatase activity (e.g., identified in a
screening assay as described herein) can be identified. Thus, to
study the effect of agents on a Kinase and Phosphatase associated
disorder, for example, in a clinical trial, cells can be isolated
and RNA prepared and analyzed for the levels of expression of
Kinase and Phosphatase and other genes implicated in the Kinase and
Phosphatase associated disorder, respectively. The levels of gene
expression (i.e., a gene expression pattern) can be quantified by
Northern blot analysis or RT-PCR, as described herein, or
alternatively by measuring the amount of protein produced, by one
of the methods as described herein, or by measuring the levels of
activity of Kinase and Phosphatase or other genes. In this way, the
gene expression pattern can serve as a marker, indicative of the
physiological response of the cells to the agent. Accordingly, this
response state may be determined before, and at various points
during treatment of the individual with the agent.
[0231] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a Kinase and Phosphatase protein, mRNA,
or genomic DNA in the pre-administration sample; (iii) obtaining
one or more post-administration samples from the subject; (iv)
detecting the level of expression or activity of the Kinase and
Phosphatase protein, mRNA, or genomic DNA in the
post-administration samples; (v) comparing the level of expression
or activity of the Kinase and Phosphatase protein, mRNA, or genomic
DNA in the pre-administration sample with the Kinase and
Phosphatase protein, mRNA, or genomic DNA in the post
administration sample or samples; and (vi) altering the
administration of the agent to the subject accordingly. For
example, increased administration of the agent may be desirable to
increase the expression or activity of Kinase and Phosphatase to
higher levels than detected, i.e., to increase the effectiveness of
the agent. Alternatively, decreased administration of the agent may
be desirable to decrease expression or activity of Kinase and
Phosphatase to lower levels than detected, i.e. to decrease the
effectiveness of the agent. According to such an embodiment, Kinase
and Phosphatase expression or activity may be used as an indicator
of the effectiveness of an agent, even in the absence of an
observable phenotypic response.
[0232] D. Methods of Treatment:
[0233] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant Kinase and Phosphatase expression or activity. With
regards to both prophylactic and therapeutic methods of treatment,
such treatments may be specifically tailored or modified, based on
knowledge obtained from the field of pharmacogenomics.
"Pharmacogenomics", as used herein, refers to the application of
genomics technologies such as gene sequencing, statistical
genetics, and gene expression analysis to drugs in clinical
development and on the market. More specifically, the term refers
the study of how a patient's genes determine his or her response to
a drug (e.g., a patient's "drug response phenotype", or "drug
response genotype".) Thus, another aspect of the invention provides
methods for tailoring an individual's prophylactic or therapeutic
treatment with either the Kinase and Phosphatase molecules of the
present invention or Kinase and Phosphatase modulators according to
that individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
[0234] 1. Prophylactic Methods
[0235] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant Kinase and Phosphatase expression or activity, by
administering to the subject a Kinase and Phosphatase or an agent
which modulates Kinase and Phosphatase expression or at least one
Kinase and Phosphatase activity. Subjects at risk for a disease
which is caused or contributed to by aberrant Kinase and
Phosphatase expression or activity can be identified by, for
example, any or a combination of diagnostic or prognostic assays as
described herein. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the Kinase
and Phosphatase aberrancy, such that a disease or disorder is
prevented or, alternatively, delayed in its progression. Depending
on the type of Kinase and Phosphatase aberrancy, for example, a
Kinase and Phosphatase, Kinase and Phosphatase agonist or Kinase
and Phosphatase antagonist agent can be used for treating the
subject. The appropriate agent can be determined based on screening
assays described herein.
[0236] 2. Therapeutic Methods
[0237] Another aspect of the invention pertains to methods of
modulating Kinase and Phosphatase expression or activity for
therapeutic purposes. Accordingly, in an exemplary embodiment, the
modulatory method of the invention involves contacting a cell with
a Kinase and Phosphatase or agent that modulates one or more of the
activities of Kinase and Phosphatase protein activity associated
with the cell. An agent that modulates Kinase and Phosphatase
protein activity can be an agent as described herein, such as a
nucleic acid or a protein, a naturally-occurring target molecule of
a Kinase and Phosphatase protein (e.g., a Kinase and Phosphatase
phosphorylation substrate), a Kinase and Phosphatase antibody, a
Kinase and Phosphatase agonist or antagonist, a peptidomimetic of a
Kinase and Phosphatase agonist or antagonist, or other small
molecule. In one embodiment, the agent stimulates one or more
Kinase and Phosphatase activities. Examples of such stimulatory
agents include active Kinase and Phosphatase protein and a nucleic
acid molecule encoding Kinase and Phosphatase that has been
introduced into the cell. In another embodiment, the agent inhibits
one or more Kinase and Phosphatase activites. Examples of such
inhibitory agents include antisense Kinase and Phosphatase nucleic
acid molecules, anti-Kinase and Phosphatase antibodies, and Kinase
and Phosphatase inhibitors. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g, by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant expression or activity of a Kinase and
Phosphatase protein or nucleic acid molecule. In one embodiment,
the method involves administering an agent (e.g., an agent
identified by a screening assay described herein), or combination
of agents that modulates (e.g., upregulates or downregulates)
Kinase and Phosphatase expression or activity. In another
embodiment, the method involves administering a Kinase and
Phosphatase protein or nucleic acid molecule as therapy to
compensate for reduced or aberrant Kinase and Phosphatase
expression or activity.
[0238] Stimulation of Kinase and Phosphatase activity is desirable
in situations in which Kinase and Phosphatase is abnormally
downregulated and/or in which increased Kinase and Phosphatase
activity is likely to have a beneficial effect. For example,
stimulation of Kinase and Phosphatase activity is desirable in
situations in which a Kinase and Phosphatase is downregulated
and/or in which increased Kinase and Phosphatase activity is likely
to have a beneficial effect Likewise, inhibition of Kinase and
Phosphatase activity is desirable in situations in which Kinase and
Phosphatase is abnormally upregulated and/or in which decreased
Kinase and Phosphatase activity is likely to have a beneficial
effect.
[0239] 3. Pharmacogenomics
[0240] The Kinase and Phosphatase molecules of the present
invention, as well as agents, or modulators which have a
stimulatory or inhibitory effect on Kinase and Phosphatase activity
(e.g., Kinase and Phosphatase gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) disorders (e.g,
proliferative disorders such as cancer) associated with aberrant
Kinase and Phosphatase activity. In conjunction with such
treatment, pharmacogenomics (i.e., the study of the relationship
between an individual's genotype and that individual's response to
a foreign compound or drug) may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a
physician or clinician may consider applying knowledge obtained in
relevant pharmacogenomics studies in determining whether to
administer a Kinase and Phosphatase molecule or Kinase and
Phosphatase modulator as well as tailoring the dosage and/or
therapeutic regimen of treatment with a Kinase and Phosphatase
molecule or Kinase and Phosphatase modulator.
[0241] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11) :983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0242] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0243] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict a drug
response. According to this method, if a gene that encodes a drug
target is known (e.g., a Kinase and Phosphatase protein or Kinase
and Phosphatase receptor of the present invention), all common
variants of that gene can be fairly easily identified in the
population and it can be determined if having one version of the
gene versus another is associated with a particular drug
response.
[0244] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2Cl9 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0245] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a Kinase and Phosphatase molecule or Kinase and
Phosphatase modulator of the present invention) can give an
indication whether gene pathways related to toxicity have been
turned on.
[0246] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment an individual. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and thus enhance therapeutic or prophylactic efficiency when
treating a subject with a Kinase and Phosphatase molecule or Kinase
and Phosphatase modulator, such as a modulator identified by one of
the exemplary screening assays described herein.
[0247] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Figures and the
Sequence Listing are incorporated herein by reference.
EXAMPLES
Example 1
Identification and Characterization of Human Kinase and Phosphatase
cDNAs
[0248] In this example, the identification and characterization of
the genes encoding human Kinases and Phosphatases is described.
[0249] Isolation of the Human Kinase and Phosphatase cDNAs
[0250] The invention is based, at least in part, on the discovery
of human genes encoding members of the Kinase and Phosphatase
family. The human Kinase and Phosphatase family members were
isolated based on a specific consensus motif or protein domain
characteristic of a Kinase or a Phosphatase family of proteins. The
search of the nucleic acid sequence database was performed with one
or more HMM motifs, a TBLASTN set, or both.
[0251] The TBLASTN set included a set of protein sequence probes
which correspond to amino acid sequence motifs that are conserved
in the protein kinase or the protein phosphatase family of
proteins. The protein families used to construct/select the TBLASTN
sets used in identifying the Kinase and Phosphatase nucleic acid
molecules of the invention are described above.
[0252] The HMM motif included a consensus sequence for a protein
domain. Such consensus sequences can be found in a database of
Hidden Markov Models (HMMs), e.g., the Pfam database, release 2.1,
(http://www.sanger.ac.uk/Software/Pfam,HMM_search). A description
of the Pfam database can be found in Sonhammer et al. (1997)
Proteins 28(3)405-420 and a detailed description of HMMs can be
found in, for example, Gribskov et al.(I990) Meth. Enzymol.
183:146-159; Gribskov et al.(I987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference. The protein families used to
construct/select the HMM motifs used in identifying the Kinase and
Phosphatase nucleic acid molecules of the invention are described
above.
[0253] The sequences of the positive clones were determined and are
set forth herein as SEQ ID NOs: 1-14.
Example 2
Expression of Recombinant Kinase and Phosphatase Proteins in
Bacterial Cells
[0254] In this example, Kinases and Phosphatases are expressed as a
recombinant glutathione-S-transferase (GST) fusion polypeptide in
E. coli and the fusion polypeptide is isolated and characterized.
Specifically, Kinases and Phosphatases are fused to GST and this
fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
Expression of the GST-Kinase and Phosphatase fusion protein in
PEB199 is induced with IPTG. The recombinant fusion polypeptide is
purified from crude bacterial lysates of the induced PEB199 strain
by affinity chromatography on glutathione beads. Using
polyacrylamide gel electrophoretic analysis of the polypeptide
purified from the bacterial lysates, the molecular weight of the
resultant fusion polypeptide is determined.
Example 3
Expression of Recombinant Kinase and Phosphatase Proteins in COS
Cells
[0255] To express the Kinase and/or Phosphatase gene in COS cells,
the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.)
is used. This vector contains an SV40 origin of replication, an
ampicillin resistance gene, an E. coli replication origin, a CMV
promoter followed by a polylinker region, and an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire Kinase and
Phosphatase protein and an HA tag (Wilson et al. (1984) Cell
37:767) or a FLAG tag fused in-frame to its 3' end of the fragment
is cloned into the polylinker region of the vector, thereby placing
the expression of the recombinant protein under the control of the
CMV promoter.
[0256] To construct the plasmid, the Kinase and Phosphatase DNA
sequence is amplified by PCR using two primers. The 5' primer
contains the restriction site of interest followed by approximately
twenty nucleotides of the Kinase and Phosphatase coding sequence
starting from the initiation codon; the 3' end sequence contains
complementary sequences to the other restriction site of interest,
a translation stop codon, the HA tag or FLAG tag and the last 20
nucleotides of the Kinase and Phosphatase coding sequence. The PCR
amplified fragment and the pCDNA/Amp vector are digested with the
appropriate restriction enzymes and the vector is dephosphorylated
using the CIAP enzyme (New England Biolabs, Beverly, Mass.).
Preferably the two restriction sites chosen are different so that
the Kinase and/or Phosphatase gene is inserted in the correct
orientation. The ligation mixture is transformed into E. coli cells
(strains HB101, DH5a, SURE, available from Stratagene Cloning
Systems, La Jolla, Calif., can be used), the transformed culture is
plated on ampicillin media plates, and resistant colonies are
selected. Plasmid DNA is isolated from transformants and examined
by restriction analysis for the presence of the correct
fragment.
[0257] COS cells are subsequently transfected with the Kinase and
Phosphatase-pcDNA/Amp plasmid DNA using the calcium phosphate or
calcium chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of
the Kinase and Phosphatase polypeptide is detected by
radiolabelling (.sup.35S-methionine or .sup.35S-cysteine available
from NEN, Boston, Mass., can be used) and immunoprecipitation
(Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988)
using an HA specific monoclonal antibody. Briefly, the cells are
labelled for 8 hours with .sup.35S-methionine (or
.sup.35S-cysteine). The culture media are then collected and the
cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1%
NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell
lysate and the culture media are precipitated with an HA specific
monoclonal antibody. Precipitated polypeptides are then analyzed by
SDS-PAGE.
[0258] Alternatively, DNA containing the Kinase and/or Phosphatase
coding sequence is cloned directly into the polylinker of the
pCDNA/Amp vector using the appropriate restriction sites. The
resulting plasmid is transfected into COS cells in the manner
described above, and the expression of the Kinase and Phosphatase
polypeptide is detected by radiolabelling and immunoprecipitation
using a Kinase and Phosphatase specific monoclonal antibody.
[0259] Equivalents
[0260] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
18 1 1868 DNA Homo sapiens All occurences of n indicate any
nucleotide 1 gcaattgatc caatatctgc aggtttctaa cacgcaagcc aggacaaaat
actcttcaag 60 gacgtgaaca ggaagctgag tgatgtctgg aaggagctct
cgctgttact tcaggttgag 120 caacgcatgc ctgtttcacc cataagccaa
ggagcgtcct gggcacagga agatcagcag 180 gatgcagacg aagacaggcg
agctttccag atgctaagaa gagataatga aaaaatagaa 240 gcttcactga
gacgattaga aatcaacatg aaagaaatca aggaaacttt gaggcagtat 300
ttaccaccaa aatgcatgca ggagatcccg caagagcaaa tcaaggagat caagaaggag
360 cagctttcag gatccccgtg gattctgcta agggaaaatg aagtcagcac
actttataaa 420 ggagaatacc acagagctcc agtggccata aaagtattca
aaaaactcca ggctggcagc 480 attgcaatag tgaggcagac tttcaataag
gagatcaaaa ccatgaagaa attcgaatct 540 cccaacatcc tgcgtatatt
tgggatttgc attgatgaaa cagtgactcc gcctcaattc 600 tccattgtca
tggagtactg tgaactcggg accctgaggg agctgttgga tagggaaaaa 660
gacctcacac ttggcaagcg catggtccta gtcctggggg cagcccgagg cctataccgg
720 ctacaccatt cagaagcacc tgaactccac ggaaaaatca gaagctcaaa
cttcctggta 780 actcaaggct accaagtgaa gcttgcagga tttgagttga
ggaaaacaca gacttccatg 840 agtttgggaa ctacgagaga aaagacacga
caagagtcaa atctacagca tatctctcac 900 ctcaggaact ggaagatgta
ttttatcaat atgatgtaaa gtctgaaata tacagctttg 960 gaatcgtcct
ctgggaaatc gccactggag atatcccgtt tcaaggctgt aattctgaga 1020
agatccgcaa gctggtggct gtgaagcggc agcaggagcc actgggtgaa gactgccctt
1080 cagagctgcg ggagatcatt gatgagtgcc cgggcccatg atccctctgt
tgcggccctc 1140 tgtggatgaa atcttaaaga aactctccca ccttttctaa
gtagtgtatc aaaatctaaa 1200 ccaaggagtc tctggacaag aagctgggag
aggcacaaac tggacatcat ctcctcttct 1260 catatccttc ggcattgggt
tatctatggg agcaaggagt gggcacgctt ctctgttaca 1320 aatagaaaac
gattccagtc atacaggaca catcccactc caaatgatat ttccaaaaac 1380
atacctctga cagtaacttt gatagatggt ttgtcaaatg tatctttctg ggtatccaca
1440 cctcttggca atgaaatttg cagctcctcc cttccataaa tgaagtctct
ttccccacca 1500 tttgaatctg ggctggcact gtgacttgat ttgatcaata
gaatgtggaa gaagtgactg 1560 tatgccagtt ccaagcctag gtttcaagag
gccttataaa tgtctgttgg aaccttaccc 1620 agccatgaac atgttgagtg
agcatgctgg agaatgagag accacatgaa gcagaaacat 1680 gctttcctag
ctgaagtcat actagcccaa ccaacatggc agctaacaca tgaatgaggc 1740
caatcaagac cagaagaacc actcaagcag atcccagccc aaattgccca ttcacacaat
1800 caggagctaa ataaattact gttgtcttaa aaaaaaaaaa aaannnnana
nnannnnaan 1860 aannaagg 1868 2 403 DNA Homo sapiens All occurences
of n indicate any nucleotide 2 tactaaaggg aataagcttg cggccgcaat
cttttttttt tttttttttt tttttttttt 60 tttttcctaa aanaaaccat
ttattgaaaa aagaaccttt ttcttaaatt tcagtaatca 120 gccttttgca
ttacaaacct gcagcttgcc aacaacaaca aaaaaggtaa nggtttcaca 180
agtcagngca ctcaacaaac aggagattaa aaacaaggna tgggatgcct gataacacta
240 cancttcttt tgaacctana anggtcaaaa cactncgctg atgtcaccgg
ttctcagtcn 300 cgcganacac ggaggaaaca naggcgatgg tccgngtnca
gtagtaangg cacttaacat 360 gcagnttntt taacctcggg gccnccagcc
ctccanaggg cct 403 3 374 DNA Homo sapiens All occurences of n
indicate any nucleotide 3 aatttggccc tcgaggccaa naattcggca
cgaggacata agccaccaat ccctgaaaga 60 ttaagccctg aaggaaagga
cttcctttct cactgccttg agagtgaccc aaagatgaga 120 tggaccgcca
gccagctcct cgaccattcg tttgtcaagg tttgcacaga tgaagaatga 180
agcctagtag aatatggact tggaaaattc tcttaatcac tactgtatgt aatatttaca
240 taaagactgt gctgagaagc aagtataagc cctttttaac cttccaagna
ctgangactg 300 cacaggtgac aagccgtcac ttntcctgct gctcctgttt
tgcctgnatg ttggcaaaaa 360 ngccctctgg aagg 374 4 545 DNA Homo
sapiens All occurences of n indicate any nucleotide 4 ggggtgcacc
atgctggcca taggggtggg gagagagcca ggcgtcaggg agcctgccgc 60
tttgagcagg ccagacagca ggggatcagg tttcagccca gcttgtattt ttggctgggt
120 gggctggggg ccgaacaggc agcgtcagct gcccatcccc agcacacacc
acactcacac 180 tacctttacc cagatagggc accatccact gcccccaccc
ccccagcccc ctgcaggcag 240 aaaggaatca ggtgctcctg ggctattgaa
tnactttagg ntttnttgga ctgtggctaa 300 ttttttggaa ggncttcttg
atccgcaacg cnggtgagnn nggcagaggg gtttggntac 360 cagnactccc
gcatnatntn angctagngc ctgagangac ccgggtctgc naccaacccg 420
gtttagggat gggggggggt ctgttgantc cncacacncc accttnttta tgtcctcaaa
480 ggctggggtc nttgggccac cacattnnta caaaggggtn ggtctataan
tcctcccncg 540 aatgc 545 5 361 DNA Homo sapiens All occurences of n
indicate any nucleotide 5 cgcgtccgag cagtgccctc naggatcctg
aagcagtaca gccaccccaa catcgtgcgt 60 ctcattggtg tctgcaccca
gaagcagccc atctacatcg tcatggagct tgtgcagggg 120 ggcgacttcc
tgaccttcct ccgcacggag ggggcccgcc tgcgggtgaa gactctgctg 180
cagatggtgg gggatgcagc tgctggcatg gagtacctgg agagcaagtg ctgcatccac
240 cgggacctgg ctgctcggaa ctgcctggtg acagagaaga atgtcctgaa
gaatcagtga 300 ctttggggat gtcccgagag gaagcccgat tgggggtcta
tgcagcctca gggggcctca 360 g 361 6 467 DNA Homo sapiens 6 gatggtttat
tccaaagctg tggacggtga acattaagac gaaagaggtg actcgcgtgg 60
aacctgaaac acggacgcct ttcttccaag aagggctgtg gcgatcaggc cactcaaggc
120 agccagcccc tcagcagggc acgatggcta atgctgcctg gaccgcagga
ctttttgttc 180 gatttcaaga caccagactc cctctccttc tcagggtcga
agacttctgg atgaggaggc 240 tgtgacagat attgcagatc ttcaccgcgt
cggggtacgt ctcccggatg tattgctcca 300 tctccaggat ggcccggccg
tgcagggtga actccccttc cttctcaatc agccacttgt 360 tttgaacaaa
cttctgcagc acctgctccg cttccttctt cctcatcttc ttgcctttaa 420
gttgatcaac caggttcaat atgtttgtgg aagacgcaaa gccggtt 467 7 1260 DNA
Homo sapiens All occurences of n indicate any nucleotide 7
aagggaataa gcttgcggcc gcatctnntt tnnntnnnnn tttntttttt tttttttttt
60 ttgcagcaac ttcccaggat ggtttattcc aaagctgtgg acggtgaaca
ttaagacgaa 120 agaggtgact cgcgtggaac ctgaaacacg gacgcctttc
ttccaagaag ggctgtggcg 180 atcaggccac tcaaggcagc cagcccctca
gcagggcacg atggctaatg ctgcctggac 240 cgcagggact ttttgttcga
tttcaagaca ccagactccc tctccttctc agggtcgaag 300 acttttggga
tctcgtgggg ccagtagtcg ttgcagtggg ggcagcgcgg ttcagcattc 360
gactggaagt acttggccac gcagggtaag tgcatcctga tcccacaggt ttcgcagctt
420 tgaccctgga tgaggaggct gtgacagata ttgcagatct tcaccgcgtc
ggggtacgtc 480 tcccggatgt attgctccat ctccaggatg gcccggccgt
gcagggtgaa ctccccttcc 540 ttctcaatca gccacttgtt ttgaacaaac
ttctgcagca cctgctccgc ttccttcttc 600 ctcatcttct tgcctttaag
ttgatcaacc aggttcaata tgtttgtgga agacgcaaag 660 ccggtttctg
agtcaataat cagttccaga gcctttctaa acaaatccag ttcattctct 720
gcaaaatccg tagccatttt ggaaattgaa gttgtagcaa gattcaccaa cgcataaatg
780 ggtctcccat catcttccgt gactcctctc tttatctcaa tatacaagga
ctccaagaca 840 ctgttaatgt tgttgatgaa gtcctccaac ttatctacgg
tggcattgcg gtcatggacc 900 ttgtagcagt gcgtctgcaa gcgcttcacg
tcccattcct ctagcacgcc atgggtcatc 960 agcaactgga ggaagcgccg
gtggacatca gtcatgacgc ccattctcct tgtgctgccc 1020 tgcatgtggg
aacgaacagg gagcccaagc gcatcccagg ccgcgctagc ggatacggcc 1080
tcgaggctgt aacataagcg gctgcggact tgtacaaagt aagaaactcc gtacacaaaa
1140 cagaggggag cacggagggc ggcgggacaa agagggtggt cggcaaaggc
tgtattttcc 1200 atagatgtaa tcacagtttg aatcgaaata caactactcc
ttcctcacca ctcagcccaa 1260 8 473 DNA Homo sapiens All occurences of
n indicate any nucleotide 8 cgcgtccgcg cggtgtatgc tgagccgctg
ccgcagcggg ctgctccacg tcctgggcct 60 tagcttcctg ctgcagaccc
gccggccgat tctcctctgc tctccacgtc tcatgaagcc 120 gctggtcgtg
ttcgtcctcg gcggccccgg cgccggcaag gggacccagt gcgcccgcat 180
cgtcgaggaa atggatcaga caatggctgc caatgctcag aagaataaat tcttgattga
240 tgggtttcca agaaatcaag acaaccttca aggatggaac aagaccatgg
atgggaaggc 300 agatgtatct ttcgttctct tttttgactg taataatgag
atttgtattg aacgatgtct 360 tgagagggga aagagtagtg gtgnnanttg
gatgacaaca nagagagctt ggaaaagaga 420 attcagncct accttcagnc
aacaaagcca atttattgac ttatattgaa gaa 473 9 3001 DNA Homo sapiens 9
cggcgccggc tcagcccgcc cctttctccc gccgcctccc cgccccgccc cgcgccgcgc
60 cggccgctgt cagctccctc agcgtccggc cgaggcgcgg tgtatgctga
gccgctgccg 120 cagccggctg ctccacgtcc tgggccttag cttcctgctg
cagacccgcc ggccgattct 180 cctctgctct ccacgtctca tgaagccgct
ggtcgtgttc gtcctcggcg gccccggcgc 240 cggcaagggg acccagtgcg
cccgcatcgt cgagaaatat ggctacacac acctttctgc 300 aggagagctg
cttcgtgatg aaaggaagaa cccagattca cagtatggtg aacttattga 360
aaagtacatt aaagaaggaa agattgtacc agttgagata accatcagtt tattaaagag
420 ggaaatggat cagacaatgg ctgccaatgc tcagaagaat aaattcttga
ttgatgggtt 480 tccaagaaat caagacaacc ttcaaggatg gaacaagacc
atggatggga aggcagatgt 540 atctttcgtt ctcttttttg actgtaataa
tgagatttgt attgaacgat gtcttgagag 600 gggaaagagt agtggtagga
gtgatgacaa cagagagagc ttggaaaaga gaattcagac 660 ctaccttcag
tcaacaaagc caattattga cttatatgaa gaaatgggga aagtcaagaa 720
aatagatgct tctaaatctg ttgatgaagt ttttgatgaa gttgtgcaga tttttgacaa
780 ggaaggctaa ttctaaacct gaaagcatcc ttgaaatcat gcttgaatat
tgctttgata 840 gctgctatca tgaccccttt ttaaggcaat tctaatcttt
cataactaca tctcaattag 900 tggctggaaa gtacatggta aaacaaagta
aattttttta tgttcttttt tttggtcaca 960 ggagtagaca gtgaattcag
gtttaacttc accttagtta tggtgctcac caaacgaagg 1020 gtatcagcta
ttttttttta aattcaaaaa gaatatccct tttatagttt gtgccttctg 1080
tgagcaaaac tttttagtac gcgtatatat ccctctagta atcacaacat tttaggattt
1140 agggatacct gcttcctctt tttcttgcaa gttttaaatt tccaacctta
agtgaatttg 1200 tggaccaaat ttcaaaggaa ctttttgtgt agtcagttct
tgcacaatgt gtttggtaaa 1260 caaactcaaa atggattctt aggagcattt
tagtgtttat taaataactg accatttgct 1320 gtagaaagat gagaaaactt
aagctttgtt ttactacaac ttgtacaaag ttgtatgaca 1380 gggcatattc
tttgcttcca agatttgggt tgggggcact aggggttcag agcctggcag 1440
aattgtcagc tttagtctga cataatctaa gggtatgggg caaggatcac atctaatgct
1500 tgtgttcctt atactctatt atatagtgtt attcatgatt cagctgatct
taacaaaatt 1560 cgtagcagtg gaaccttgaa atgcatgtgg ctagatttat
gctaaaatga ttctcagtta 1620 gcattttagt aacacttcaa aggttttttt
ttgtttgttt tctagactta ataaaagctt 1680 aggattaatt agaagaagca
atctagttaa atttcccatt tgtattttat tttcttgaat 1740 acttttttca
tagttatttg tttaaaaaga tttaaaaatc attgcacttt ggtcagaaaa 1800
ataataaata tatcttataa atgtttgatt cccttccttg ctatttttat tcagtagatt
1860 tttgtttggc atcatgttga agcaccgaaa gataaatgat ttttaaaagg
ctatagagtc 1920 caaaggaata ttcttttaca ccaattcttc ctttaaaaat
ctctgaggaa tttgttttcg 1980 ccttactttt ttttcttctg tcacaatgct
aagtggtatc cgaggttctt aatatgagat 2040 ttaaaatctt aaaatgtttc
ttattttcag cacttacatc atttggtaca cagggtcaaa 2100 tagggcaaat
aattttgtct ttgtataata gatttgatat ttaaagtcac tggaaatagg 2160
acaagttaat ggatgttttt atattttaat agaatcattt atttctatgt gttatgaaat
2220 tcacttaatg ataaattttt caacatactt gccattagaa aacaaagtat
tgctaagtac 2280 tataacatat tggccactaa aattcatatt gagattatct
tggtttcttg gaagagatag 2340 gaatgagttc ttatctagtg ttgcaggcca
gcaaatacag aggtggttta atcaaacagc 2400 tctagtatga agcaagagta
aagactaagg tttcgagagc attcctactc acataagtga 2460 agaaatctgt
cagataggaa tctaaatatt tatagtgaga ttgtgaaagc aaccttaaag 2520
ttttgaagaa gactgatgag actaggtgct ttgcttcctt tcatcaggta tctttctgtg
2580 gcatttgaga acagaaacca agaaacatgg taattactaa attatgaggc
tttgcttttt 2640 gtttgctttt aagtagaaaa acatgttggc aacattgagt
tttggagttg attgagataa 2700 tatgacttaa ctagttttgt cattccattt
gttaaagata cagtcaccaa gaatgttttg 2760 agttttttga aagaccccaa
tttaagcctt gcttattttt aaattatttc cattcagtga 2820 tgttggatgt
atatcagtta tttagtaaat aatctcaata aattttgtgc tgtggccttt 2880
gctaaaaaaa aaaaaaaata ttggtgactg tatctttaac aaatggaatg acaaaactag
2940 ttaagtcata ttatctcaat caactccaaa actcaatgtt gccaacatgt
ttttctactt 3000 a 3001 10 526 DNA Homo sapiens All occurences of n
indicate any nucleotide 10 ncncgtccgg tgacctgaag tcggacaaca
ttctggtgtg gtcccttgac gtcaaggagc 60 acatcaacat caagctatct
gactacggga tttcgaggca gtcattccat gagggcgccc 120 taggcgtgga
gggcactcct ggctaccagg ccccagagat caggcctcgc attgtatatg 180
atgagaaggt agatatgttc tcctatggaa tggtgctcta cgagttgctg tcaggacagc
240 gccctgcact gggccaccac cagctccaga ttgccaagaa gctgtccaag
ggcatccgcc 300 cggttctggg gcagccggag gaagtgcagt tccggcgact
gcaggcgctc atgatggagt 360 gctgggacac taagccagag aagcgaccac
tggccctgtc ggtggtgaag ccagatgaag 420 gacccgactt ttgccacctt
catgtatgaa ctgtgctgtg ggaagcagac agccttcttc 480 ttcatnccag
ggccaggagt acactgtggg tgttttggga tggaaa 526 11 683 DNA Homo sapiens
All occurences of n indicate any nucleotide 11 tttttttttt
caataacaaa ggtccagtat tacccacaac aaagacaaat attttcaaca 60
tagaacaata agagatattg ataccctatg agcttgttac atctctgtca ttttacacat
120 tgagatcaaa atccaaacac caggaggccc tctggtaaaa gagtgctggc
tgcctaccca 180 acattctccc ctaatgtctt agtgtcagaa cccctttgtt
attagggata gtcacgtacc 240 cagcaaataa gccacatctc ccagcctcca
ttccaggtag gggtgggtgg ttagtgagat 300 ggaagcagaa gtcattgggt
ggagcttttg ggaaagctct ttaaaagcgc cctttgctct 360 tctccccttt
cctccatttt cccttcccta aacacaaaat aggcagctag agctccagta 420
accatcttgt agcaaaccta acattggaag ccatttgtca aggttggcag ggcagagata
480 cagcctctga gtatctgatg acctgtcctg ccagtcctgg actccgaaac
attatttttc 540 ttttatgtgc tggaaaaaat aaacctgctt ctcgttgaag
ccattgntag tttgagtcgc 600 tcttcctagc agctgaaggt aattccttac
taataccagc tggcctcagt agtttcacag 660 gtccttccgg acgcggggtc gac 683
12 1448 DNA Homo sapiens All occurences of n indicate any
nucleotide 12 tcagatccca atctcccccc aagtatctcg tcacaaatca
cactatcgta atcgagaaca 60 ctttgctact atacggacag catcactggt
tacgaggcaa atgcaagaac atgagcagga 120 ctctgagctt agagaacaaa
tgtctggcta taagcggaat gaggcgacaa catcaaaagc 180 aactgatgnc
tctggaaaac aagctaaagg ctgagatgga tgaacatcgc ctcagattag 240
acaaagatct tgaaactcag cgtaacaatt ttgctgcaga aatggagaaa cttatcaaga
300 aacaccaggc tgctatggag aaagaggcta aagtgatgtc caatgaagag
aaaaaatttc 360 agcaacatat tcaggcccaa cagaagaaag aactgaatag
ttttctcgag tcccagaaaa 420 gagagtataa acttcgaaaa gagcagctta
aagaggagct aaatgaaaac cagagtaccc 480 ccaaaaaaga aaaacaggag
tggctttcaa agcagaagga gaatatacag catttccaag 540 cagaagaaga
agctaccttc ttcgacgtca aagacaatac ctagagctgg aatgcccgtc 600
gcttcaagag aagaatgtta cttgggcgtc ataacttaga gcaggacctt gtcagggagg
660 agttaaacaa aagacagact cagaaggact tagagcatgc catgctactc
cgacagcatg 720 aatctatgca agaactggag ttccgccacc tcaacacaat
tcagaagatg cgctgtgagt 780 tgatcagatt acagcatcaa actgagctca
ctaaccagct ggaatataat aagcgaagag 840 aacgagaact aagacgaaag
catgtcatgg aagttcgaca acagcctaag agtttgaagt 900 ctaaagaact
ccaaataaaa aagcagtttc aggatacctg caaaatccaa accagacagt 960
acaaagcatt aagaaatcac ctgctggaga ctacaccaaa gagtgagcac aaagctgttc
1020 tgaaacggct caaggaggaa cagacccgga aattagctat cttggctgag
cagtatgatc 1080 acagcattaa tgaaatgctc tccacacaaa ccgtgggttt
gctttttttg gggcaaaaca 1140 aatttagtgc cccttttctt cccccacctg
aacgaaatca cagcaattaa agtactagtt 1200 ggaaatgata gctctcccga
gcttatcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac 1260 aacatggtga
aaccctgtct ctgctgaaag tacaaaaatt agttgggtgt ggcggcacat 1320
gcctgtggtc ccagctactc ggggggctga agcaggagaa tcccttgaac ccaggaggca
1380 gaggttgcag tgagtcaaga acacaccaca gcactccagc ccgggtgaca
gagcaagacc 1440 ctgtctca 1448 13 521 DNA Homo sapiens 13 tctcggcccg
gctgcgccag agtccgcgcg atggagcccc ggccgcggcg gcggcgcagg 60
agtcgccccc tggtcgccgc cttcctgcga gacccgggct cgggccgcgt gtacaggcgc
120 gggaagctga tcggcaaggg cgccttcagc cgctgctaca agctgacaga
catgtccacc 180 agcgccgtgt tcgccctcaa ggtggtgccg tgtggcgggg
ctggggccgg gtggcttcgc 240 ccgcagggaa aggtggagcg tgagattgcc
ctgcatagcc gcctgcgacc ccgcaacatc 300 gtggctttcc acggacactt
tgctgaccgc gaccacgtgt acatggtgct ggagtactgc 360 agccgccagt
ctttggccca cgtgctgagg gcgcggcaga tcctgacgga gccagaagtg 420
cgcgactacc tgcggggcct ggtcagcggc ctgcgctacc tgcaccagcg gtgcatcctg
480 caccgcgacc tgaagctcag taacttcttc cttaacaaga a 521 14 481 DNA
Homo sapiens 14 ggctgatttt ggtcttgccc gggccaagtc cattcccagc
cagacatact cttcagaagt 60 cgtgaccctc tggtaccggc cccctgatgc
tttgctggga gccactgaat attcctctga 120 gctggacata tggggtgcag
gctgcatctt tattgaaatg ttccagggtc aacctttgtt 180 tcctggggtt
tccaacatcc ttgaacagct ggagaaaatc tgggaggtgc tgggagtccc 240
tacagaggat acttggccgg gagtctccaa gctacctaac tacaatccag aatggttccc
300 actgcctacg cctcgaagcc ttcatgttgt ctggaacagg ctgggcaggg
ttcctgaagc 360 tgaagacctg gcctcccaga tgctaaaagg ctttcccaga
gaccgcgtct ccgcccagga 420 agcacttgtt catgattatt tcagcgccct
gccatctcag ctgtaccagc ttcctgatga 480 g 481 15 86 PRT Homo sapiens
ANY 13 OF THE Xaa's AT POSITIONS 42-59 MAY BE ABSENT-INTENDED TO
EQUAL A RANGE OF 5-18 AMINO ACIDS 15 Leu Ile Val Gly Pro Gly Pro
Phe Tyr Trp Met Gly Ser Thr Asn His 1 5 10 15 Ser Gly Ala Pro Trp
Leu Ile Val Cys Ala Thr Pro Asp Xaa Gly Ser 20 25 30 Thr Ala Cys
Leu Ile Val Met Phe Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Ile Val Met Phe 50 55
60 Tyr Trp Cys Ser Thr Ala Arg Ala Ile Val Pro Leu Ile Val Met Phe
65 70 75 80 Ala Gly Cys Lys Arg Lys 85 16 46 PRT Homo sapiens AT
POSITIONS 8,11,20,21 Xaa = ANY AMINO ACID 16 Leu Ile Val Met Phe
Tyr Cys Xaa His Tyr Xaa Asp Leu Ile Val Met 1 5 10 15 Phe Tyr Lys
Xaa Xaa Asn Leu Ile Val Met Phe Tyr Cys Thr Leu Ile 20 25 30 Val
Met Phe Tyr Cys Thr Leu Ile Val Met Phe Tyr Cys Thr 35 40 45 17 47
PRT Homo sapiens AT POSITIONS 8,11,24, AND 25 Xaa = ANY AMINO ACID
17 Leu Ile Val Met Phe Tyr Cys Xaa His Tyr Xaa Asp Leu Ile Val Met
1 5 10 15 Phe Tyr Arg Ser Thr Ala Cys Xaa Xaa Asn Leu Ile Val Met
Phe Tyr 20 25 30 Cys Leu Ile Val Met Phe Tyr Cys Leu Ile Val Met
Phe Tyr Cys 35 40 45 18 33 PRT Homo sapiens AT POSITIONS 29,30, AND
31 Xaa = ANY AMINO ACID 18 Leu Ile Val Met Phe Tyr Trp Leu Ile Val
Met Phe
Tyr Trp Leu Ile 1 5 10 15 Val Met Phe Tyr Trp Asp Gly Phe Tyr Ile
Pro Arg Xaa Xaa Xaa Asn 20 25 30 Gln
* * * * *
References