Note: Descriptions are shown in the official language in which they were submitted.
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MAMMALIAN CHK1 EFFECTOR CELL-CYCLE CHECKPOINT
PROTEIN KINASE MATERIALS AND METHODS
FIELD OF THE INVENTION
The present invention generally cell cycle checkpoint protein kinases which
are essential to cellular DNA damage responses and coordinating cell cycle
arrest. The
checkpoint kinases play a role in the surveillance and response to DNA damage
that
occurs as a result of replication errors, DNA mismatches, radiation treatment,
or
chemotherapy. These checkpoint kinases are required in regulatory pathways
that lead
to cell cycle arrest and apoptosis following DNA damage, giving the cell
notice and time
to correct lesions prior to the initiation of DNA replication or chromosome
separation.
More particularly, the present invention relates to novel mammalian effector
(Chk 1 )
checkpoint protein kinases, polynucleotides encoding the same, and methods and
materials
for assaying and modulating the enzymatic activity of the kinases.
BACKGROUND
The cell cycle is structurally and functionally conserved in its basic process
and mode of regulation across all eukaryotic species. The process of
eukaryotic cell
growth and division is the somatic (mitotic} cell cycle which consists of four
phases, the
G1 phase, the S phase, the G2 phase, and the M phase. The Gl, S, and G2 phases
are
collectively referred to as interphase of the cell cycle. During the Gl (gap)
phase,
biosynthetic activities of the cell progress at a high rate. The S (synthesis)
phase begins
when DNA synthesis starts and ends when the DNA content of the nucleus of the
cell has
been replicated and two identical sets of chromosomes are formed. The cell
then enters
the G2 (gap) phase which continues until mitosis starts. In mitosis, the
chromosomes pair
and separate and two new nuclei form, and in cytokinesis, the cell itself
splits into two
daughter cells each receiving one nucleus containing one of the two sets of
chromosomes.
Mitosis (the M phase of the cell cycle) is immediately followed by
cytokinesis.
Cytokinesis terminates the M phase and marks the beginning of interphase of
the next cell
cycle. The sequence in which the events in the cell cycle proceed is tightly
regulated such
that the initiation of one cell cycle event is dependent on the completion of
the prior cell
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cycle event. This allows fidelity in the duplication and segregation of
genetic material
from one generation of somatic cells to the next.
Meiosis is the form of cell division that produces germ cells in higher
eukaryotes. In contrast to mitosis, where mitotic cell division results in
genetically
identical cells containing two of each chromosome, meiotic cell division
results in cells
containing one copy of each chromosome. In addition, in meiosis homologous
chromosomes pair and exchange genetic material. Meiosis consists of two stages
of cell
division, meiosis I and meiosis II, In meiosis I, maternal and paternal
chromosomes
duplicate and homologous chromosomes pair together (synapsis). The cell then
undergoes division in which homologous pairs of duplicate chromosomes separate
and
enter individual cells resulting in two diploid daughter cells. The daughter
cells then enter
meiosis II. In meiosis II, the chromosomes align without further replication
and sister
chromatids separate, as in mitosis, to produce haploid cells. The sequence of
events in
both meiosis I and meiosis II are interphase, prophase, metaphase, anaphase,
and
telophase.
The first stage of meiosis I is interphase I in which each chromosome is
replicated. The two copies of the replicated chromosome are called sister
chromatids.
Five sequential stages then define the first meiotic prophase. During
leptotene, the newly
replicated sister chromatids are in close apposition so that they may
associate and undergo
recombination. During zygotene, a proteinaceous structure termed the
synaptonemal
complex forms between maternal sister chromatids and paternal sister
chromatids resulting
in a bivalent (four chromatids). During pachytene, recombination between two
sister
chromatids (i.e. exchange of genetic material between maternal and paternal
chromosomes) begins. The next stage, diplotene, is marked by the disassembly
of the
protein axes and the two sister chromatids begin separating. Diakinesis, the
final stage,
is characterized by detachment of the chromosomes from the nuclear envelope
and each
bivalent is clearly seen to contain four separate chromatids, with each pair
of sister
chromatids linked at their centromeres. Thus, early in meiosis during the
"reduction
division" process, sister chromatids pair and undergo reciprocal recombination
at some
regions. Programmed DNA strand breaks initiate recombination. [Cao et al.,
Cell,
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88:3 75-3 84 ( 1997)) . The changes observed during the first meiotic prophase
facilitates
the genetic reassortment that assures genetic viability.
The process of monitoring genome integrity and preventing cell cycle
progress in the event of DNA damage has been described as "cell cycle
checkpoint"
[Hartwell and Weinert Science, 246:629-634 (1989); Weinert et al., Genes and
Dev.,
8:6S2 ( 1994)). Cell cycle checkpoints consist of signal transduction cascades
which
couple DNA damage detection to cell cycle progression. In meiosis, cell cycle
checkpoints control programmed DNA breaks, ensuring the proper segregation of
a
complete haploid set of chromosomes to each gamete.
Failure of cell cycle checkpoints predisposes individuals to or directly
causes many disease states such as cancer, ataxia telangiectasia, embryo
abnormalities, and
various immunological defects associated with aberrant B and T cell
development. The
latter are associated with pathological states such as lupus, arthritis and
autoimmune
diseases. Intense research efforts have therefore focused on identifying cell
cycle
checkpoints and the proteins essential for the fiznction of the checkpoints.
It has been reported that cell cycle checkpoints comprise at least three
distinct classes of polypeptides which act sequentially in response to cell
cycle signals or
defects in chromosomal mechanisms. [Can, A.M., Science, 271:314-315 (1996)).
The
first class is a family of proteins which detect or sense DNA damage or
abnormalities in
the cell cycle. These sensors include Atm and Atr [Keegan et al., Genes and
Devel.,
10:2423-2437 (1996)]. The second class of polypeptides amplify and transmit
the signal
detected by the detector and is exemplified by Rad53 [Alen et al. (1994) Genes
Dev.
8:2416-2488]. Finally, the cell cycle checkpoint effects a cellular response,
e.g. arrest of
mitosis/meiosis, apoptosis through cell cycle effectors.
Genetic analysis in the yeasts Schizosaccharomyces pombe and
Saccharomyces cerevisiae has identified a number of checkpoint genes important
for
mitotic arrest and DNA repair responses to IR. For a review, see Carr and
Hoekstra,
Trends in Cell Biology, 5: 32-40 ( 1995). One such gene, identified in yeasts,
is required
for a DNA damage checkpoint which arrests mitosis at the G2 phase, as well as
a related
checkpoint which monitors the completion of DNA synthesis and arrests the cell
cycle at
the S phase. The gene is named rad3 in S. pombe [Seaton et al., Gene, 119: 83-
89 (l992)
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Bentley et al., ( 1996) EM BO J. 15: 6641-6651 ], MEC 1 /ESR1 in S. cerevisiae
[Kato and
Ogawa, Nuc. Acids. Res., 22(1 S): 3104-3112 (l994)], but is hereinafter
referred to as
rad3. Cells having mutations in rad3 fail to either sense or appropriately
respond to DNA
damage and subsequently lose viability more rapidly than wild type cells after
exposure
to clastogenic agents or events (e.g., IR, DNA damaging agents, and mutations
affecting
chromosomal integrity). See Weinert et al., GENES & DEVELOPMENT, 8: 652-665
(1994) and Al-Khodairy et al., EMBO J., 11(4): 1343-1350 (1992). Rad3 thus
appears
to be a checkpoint detector of DNA damage (Carr, 1996). In addition rad3
appears to
function in vivo as a multimer. [Bentley et al., 1996].
The product of the rad3 gene is an approximately 270 kD protein that is
a member of a growing family of high molecular weight mammalian checkpoint
kinases.
See Hunter, Cell, 83: 1-4 (1995) for a discussion of this family of kinases.
This family
includes ecll (S. cerevisiae) mei-41 (Drosophila melanogaster), torl (S.
cerevisiae), tort
(S. cerevisiae}, Frap (Human), tell (S. cerevisiae), DNA-Pk (Human) Atr
(human) and
1 S Atm (human). These proteins have been identified as members of a family
based on
sequence homology and complementation studies.
The human homolog of rad3, Atr (Ataxia Telangiectasia and rad3 related)
was identified in Bently et al., EMBO J., 15:6641-66S1 (l996). Bently et al.,
showed that
recombinant Atr can heteromultimerize with rad3 when expressed in S. pombe. In
addition, recombinant Atr expression complemented S. cerevisiae mecl mutants.
The primary structures of the catalytic domains found in members of this
kinase family are closely related to well characterized phosphatidylinositol
kinases. This
structural relationship initially suggested that these mammalian checkpoint
kinases might
be capable of phosphorylating lipids. However, when the substrate specificity
of the
mammalian checkpoint kinases is examined, these enzymes appear to fi~nction as
protein
kinases and have yet to be demonstrated to phosphorylate
phosphatidylinositides.
Atm (Ataxia Telangiectasia Mutated), another member of this family was
identified through the analysis of the human disease syndrome ataxia-
telangiectasia (AT)
[Savitsky et al., Science, 268:1749-1753 (1995) and Savitsky et al., Human
Molecular
Genetics, 4(l1):2025-2032 (1995)]. Patients with AT exhibit a diverse set of
clinical
symptoms, including predisposition to a variety of tumor types. Fibroblasts
from AT
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patients are radiosensitive and fail to undergo mitotic arrest following
treatment with IR.
Mutant mice lacking Atm show gonadal atrophies, meiotic abnormalities and
severe
chromosome fragmentation [Ashley et al., Proc. Nat. Acad. Sci. USA, 93:13084 (
1996)J.
This is reminiscent of the S. pombe stains with rad3 defects where cells fail
to sense or
respond appropriately to DNA damage.
This family of kinases thus appear to function as detectors for defects of
various cell-cycle transitions [Carr, 1996J.
Recently it was shown that the mammalian Atm and Atr proteins associate
with chromosomes during pachynema of meiotic prophase and may monitor strand
disruptions that occur during meiotic chromosome synapsis and recombination.
Localization of Atm and Atr kinases shows complementary patterns of foci
during
zygonema and pachynema, commensurate with different roles in monitoring the
DNA
structure during meiotic recombination [Keegan et al., Genes Dev., 10:2423 (
1996)J.
Checkpoint proteins clearly play a role in signal transduction cascades.
The detectors Atm and Atr are protein kinases that comprise a early step in
the signal
transduction cascade. It is thought that signally is amplified by protein
kinases such as
Rad53 which acts to transduce a signal from the detectors to the effectors.
To date, p53 and S. pombe Chkl and weel have been identified as effector
checkpoint proteins.
The S. pombe Chkl gene was isolated based on its genetic interaction with
the cdc2.r4 allele [Al-Khodairy et al., Mol. Biol. Cell, 5:147-160 (1994)J,
and by
complementation of the radiation sensitivity of a rad27 mutant. cdc2 encodes a
protein
kinase subunit that associates with cyclins to form active protein kinase
complexes that
induce passage through mitosis. [Broek et al., Nature, 349:388-393 (1991J. S.
pombe
Chkl appears to effect the mitotic arrest following DNA damage and S. pombe
Chkl
deletion mutants fail to undergo cell-cycle arrest after irradiation [Walworth
et al. ,
Nature, 3 63 :3 68 ( 1993 ), Al-Khodairy et al., Mol. Biol. Cell., 5:147-160 (
1994), Carr,
A.M., Semin. Cell Biol., 6:65-72 (1995)J. However, Chkl is not required for
the other
checkpoint protein-mediated DNA cellular responses to blocks to DNA
replication.
Walworth and Bernards Science, 271:353-356 (1996) demonstrated that in vivo
activity
of Chkl is regulated by phosphorylation of Chkl. By examining the
phosphorylation
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status of Chkl in various S. pombe strains harboring mutations in checkpoint
genes,
Walworth and Bernards, Science, 27I:353-356 (1996) demonstrated that Chkl acts
downstream of these checkpoint proteins. Chkl phosphorylation was abolished or
greatly
diminished in S. pombe rad 1, rad 3, rad 9, rad 17, and rad26 mutants.
In addition, S. pombe Chkl appears to function during the G1 and G2
phases of mitosis. Carr et al., Curr. Biol., 5:1 l79-1190 (1995) demonstrated
that Chkl
deficient cells failed to enter the S phase indicating that Chk 1 is a G 1
checkpoint kinase.
It was demonstrated in O'Connell et al., EMBO Journal, 16:545-554 ( 1997) that
Chk 1
phosphorylated weel, a checkpoint kinase involved in G2 cell cycle arrest.
The Drosophila homolog of S. pombe Chkl was identified as "Grp" in
Sibon et al., Nature, 388:93-97 (1997). Grp is required for cell cycle control
at the
mid-blastula transition (MBT) in which the maternal component of the DNA-
replication
machinery slows DNA synthesis and induces a checkpoint-dependent delay in cell
cycle
progression during embryogenesis.
The C. elegans homolog of Chkl was first reported as an EST in Genbank
Accession No. U44902. The S. cerevisiae homolog of Chkl was identified as a
probable
ser/thr protein kinase in Genbank Accession No. 585344.
To date, there has been no identification of a mammalian effector
checkpoint (Chkl) protein kinase. There thus exists a need in the art for
identification of
the mammalian effector proteins that are involved in the cell cycle
checkpoints in order
to develop therapies for the human disease states associated with defective
cell cycle
checkpoints and for the isolation of polynucleotides encoding those proteins
which in
themselves may be useful as therapeutics or which would enable the development
of
therapeutically usefial modulators of the proteins encoded by the
polynucleotides.
SUMMARY OF THE INVENTION
The present invention provides novel human mammalian effector cell cycle
checkpoint, Chkl, kinases and polynucleotides encoding the same.
In one of its aspects, the present invention provides purified and isolated
polynucleotides (e.g., DNAs and RNAs, both coding and non-coding strands
thereof)
encoding the human and mouse effector cell cycle checkpoint kinase and
polynucleotides
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encoding other mammalian checkpoint kinases that exhibit 50% or greater amino
acid
identity to the polynucleotide region encoding the human Chkl kinase domain
(amino
acids 14 to 264 of SEQ ID NO.: 2). Preferably, the polynucleotides encode a
checkpoint
kinase that exhibits 70% or greater amino acid identity to amino acids 14 to
264 of SEQ
m NO.: 2. Even more preferably, the polynucleotides encode a checkpoint kinase
that
exhibits 90% or greater amino acid identity to amino acids 14 to 264 of SEQ B7
NO.: 2.
Polynucleotides contemplated by the invention include genomic DNAs, RNAs,
cDNAs
and wholly or partially chemically synthesized DNAs. Preferred polynucleotides
of the
invention comprise the human Chkl DNA sequence set out in SEQ ID NO.: 1, the
mouse
Chkl DNA sequence set out in SEQ 117 NO.: 3, and DNA sequences which hybridize
to
the noncoding strands thereof under stringent conditions or which would
hybridize but for
the redundancy of the genetic code. Exemplary stringent hybridization
conditions are as
follows: hybridization at 65 ° C in 3X SSC, 20 mM NaP04 pH 6.8 and
washing at 65 °
C in 0.2X SSC. It is understood by those of skill in the art that variation in
these
conditions occurs based on the length and GC nucleotide base content of the
sequences
to be hybridized. Formulas standard in the art are appropriate for determining
exact
hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning,
Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). A
polynucleotide vector encoding human Chkl (plasmid pGEMT-Chklhu ) was
deposited
with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD
20852
on date August 27, 1997 under Accession No. ATCC98520.
The DNA sequence information provided by the present invention makes
possible the identification and isolation of DNAs encoding mammalian
checkpoint related
molecules by well-known techniques such as DNA/DNA hybridization as described
above
and polymerase chain reaction (PCR) cloning. As one series of examples,
knowledge of
the sequence of a cDNA encoding a mammalian Chkl of the invention makes
possible the
isolation by DNA/DNA hybridization of genomic DNA sequences encoding the
kinase and
expression control regulatory sequences such as promoters, operators and the
like.
Similarly, knowledge of a partial cDNA sequence makes isolation of a complete
cDNA
possible. DNA/DNA hybridization procedures carried out with DNA sequences of
the
invention under stringent conditions are likewise expected to allow the
isolation of DNAs
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' encoding allelic variants of the kinase non-human species enzymes homologous
to the
mammalian Chk 1 kinase and other structurally related proteins sharing one or
more of the
enzymatic activities, or abilities to interact with members or regulators, of
the cell cycle
checkpoint pathway in which mammalian Chkl participates. Polynucleotides of
the
S invention when detectably labeled are also useful in hybridization assays to
detect the
capacity of mammalian cells to synthesize kinases of the invention. The DNA
sequence
information provided by the present invention also makes possible the
development, by
homologous recombination or "knockout" strategies [see, Capecchi, Science,
244:
1288-1292 (l989}], of rodents that fail to express a functional kinase or that
express a
variant thereof. Such rodents and their cells are useful as models for
studying the
activities of mouse and kinase modulators in vivo. Polynucleotides of the
invention may
also be the basis for diagnostic methods useful for identifying a genetic
alterations) in the
mammalian Chkl gene locus that underlies a disease state or states. Also made
available
by the invention are anti-sense polynucleotides relevant to regulating
expression of Chkl
by those cells which ordinarily express the same.
For example, primers designed from the Chk1 cDNA are useful for reverse
transcriptase PCR analysis of mRNA samples from tumor cells to detect the
presence or
absence of Chk 1 mRNA. Further, sequence information from the Chk 1 genomic
clone
can be used for single stranded conformational polymorphism (SSCP) analysis of
genomic
ZO DNA prepared from tumor cells to detect alterations or mutations of the
Chkl gene.
Likewise, the Chkl cDNA and/or the Chkl genomic clone can be used in
fluorescence in
situ hybridization (FISH) analysis to detect alterations in the Chkl gene.
The invention also provides autonomously replicating recombinant
constructions such as plasmid and viral DNA vectors incorporating
polynucleotides of the
invention, especially vectors in which the polynucleotides are functionally
linked to an
endogenous or heterologous expression control DNA sequence and a transcription
terminator.
According to another aspect of the invention, host cells, especially
unicellular host cells such as procaryotic and eukaryotic cells, are stably
transformed or
transfected with DNAs of the invention in a manner allowing expression of a
mammalian
Chkl kinase therein. Host cells of the invention are conspicuously useful in
methods for
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the large scale production of Chkl wherein the cells are grown in a suitable
culture
medium and the desired enzymes are isolated from the cells or from the medium
in which
the cells are grown.
Chkl products having part or a11 of the amino acid sequence set out in
S SEQ ID NO.: 2 or SEQ >I7 NO.: 4 are contemplated. Use of mammalian host
cells is
expected to provide for such post-translational modifications (e.g.,
myristoylation,
glycosylation, truncation, lipidation and tyrosine, serine or threonine
phosphorylation) as
may be needed to confer optimal biological activity on recombinant expression
products
of the invention. The enzyme products of the invention may be full length
polypeptides,
fragments or variants. Variants comprise Chkl products wherein one or more of
the
specified (i. e., naturally encoded) amino acids is deleted or replaced or
wherein one or
more nonspecified amino acids are added: ( 1 ) without loss of the protein
kinase activity
specific to Chkl; or (2) with disablement of the protein kinase activity
specific to Chkl;
or (3) with disablement of the ability to interact with members or regulators
of the cell
cycle checkpoint pathway. Substrates of Chk 1 and proteins which interact with
Chk 1 may
be identified by various assays.
Substrates of Chk I may be identified by incorporating test compounds in
assays for kinase activity. Chkl kinase is resuspended in kinase buffer and
incubated
either in the presence or absence of the test compound (e.g., myelin basic
protein, casein,
histone H1, or appropriate substrate peptide). The amount of phosphate
transferred by
the kinase to the test compound are measured by autoradiography or
scintillation
counting. Transfer of phosphate to the test compound is indicative that the
test
compound is a substrate of the kinase.
Yet another aspect of this invention provides a diagnostic assay for
detecting and quantifying the presence of Chk 1 in a biological sample. A
biological
sample suspected of comprising Chkl is utilized in a kinase assay. As
described herein,
the presence of Chkl is identified by the phosphorylation of a substrate
protein, e.g.
myelin b protein, or the detection of a self phosphorylated product. The
phosphorylated
product of the kinase reaction can be detected by for example, autoradiography
or
scintillation counting.
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' Interacting proteins may be identified by the following assays.
A first assay contemplated by the invention is a two-hybrid screen. The
two-hybrid system was developed in yeast [Chien et al., Proc. Natl. Acad. Sci.
USA, 88:
9578-9582 (1991)] and is based on functional in vivo reconstitution of a
transcription
factor which activates a reporter gene. Specifically, a polynucleotide
encoding a protein
that interacts with Chkl is isolated by: transforming or transfecting
appropriate host cells
with a DNA construct comprising a reporter gene under the control of a
promoter
regulated by a transcription factor having a DNA binding domain and an
activating
domain; expressing in the host cells a first hybrid DNA sequence encoding a
first fusion
of part or all of Chkl and either the DNA binding domain or the activating
domain of the
transcript ion factor; expressing in the host cells a library of second hybrid
DNA sequences
encoding second fixsions of part or a11 of putative Chk 1 binding proteins and
the DNA
binding domain or activating domain of the transcription factor which is not
incorporated
in the first firsion; detecting binding of an Chk 1 interacting protein to Chk
1 in a particular
host cell by detecting the production of reporter gene product in the host
cell; and
isolating second hybrid DNA sequences encoding the interacting protein from
the
particular host cell. Presently preferred for use in the assay are a lexA
promoter to drive
expression of the reporter gene, the lacZ reporter gene, a transcription
factor comprising
the lexA DNA binding domain and the GAL4 transactivation domain, and yeast
host cells.
Other assays for identifying proteins that interact with Chk 1 may involve
immobilizing Chk 1 or a test protein, detectably labeling the nonimmobilized
binding
partner, incubating the binding partners together and determining the amount
of label
bound. Bound label indicates that the test protein interacts with Chkl.
Another type of assay for identifying Chkl interacting proteins involves
immobilizing Chkl or a fragment thereof on a solid support coated (or
impregnated with)
a fluorescent agent, labeling a test protein with a compound capable of
exciting the
fluorescent agent, contacting the immobilized Chkl with the labeled test
protein, detecting
light emission by the fluorescent agent, and identifying interacting proteins
as test proteins
which result in the emission of light by the fluorescent agent. Alternatively,
the putative
interacting protein may be immobilized and Chkl may be labeled in the assay.
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' Also comprehended by the present invention are antibody products (e.g.,
monoclonal and polyclonal antibodies, single chain antibodies, chimeric
antibodies,
CDR-grafted antibodies and antigen-binding fragments thereof) and other
binding proteins
(such as those identified in the assays above) which are specific for the Chkl
kinases of
the invention. Binding proteins can be developed using isolated natural or
recombinant
enzymes. The binding proteins are useful, in turn, for purifying recombinant
and naturally
occurring enzymes and identifying cells producing such enzymes. Assays for the
detection
and quantification of proteins in cells and in fluids may involve a single
antibody substance
or multiple antibody substances in a "sandwich" assay format to determine
cytological
analysis of Chkl protein levels. The binding proteins are also manifestly
usefi~l in
modulating (i.e.,blocking, inhibiting, or stimulating) enzyme/substrate or
enzymelregulator
interactions. Anti-idiotypic antibodies specific for mammalian checkpoint
kinase binding
proteins are also contemplated.
It is fizrther contemplated that antibodies against Chkl can be used in
diagnosis of Atm function. Because Chkl protein levels are low or absent in AT
patients
Chkl levels can be used as a marker in the development of compounds that
inhibit Atm
fixnction. Because expression of Chkl in AT cells is low or non-existent,
inhibition of
Atm function should decrease Chkl expression. This decrease can be monitored
by
examining the expression levels of Chkl .
The invention contemplates that mutations in the Chk 1 gene that result in
loss of normal fixnction of the Chk 1 gene product underlie human disease
states in which
failure of a cell cycle checkpoint is involved. Gene therapy to restore Chk 1
activity would
thus be indicated in treating those disease states (for example, testicular
cancer). Delivery
of a functional Chkl gene to appropriate cells is effected in vivo or ex vivo
by use of viral
vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus) or ex vivo
by use of
physical DNA transfer methods (e.g., liposomes or chemical treatments). For
reviews of
gene therapy technology see Friedmann, Science, 244: 127S-1281 (1989); Verma,
Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).
Alternatively, it is contemplated that in other human disease states
preventing the
expression of or inhibiting the activity of Chkl will be useful in treating
the disease states.
It is contemplated that antisense therapy or gene therapy could be applied to
negatively
*rB
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regulate the expression of Chkl. Antisense nucleic acids (preferably 10 to 20
base pair
oligonucleotides) capable of specifically binding to Chkl expression control
sequences or
Chkl RNA are introduced into cells (e.g., by a viral vector or colloidal
dispersion system
such as a Iiposome). The antisense nucleic acid binds to the Chkl target
sequence in the
cell and prevents transcription or translation of the target sequence.
Phosphothioate and
methylphosphate antisense oligonucleotides are specifically contemplated for
therapeutic
use by the invention. The antisense oligonucleotides may be further modified
by
poly-L-lysine, transfernn polylysine, or cholesterol moieties at their 5 end.
Checkpoint signal transduction results in transcriptional regulation. One
example of transcriptional regulation is MyoD muscle regulation. Chkl
expression
suppresses the ability of MyoD to induce muscle gene transcription and
suppresses the
ability of MyoD to induce myogenesis (see Example 7). It is contemplated that
another
aspect of this invention is to regulate Chkl levels in order to effect the
differentiation and
proliferation of stem cells such as those involved in muscle proliferation.
Agents that modulate Chkl protein kinase activity may be identified by
incubating a test compound with Chkl immunopurified from cells naturally
expressing the
mamrnafian checkpoint protein kinase, with Chk 1 obtained from recombinant
procaryotic
or eukaryotic host cells expressing the enzyme, or with purified Chkl, and
then
determining the effect of the test compound on Chkl protein kinase activity.
The activity
of the checkpoint protein kinase can be measured by determining the amount of
32P-phosphate transferred by the protein kinase from gamma-32P-ATP to either
itself
(autophosphorylation) or to an exogenous substrate such as a lipid or protein.
The
amount of phosphate incorporated into the substrate is measured by
scintillation counting
or autoradiography. An increase in the amount of phosphate transferred to the
substrate
in the presence of the test compound compared to the amount of phosphate
transferred
to the substrate in the absence of the test compound indicates that the test
compound is
an activator of the Chkl protein kinase. Conversely, a decrease in the amount
of
phosphate transferred to the substrate in presence of the test compound
compared to the
moles of phosphate transferred to the substrate in the absence of the test
compound
indicates that the modulator is an inhibitor of the Chkl protein kinase.
*rB
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In a presently preferred assay, a Chkl-specific antibody linked to agarose
beads is incubated with a cell lysate prepared from host cells expressing the
protein kinase.
The beads are washed to remove proteins binding nonspecifically to the beads
and the
beads are then resuspended in kinase bui~er. The reaction is initiated by the
addition of
gamma-32P-ATP and an appropriate exogenous substrate such as lipid or peptide.
The
activity of the protein kinase is measured by determining the moles of 32P-
phosphate
transferred either to the protein kinase itself or the added substrate.
In a preferred embodiment the host cells lack endogenous Chkl and/or
ATM protein kinase activity. The selectivity of a compound that modulates the
protein
kinase activity of Chkl can be evaluated by comparing its activity on Chkl to
its activity
on other known mammalian checkpoint protein kinases. The combination of the
recombinant Chkl products of the invention with other recombinant mammalian
checkpoint kinase products in a series of independent assays provides a system
for
developing selective modulators of Chkl.
Furthermore, combinatorial libraries, peptide and peptide mimetics, defined
chemical entities, oligonucleotides, and natural product libraries may be
screened for
activity as modulators in assays such as those described below.
For example, an assay for identifying modulators of Chkl kinase activity
involves incubating a Chkl protein kinase preparation in kinase buffer with
gamma-32P-ATP and an exogenous kinase substrate, both in the presence and
absence
of a test compound, and measuring the amount of phosphate transferred to the
substrate.
An increase in the amount of phosphate transferred to the substrate in
presence of the test
compound compared to the amount of phosphate transferred to the substrate in
the
absence of the test compound indicates that the test compound is an activator
of the Chkl
kinase. Conversely, a decrease in the amount of phosphate transferred to the
substrate in
presence of the test compound compared to the amount of phosphate transferred
to the
substrate in the absence of the test compound indicates that the modulator is
an inhibitor
of said Chkl protein kinase.
Moreover, assays for identifying compounds that modulate interaction of
Chkl with other proteins may involve: transforming or transfecting appropriate
host cells
with a DNA construct comprising a reporter gene under the control of a
promoter
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regulated by a transcription factor having a DNA-binding domain and an
activating
domain; expressing in the host cells a first hybrid DNA sequence encoding a
first fusion
of part or a11 of Chkl and the DNA binding domain or the activating domain of
the
transcription factor; expressing in the host cells a second hybrid DNA
sequence encoding
part or all of a protein that interacts with Chkl and the DNA binding domain
or activating
domain of the transcription factor which is not incorporated in the first
fizsion; evaluating
the elect of a test compound on the interaction between Chk 1 and the
interacting protein
by detecting binding of the interacting protein to Chk 1 in a particular host
cell by
measuring the production of reporter gene product in the host cell in'the
presence or
absence of the test compound; and identifying modulating compounds as those
test
compounds altering production of the reported gene product in comparison to
production
of the reporter gene product in the absence of the modulating compound.
Presently
preferred for use in the assay are a lexA promoter to drive expression of the
reporter gene,
the lacZ reporter gene, a transcription factor comprising the lexA DNA binding
domain
and the GAL4 transactivation domain, and yeast host cells.
Another type of assay for identifying compounds that modulate the
interaction between Chkl and an interacting protein involves immobilizing Chkl
or a
natural Chkl interacting protein, detectably labeling the nonimmobilized
binding partner,
incubating the binding partners together and determining the effect of a test
compound on
the amount of label bound wherein a reduction in the label bound in the
present of the test
compound compared to the amount of label bound in the absence of the test
compound
indicates that the test agent is an inhibitor of Chk 1 interaction with
protein. Conversely,
an increase in the binding in the presence of the test compound compared to
the amount
label bound in the absence of the compound indicates that the putative
modulator is an
activator of Chkl interaction with the protein.
Yet another method contemplated by the invention for identifying
compounds that modulate the binding between Chkl and an interacting protein
involves
immobilizing Chkl or a fragment thereof on a solid support coated (or
impregnated with)
a fluorescent agent, labeling the interacting protein with a compound capable
of exciting
3 0 the fluorescent agent, contacting the immobilized Chk 1 with the labeled
interacting protein
in the presence and absence of a test compound, detecting light emission by
the
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fluorescent agent, and identifying modulating compounds as those test
compounds that
affect the emission of light by the fluorescent agent in comparison to the
emission of light
by the fluorescent agent in the absence of the test compound. Alternatively,
the Chk 1
interacting protein may be immobilized and Chk I may be labeled in the assay.
Modulators of Chk 1 may affect its protein kinase activity, its localization
in the cell, and/or its interaction with members of the cell cycle checkpoint
pathway. Chkl
modulators may be formulated in compositions comprising pharmaceutically
acceptable
Garners. Such compositions may additionally include chemotherapeutic agents.
Dosage
amounts indicated would be sufficient to result in modulation of Chkl activity
in vivo.
Selective modulators may include, for example, polypeptides or peptides which
specifically bind to Chkl or Chkl nucleic acid, oligonucleotides which
specifically bind
to Chk 1 or Chkl nucleic acid, and/or other non-peptide compounds (e.g.,
isolated or
synthetic organic molecules) which specifically react with Chk I or Chk 1
nucleic acid.
Mutant forms of Chkl which affect the enzymatic activity or cellular
localization of
wild-type Chkl are also contemplated by the invention.
DETAILED DESCRIPTION
The present invention is illustrated by the following examples. Example
1 details the isolation of polynucleotides encoding mammalian Chkl kinases and
chromosomal mapping of the human Chkl DNA. Example 2 describes the recombinant
expression ofDNAs encoding mammalian Chkl. Example 3 describes the preparation
of
antibodies to Chkl. Northern blots showing tissue and cell distribution of
Chkl are
described in Example 4. Example 5 reports the results of immunohistological
and western
blot studies of Chkl expression. Example 6 describes biochenucal and
biological activities
of murine Chkl.
Ezampte 1
A. Isolation of Human Chkl cDNA
A ChkIHu cDNA was identified by screening EST sequences for similarity
to S. pombe Chkl. An EST (H67490) with homology to the COOH-terminus of Chkl
was identified and cloned and used to build a contig showing limited homology
to the
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COOH-terminal 120 amino acids. This contig was extended by RACE PCR to give a
clone of 173 Sbp. The RACE PCR fragment was used to probe cDNA libraries to
generate the final sequence. Library screening of 1 x 106 independent cDNA
clones from
a human testis unizap library (Clontech) with RACE derived sequence
information at high
stringency in Express hybridization solution (Ciontech) yielded eleven
overlapping
sequences that were used to assemble the full length ChklHu.
The full length human Chkl cDNA was subcloned into pGEMT. The
plasmid containing the full length human cDNA is identified as pGEMT-ChkIHU
pGEMT-ChkIHU was deposited with American Type Culture Collection, 12301
Parklawn Drive, Rockville, MX 20852 on date 27th August 199? under Accession
No.
ATCC 98520. The full length cDNA and deduced amino acid sequences of human
Chkl
(ChklHu) are provided in SEQ 117 NOs.: 2 and 4 respectively. The fizll length
DNA and
deduced amino acid sequences of human Chk 1 (Chk 1 Hu) are provided in SEQ B~
NOs.
1 and 2, respectively.
B. Isolation of a Murine Chkl cDNA
The ChklMo was identified by library screening of a mouse testis cDNA
library using a degenerate human Chkl specific probe. The partial cDNA and
deduced
amino acid sequences of mouse Chk 1 (Chk 1 Mu) are provided in SEQ ID NO. : 3
and 4,
respectively.
C. Structural Analysis of ChklHu and ChklMu
The ChklHu and ChklMu cDNAs encode a protein of approximately 70 kD. The
kinase domain of ChklHu is 161 to 264 of SEQ ID NO.: 2. The kinase domain of
ChklMu comprises amino acids 1 to 61 of SEQ ID NO.: 4. The protein kinase
domains
of human and mouse Chk I as disclosed herein and are approximately 90%
identical at the
amino acid level. Relative to C. elegans, S. pombe, and S. cerevisiae Chkl-
like proteins,
the human form is approximately 56%, 47%, and 37% identical to the protein
kinase
domains, respectively. Figure 1 compares the amino acid sequences of Chk 1
homologs
from human, mouse, C. elegans, S. pombe and S. cerevisiae. In Figure 1, amino
acid
residues identical among a11 species are boxed. Roman numerals indicate
subdomains
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conserved among the homologs. Subdomains V through IX comprise the substrate
recognition site and contain the greatest frequency of conserved residues.
This is
determined by homology with other known kinases (Hanks et al., Science 241:42-
52,
1988).
D. Isolation of a Murine Chkl Genomic Clone
A mouse genomic clone encoding Chkl was obtained using PCR screening
of a P 1 mouse l29 library. The PCR primers used to screen the mouse 129 p 1
library
were mmChk2 (ACG TGG ACA AAC TGG TTC AGG) (SEQ U~ NO.: 5) and mmChk
21: CTG ATA GCC CAA CTT CTC GAA SEQ ID NO.: 6). These primers were used
to generate an amplicon of 208 by corresponding to nucleotide X to X of SEQ ID
NO.:
4. The amplicon was used to identify a clone of approximately 81-100 kb. An
EcoRl
restriction digest of the genomic clone was performed and the restriction
products were
subcloned into a vector with zeocin as the selectable marker. The vector was
obtained
1 S from Invitrogen p. zero 1.1 (2.8 kb).
The Eco RI restriction digests were resolved on a 0.8% agarose gel and
transferred to nitrocellulose according to standard procedures. The
nitrocellulose blot
was probed with a 208bp amplicon to confirm that a genomic clone was
identified.
E. Chromosomal Mapping of the Human Chkl Gene
The Stanford G3 Radiation Hybrid panel was used to map the location of
Chk I . PCR reactions using two oligonucleotides in the 3' untranslated region
of the
human cDNA library derived Chkl DNA fragment yielded a unique PCR amplicon
with
primer I(Chkl 30mer3'UT) GGCTCTGGGGAATCCTGGTGAATATAGTGCTGC
(SEQ ID NO.: ? and primer 2 (Chkl 30mer 3'UT)
TCCCCTGAAACTTGGTTTCCACCAGATGAG (SEQ D7 NO. 8. For sublocalization,
chromosome 11 radiation hybrid DNA samples obtained from Research Genetics
(Huntsville, Alabama) were analyzed and the results were decoded by the RH
server at
httpalshgc.stanford.edu/. ChklHu localized to marker D1154610 which has been
mapped
3 0 to the telomeric region of 11 q at 23.3. This region has been identified
as the site of tumor
*rB
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suppressor genes implicated in ovarian, breast, lung, colon and cervix cancer
and
melanoma. [Gabra et al., Cancer Research, 56:950-954 (1996}].
Example 2
Chkl was expressed in recombinant host cells.
A. Expression and Kinase Activity of Chkl
Glutathione-S-Transferase Fusion Protein
DNA encoding Chk 1 glutathione-S-transferase fizsion protein
(GST-ChklHu) was also prepared. A DNA encoding GST-ChklHu was cloned into
pGEX KGH using NdeI Sa 1 I restriction sites introduced into Chk 1 Hu. The
internal Nde
I sites were eliminated by silent mutagenesis. E. coli F DhSa transformed with
GST-Chkl
were used to prepare recombinant proteins. To 200 m1 of culture, SmM IPTG was
added
and cells were grown for four hours at 37 C. The culture was harvested and
washed in
STE buffer (IOmM Tris pH 8, 150 mM NaCl, and 1mM EDTA). The cells were
resuspended in 6 mol STE with 1mM PMSF and 100mg lysozyme. The cultures were
incubated on ice for fifteen minutes, and 5 mM DTT and 1.5% Sarkosyl was added
and
the cells were sonicated. The debris was pelleted, and the supernatant was
made to 2%
Triton X-100. Glutathione agarose beads were added and the beads were pelleted
in a
benchtop centrifuge and bound proteins were eluted with elution buffer (SOmM
Tris pH
8.0, 15 0 mM NaC 1, 1 mM PMSF, and 10 mM glutathione.
Kinase assays were performed by incubating GST-ChklHu in kinase buffer (25M
HEPES, pH 7.7; 50 mM KC 1; 10 mM MgC 12; 0.1 % NP-40; 2% glycerol; 1 mM DTT,
S OuM ATP) with or without l mg os substrate protein, and incubated in kinase
buffer
containing lOmCi [g-32P] ATP (3000 Ci/mmol) for twenty minutes at 37 C. The
reactions were stopped with 20 ul 2 X SDS sample buffer prior to separation on
6%
PAGE. Kinase reactions were then transferred to Immobilon, exposed to film,
then
subsequently probed to detect precipitated protein.
The glutathione affinity purified fi.~sion was abie to autophosphorylate and
phosphorylate substrate proteins such a s myeline basic protein showing that
Chkl is
active as a protein kinase, independent of regulatory subunits.
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Example 3
Antibodies specific for mammalian Chkl proteins were generated as
follows.
A. Generation of Polyclonal Antibodies
Polyclonal antibodies were generated against a mouse Chk 1 polypeptide
fragment. CLKETFEKLGYQWKK (amino acids 191 to 203 of SEQ ID NO.: 3) was
coupled to Keyhole Lympet Hemocyanin (KLH) via the N-terminally added cysteine
and
a rabbit was injected with 150 mg per injection. To affinity purify the rabbit
sera, 3 mls
of thiol coupling gel (TCGeI Quality Controlled Biochemicals) (St. Louse,
Missouri) that
was equilibrated with degassed TEB (50 mM Tris, 5 mM EDTA-Na, pH 8.5) was
mixed
with 1.25 mg of HPLC purified peptide. The coupled resin was loaded into an
econo
column (Bio Rad) (Hercules, California) and was washed with 10 column volumes
of
TEB. The resin was treated blocking buffer (50 mM cysteine in TEB buffer per
ml of gel)
and was sequentially washed with 20 column volumes of salt buffer. The column
was
washed with 20 column volumes of salt buffer (500 mM NaCl in 50 mM NaH2P04, pH
6:5) and 10 column volumes of phosphate buffer (50 mM NaH2P04, pH 6.5 ).
Twenty
mls of rabbit serum was loaded onto the column and was washed with 10 column
volumes
of salt buffer and the antibody was eluted with glycine buffer ( 100mM Glycine-
HCL, pH
2.5). One ml fractions were collected in 50 ul of I.OM Tris, (pH 9.5).
Fractions
containing antibody were pooled and dialyzed in storage buffer ( l OmM
NaH2P04, 20mM
MgCI, pH 7.0) and stored at -20 as a Chkl#2-3. The polyclonal antisera, a
Chkl#2-3
was able to immunoprecipitate mouse Chkl protein from mouse testes extract as
described in Example 6. In addition, aChk 1 #2-3 recognized recombinant human
Chk 1 and
the ChklHu glutathione-S-transferase fusion protein expressed in E. coli.
B. Generation of Monoclonal Antibodies
Monoclonal antibodies are prepared by immunizing Balb/c mice
subcutaneously with Chkl, Gst-Chkl or a Chkl fragment in complete Freund's
adjuvant
(CFA). Subsequent immunizations in CFA or incomplete Freund's adjuvant is
performed
to increase immune response.
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The spleen of the immunized animal is removed aseptically and a single-cell
suspension is formed by grinding the spleen between the frosted ends of two
glass
microscope slides submerged in serum free RPMI 1640, supplemented with 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 mg/ml
streptomycin
(RPMI) (Gibco, Canada). The cell suspension is filtered through sterile 70-
mesh Nitex
cell strainer (Becton Dickinson, Parsippany, New Jersey), and washed twice by
centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum
free RPMI.
Thymocytes taken from naive Balb/c mice are prepared in the same manner.
Two x 108 spleen cells are combined with 4 x 107 NS-1 cells (kept in log
phase in RPMI with 11% fetal bovine serum (FBS) for three days prior to
fusion),
centrifuged and the supernatant is aspirated. The cell pellet is dislodged and
2 m1 of 37
C PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer Mannheim) is added while
stirring over the course of one minute, followed by the addition of 14 ml of
serum free
RPMI over seven minutes. Additional RPMI can be added and the cells are
centrifuged
at 200 g for 10 minutes. After discarding the supernatant, the pellet is
resuspended in 200
ml RPMI containing 15% FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16
mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x
106
thymocytes/ml. The suspension is dispensed into ten 96-well flat bottom tissue
culture
plates (Corning, United Kingdom) at 200 ml/well. Cells are fed on days 2, 4,
and 6 days
post-fusion by aspirating 100 m1 from each well with an 18 G needle (Becton
Dickinson),
and adding 100 lnl/well plating medium containing 10 U/ml IL-6 and lacking
thymocytes.
When cell growth reaches 60-80% confluence (day 8-10), culture
supernatants are taken from each well and screened for reactivity to Chkl by
ELISA.
ELISAs are performed as follows. Immulon 4 plates (Dynatech, Cambridge,
Massachusetts) are coated at 4 C with 50 ml/well with 100ngiwell of Chkl in 50
mM
carbonate buffer, pH 9.6. Plates are washed with PB S with 0.05%, Tween 20 (PB
ST)
and blocked 30 minutes at 37 C with 0.5% Fish Skin Gelatin. Plates are washed
as
described above and 50 m1 culture supernatant is added. After incubation at 37
C for 30
minutes, 50 m1 of horseradish peroxidase conjugated goat anti-mouse IgG(fc)
(Jackson
ImmunoResearch, West Grove, Pennsylvania) [diluted 1:10,000 in PBST] is added.
Plates are incubated at 37 C for 30 minutes, washed with PBST and 100 ml of
substrate,
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consisting of 1 mg/ml TMB (Sigma) and O.15m1/ml 30% H202 in 100 mM Citrate, pH
4.5, is added. The color reaction is stopped with the addition of 50 ml of 15%
H2S04.
A450 is read on a plate reader (Dynatech).
S Example 4
Northern analysis was performed to determine tissue distribution of Chkl
in mouse and human tissue. Oligonucleotides mmchkl:
GTTGAGACTCCATCATCAAGG (SEQ ID NO.: 9) and mmChkl':
TCTGGCTGGGAACTAGAGAAC (SEQ D7 NO.: 10) were used to generate an
amplicon of 220bp, identified as mmChk 1 + 1 ~, mmChk 1 + 1' and mmChk2+2'
(Example 1 )
were used as probes for northern analysis. The conditions for PCR were as
follows: One
cycle of eight minutes at 94 ° C then forty cycles of 94 ° C for
twenty seconds, S 8 ° C for
twenty seconds, 72 ° C for twenty seconds. The products were analyzed
on a 4% agarose
gel.
1 S The amplicons were labeled with 32P-ATP for Northern analysis. A nylon
membrane containing 2mg of size fractionated poly (A)+ RNA from human and
mouse
tissue sources including human heart, brain, placenta, lung, liver, skeletal
muscle, kidney,
pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and
peripheral
blood leukocytes, and mouse heart, brain, spleen, lung, liver, skeletal
muscle, kidney, and
testis (Clonetech Laboratories, Palo Alto, California) was probed with the
labeled
ampiicons as recommended by the manufacturer except that the final wash was
performed
at 55 °C to minimize the possibility of cross-hybridization to related
sequences.
In mouse tissue, Chkl expression was observed in lung, spleen, and testes
in the mouse. In human tissue, expression was seen in thymus, lung, prostate,
and testes.
However, testes RNA samples from both mouse and human show approximately two
to
four fold higher levels of RNA expression than other tissues.
To determine Chkl expression in developing mouse embryos, mRNA
obtained from total embryos at day 7, 11, 15, and 17 were probed with labeled
mChkl+1'
and mmChk2+2'. The Northern blots showed that Chkl expression peaked at day 11
of
embryogenesis.
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Example 5
Because of the roles Atr and Atm play in meiosis and their association with
meiotic chromosomes, the expression of ChklMo protein was examined by
immunohistological characterization of cross-sections of mouse testes. In
testes,
pre-meiotic cells surround the surface of the seminiferous tubule and
progression towards
the interior lumen of the tubule corresponds to progressively later stages of
meiosis and
sperm maturation [Moms et al., J. Histochem. Cytochem, 25:480 ( 1977)].
Testes from normal and mutant mice (discussed infra) as described in
Heiner et al., Cancer Res., 57:1664-1667 (1997) were obtained and preserved in
Tissue
Tek OCT compound, a tissue freezing media, containing 10.24% w/w polyvinyl
alcohol,
4.26% w/w polyethylene glycol, and 85.50% w/w nonreacting ingredients. Both
wildtype
and mutant mice were tested and analyzed by immunohistochemistry using aChkl#2-
3,
a polyclonal antibody. Six micron cryosections were placed in a 50 C oven to
dry and
fixed in 4 C acetone for two minutes. The slides were incubated with aChk 1 #2-
3 antisera
at a 1:100 dilution for thirty minutes. The secondary, goat anti-rabbit
biotinylated
antibody was applied to each section at a dilution of l:200 and incubated for
fifteen
minutes at 37 C. The tertiary antibody, goat anti biotin was applied to each
section at a
dilution of 1:200 and incubated for fifteen minutes at 37 C. After each
incubation, the
slides were rinsed with 1 X TBS. DAB horseradish-peroxidase substrate was used
to
detect positive signal in the samples. The reaction was stopped in water and
counterstained with Gill's Hematoxylin.
The testes of ChklMo, atm+/+ p53+/+ (A), atm+/+ p53+/- (B) atm-/-
p53+/+ (C), atm-/- p53+/- (D), and, atm-/- p53-/- (E) were
immunohistologically
characterized using anti-Chkl antiserum. Testes cryosections were stained with
afI~lnity-purified anti-Chkl antiserum (aChkl #2-3) and with an anti-ATR
monoclonal
(224C). The specificity of staining was confirmed by examining pre-immune
serum and
by specific and non-specific peptide block experiments. Affinity purified Chkl
polyclonal
antisera (Chk 1 #2-3 of Example 3 ) was used to determine the localization.
In contrast to staining patterns reported for Atr, ChklMo shows temporal
increases and decreases in nuclear staining in normal mice. ChklMo is most
highly
expressed at pachynema in primary spermatocytes, indicating that ChklMo may
act
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downstream of Atr function during meiotic prophase, after Atr, which acts
earlier during
zygonema. Since Atm and p53 have checkpoint properties and may act at an
earlier phase
in signaling relative to Chk 1 Hu/Mo, the localization of Chk 1 Mo by
histological analyzes
was examined in atm-/-p53+/+, atm-/-p53-/- and atm-/-p53+/- mice [Done hower
et al.,
Nature, 3 56:215 ( 1992); Kuerbitz et al., Proc. Natl. Acad. Sci. USA, 89:7491
( 1992);
Westphal et al., Nat. Gen., 16:397-401 (I997)]. ChklMo accumulation and
localization
was independent of p53 status but dependent on Atm, suggesting that Chk 1 Mo
also acts
downstream of Atm in meiotic prophase.
To further analyze the role of Chkl in mammalian meiosis, the temporal
and spatial distribution of Chk 1 in surface spread preparations of
spermatocytes was
determined.
For the meiotic preparations, surface spreads of spermatocytes from fifteen
to twenty-one day old mice (C57-b1/6) were prepared and antibody incubation
and
detection procedures were prefonmed as described in Ashley et al., Chromosoma,
104:19
( 1995). Antibody incubation and detection procedures were a modification of
the
protocol of Moens et al. as described previously in Ashley et al. Since the
antibodies
against Chk 1 and Sep3 (control) were both raised in rabbit, spermatocytes
were labeled
and imaged sequentially. Goat-anti rabbit IgG-rhodamine-conjugated and goat-
anti rabbit
IgG-FITC-conjugated (Pierce) secondary antibodies were used for detection. All
preparations were counterstained with 4', 6' diamino-2-phenylindole (DPAI,
Sigma) and
mounted in a DABCO (Sigma) antifade solution. The preparations were examined
on a
Zeiss Axioskop (63-X and 100-X, 1.2 Plan Neoflour oil-immersion objective).
Each
fluorochrome (FITC, rhodamine and DAPI) image was captured separately as an 8-
bit
source image using a computer assisted cooled CCD camera (Photometrics CH220)
and
the separate images 24-bit pseudocolored and merged with custom software
developed
by Tim Rand [Ried et al., Proc. Natl. Acad. Sci. USA, 89:1388 (1992)].
Zygotene spermatocytes were stained with an antiserum against Scp3, a
component of the axial element, which forms between the sister-chromatids. The
same
zygotene spermatocytes were labeled with a-Chk 1 #23 . Chk 1 is present along
the
synaptonemal complexes (SC) of synapsed homologous chromosomes. Chromosomes
that are in the process of synapsing have Chkl staining, but no staining is
observed on the
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' unsynapsed axial elements. As meiosis proceeds into pachynema, ChklMo
remains
associated with autosomal synaptonemal complexes in a focal staining pattern
similar to
ATM.
A pachytene spermatocyte was labeled with both anti-Chkl and a mouse
monoclonal antibody against Atr (224C). Although Chk 1 initially appears in a
focal
pattern, in pachynema Chkl seems to accumulate along the SCs. In addition,
Chkl foci
appear along the unsynapsed axial elements of the X and Y chromosomes in mid
pachynema where it colocalizes with Atr. Chkl remains on the SCs throughout
pachynema and disappears when the homologous chromosomes disassociate in
diplonema.
Progression of meiotic prophase I appears to be normal in p53-/- mice. In
an early pachytene spermatocyte labeled for Chkl and Scp3, Chkl is present
along the
SCs and the synapsed region of the sex chromosomes. However, Chkl does not
appear
along the unsynapsed axial element of the X chromosome in early pachytene. In
atm-l-
mice, progression of meiosis is disrupted as the SCs begin to fragment
following synapsis.
Although homologous chromosomes synapse in atm-/- sperrnatocytes, no Chkl is
detected along the synapsed bivalents or fragmented SCs.
Thus, in normal mice, Chkl appears in a focal pattern along the
synaptonemal complexes of synapsing homologous chromosomes in zygonema, in a
pattern similar to that of Atm. Chkl accumulates along the SCs as meiosis
progresses
into pachynema, and by mid-pachynema Chk 1 coats the entire SCs. In early
pachynema,
Chk 1 is present along the synapsed region of the XY bivalent. However, by
mid-pachynema, Chk 1 foci also appear along the unsynapsed axes of both the X
and Y
chromosomes, where it colocalizes with Atr. Atr is found in foci along the
unsynapsed
axes of the sex chromosomes early, and later coats the entire X and Y axes
throughout
pachynema.
Meiosis appears to be unaffected in p53 deficient mice, as is demonstrated
by histological analysis and immunostaining of surface spread spermatocytes
with Scp3
and Chk 1. In contrast, atm-/- mice are sterile as the result of progressive
fragmentation
of meiotic chromosomes following synapsis, leading to apoptosis.
Immunolocalization
of Chkl in atm-/- spermatocytes indicates a lack of Chkl on the 5Cs,
suggesting a role
of Chkl downstream of Atm in mammalian meiosis. To determine if the lack of
Chkl
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staining in atm-/- nuclei and the lack of Chk 1 Mo protein on meiotic prophase
chromatin
was reflected by the level of ChklMo, Western analysis of testes extracts was
performed
according to Keegan et al., Genes Dev., 10:2423 ( 1996). Chk 1 protein was
present in an
Atm-dependent fashion, suggesting that synthesi s or stability of Chk 1 Mo
depends on the
Atm protein. MTE of mice lacking Atm did not stain for Chk 1 indicating that
atm-1- mice
did not express Chkl . In contrast, MTE of wild type mouse or from mice in
which p53
expression was disrupted showed Chkl staining.
Example 6
The kinase activity of mouse Chk 1 and its ability to associate with Atr
were also demonstrated.
A. Kinase Activity of Murine Testes Chkl
Antibody aChkl#2-3 also used to immunoprecipitate Chkl from mouse
testes extract (MTE). Approximately 30 decapsulated testes were ground in a
mortar
with liquid nitrogen and the grounds were transferred to a 15 m1 dounce
homogenizer.
Fifteen mls of lysis buffer (SOmM NaP04, pH 7.2, 0.5% TritonX-100, 2mM EDTA,
2mM
EGTA, ZSmM NaF, 25mM 2-glycerophosphate, 1mM phenylmethylsulfonyl fluoride
(PMSF), 1 mg/ml. leupeptin, 1 mg/ml pepstatin A, and 2mM DTT) was added and
the
extract was dounced 30 times with a loose pestle and 20 times with a tight
pestle. After
a low speed spin, the supernatant assayed using BCA for protein concentration
determination.
For Chkl immunoprecipitations, 400mg of MTE extract was incubated
with either l0mg of affinity purified Chkl#2-3 or with approximately l Omg of
preimmune
serum for thirty minutes on ice. Protein A-agarose slurry (Pierce) was added
and the
mixture was incubated for thirty minutes at 4 C. The immune-complex bound
slurry was
washed three times in TSAT and one time with kinase buffer (25uM HEPES, pH
7.7; 50
mM KC 1; 10 mM MgC 12; 0.1 % NP-40; 2% glycerol; 1 mM DTT, SOuM ATP), and
incubated in kinase buffer containing lOmCi [g-32P] ATP (3000 Ci/mmol) for
twenty
minutes at 37 C. The reactions were stopped with 20 ul 2 X SDS sample buffer
prior to
CA 02270911 1999-OS-04
WO 99I11795 PCT/US98/18558
-26-
separation on 6% PAGE. Kinase reactions were then transferred to Immobilon,
exposed
to film, then subsequently probed to detect precipitated protein.
To determine if the immunoprecipitated Chk 1 from MTE could
self phosphorylate, 2X kinase buffer and 10 mCi g32-P-data (3000 Ci/mM) were
added
to the immunoprecipitate. The phosphorylation reactions were incubated at 30 C
for
fifteen minutes. The reactions were electrophoresed on a 6% PAGE gel,
transferred to
immobilon P, and exposed to X-ray film. The blots showed that
immunoprecipitated
Chk 1 was able to self phosphorylate as was the Chk 1-GST fusion protein
(Example 2).
Mouse IgG and Chkl preimmune sera did not immunoprecipitate ChklMo.
C. Association of Chkl and Atr
To determine if Chkl and Atr can associate in meiotic cells, 460 mg of
MTE was immunoprecipitated with anti-Atr monoclonal antibody (aAtr-224C) under
conditions as described above. The Atr immunoprecipitate was electrophoresed
on a 6%
or 8% PAGE, electroblotted onto immoblin P membrane and was probed with anti-
Chk 1
antibody (aChkl #Z-3). The blots showed that Chkl co-precipitates with Atr
indicating
that Atr and Chkl associate in meiotic cells. In addition, the Chkl that
immunoprecipitates with Atr was able to self phosphorylate.
Numerous modifications and variations in the practice of this invention are
expected to occur to those of skill in the art. Only such limitations that
appear in the
appended claims should be placed on the invention.
CA 02270911 1999-OS-04
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Medical Research Council
(ii) TITLE OF INVENTION: MAMMALIAN CHK1 EFFECTOR CELL-CYCLE
CHECKPOINT PROTEIN KINASE MATERIALS AND METHODS
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Medical research Council
(B) STREET: 20, Park Crescent
(C) CITY: London
(D) STATE:
(E) COUNTRY: UK
(F) ZIP: W1N 4AL
(v) COMPUTER READABLE FORM:
(A) MBDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NO1~ER:
(B) FILING DATE:
(C) CLASSIFICATION:
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQQENCE CHARACTERISTICS:
(A) LENGTH: 1933 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
"Human Chkl"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 34..l461
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCCGGACAGT CCGCCGAGGT GCTCGGTGGA GTC ATG GCA GTG CCC TTT GTG GAA 54
Met Ala Val Pro Phe Val Glu
1 5
GAC TGG GAC TTG GTG CAA ACC CTG GGA GAA GGT GCC TAT GGA GAA GTT 102
Asp Trp Asp Leu Val Gln Thr Leu Gly Glu Gly Ala Tyr Gly Glu Val
15 20
SU8ST1TUTE SHEET (RULE 26)
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WO 99/11795 PCT/US98/18558
-2-
CAA CTT GCT GTG AAT AGA GTA ACT GAA GAA GCA GTC GCA GTG AAG ATT 150
Gln Leu Ala Val Asn Arg Val Thr Glu Glu Ala Val Ala Val Lys Ile
25 30 35
GTA GAT ATG AAG CGT GCC GTA GAC TGT CCA GAA AAT ATT AAG AAA GAG 198
Val Asp Met Lys Arg Ala Val Asp Cys Pro Glu Asn Ile Lye Lys Glu
40 45 50 55
ATC TGT ATC AAT AAA ATG CTA AAT CAT GAA AAT GTA GTA AAA TTC TAT 246
Ile Cys Ile Asn Lys Met Leu Asn His Glu Asn Val Val Lys Phe Tyr
60 65 70
GGT CAC AGG AGA GAA GGC AAT ATC CAA TAT TTA TTT CTG GAG TAC TGT 294
Gly His Arg Arg Glu Gly Asn Ile Gln Tyr Leu Phe Leu Glu Tyr Cys
75 80 85
AGT GGA GGA GAG CTT TIT GAC AGA ATA GAG CCA GAC ATA GGC ATG CCT 342
Ser Gly Gly Glu Leu Phe Asp Arg Ile Glu Pro Asp Ile Gly Met Pro
90 95 l00
GAA CCA GAT GCT CAG AGA TTC TTC CAT CAA CTC ATG GCA GGG GTG GTT 390
Glu Pro Asp Ala Gln Arg Phe Phe Hie Gln Leu Met Ala Gly Val Val
105 1l0 115
TAT CTG CAT GGT ATT GGA ATA ACT CAC AGG GAT ATT AAA CCA GAA AAT 438
Tyr Leu His Gly Ile Gly Ile Thr His Rrg Asp Ile Lys Pro Glu Asn
120 l25 l30 135
CTT CTG TTG GAT GAA AGG GAT AAC CTC AAA ATC TCA GAC TTT GGC TTG 486
Leu Leu Leu Asp Glu Arg Asp Asn Leu Lys Ile Ser Asp Phe Gly Leu
l40 145 l50
GCA ACA GTA TZ'T CGG TAT AAT AAT CGT GAG CGT TTG TTG AAC AAG ATG 534
Ala Thr Val Phe Arg Tyr Asn Asn Arg Glu Arg Leu Leu Asn Lys Met
155 160 165
TGT GGT ACT TTA CCA TAT GTT GCT CCA GAA CTT CTG AAG AGA AGA GAA 582
Cys Gly Thr Leu Pro Tyr Val Ala Pro Glu Leu Leu Lys Arg Arg Glu
l70 l75 l80
TTT CAT GCA GAA CCA GTT GAT GTT TGG TCC TGT GGA ATA 630
GTA CTT ACT
Phe His Ala Glu Pro Val Asp Val Trp Ser Cys Gly Ile
Val Leu Thr
l85 l90 l95
GCA ATG CTC GCT GGA GAA TTG CCA TGG GAC CAA CCC AGT 678
GAC AGC TGT
Ala Met Leu Ala Gly Glu Leu Pro Trp Asp Gln Pro Ser
Asp Ser Cys
200 205 210 2l5
CAG GAG TAT TCT GAC TGG AAA GAA AAA AAA ACA TAC CTC 726
AAC CCT TGG
Gln Glu Tyr Ser Asp Trp Lys Glu Lys Lys Thr Tyr Leu
Asn Pro Trp
220 225 230
AAA AAA ATC GAT TCT GCT CCT CTA GCT CTG CTG CAT AAA 774
ATC TTA GTT
Lys Lys Ile Asp Ser Ala Pro Leu Ala Leu Leu His Lye
Ile Leu Val
235 240 245
GAG AAT CCA TCA GCA AGA ATT ACC ATT CCA GAC ATC AAA AAA GAT AGA 822
SUBSTITUTE SHEET (RULE 26)
CA 02270911 1999-OS-04
WO 99/11795 PCT/US98/18558
-3-
Glu Aen Pro Ser Ala Arg Ile Thr Ile Pro Asp Ile Lys Lys Asp Arg
250 255 260
TGG TAC AAC AAA CCC CTC AAG AAA GGG GCA AAA AGG CCC CGA GTC ACT 870
Trp Tyr Asn Lys Pro Leu Lys Lys Gly Ala Lys Arg Pro Arg Val Thr
265 270 275
TCA GGT GGT GTG TCA GAG TCT CCC AGT GGA TTT TCT AAG CAC ATT CAA 9l8
Ser Gly Gly Val Ser Glu Ser Pro Sex Gly Phe Ser Lye His Ile Gln
280 285 290 295
TCC AAT TTG GAC TTC TCT CCA GTA AAC AGT GCT TCT AGT GAA GAA AAT 966
Ser Asn Leu Asp Phe Ser Pro Val Asn Ser Ala Ser Ser Glu Glu Asn
300 305 3l0
GTG AAG TAC TCC AGT TCT CAG CCA GAA CCC CGC ACA GGT CTT TCC TTA 1014
Val Lys Tyr Ser Ser Ser Gln Pro Glu Pro Arg Thr Gly Leu Ser Leu
315 320 325
TGG GAT ACC AGC CCC TCA TAC ATT GAT AAA TTG GTA CAA GGG ATC AGC 1062
Trp Asp Thr Ser Pro Ser Tyr Ile Asp Lys Leu Val Gln Gly Ile Ser
330 335 340
TTT TCC CAG CCC ACA TGT CCT GAT CAT ATG CTT TTG AAT AGT CAG TTA 1I10
Phe Ser Gln Pro Thr Cys Pro Asp His Met Leu Leu Asn Ser Gln Leu
345 350 355
CIT GGC ACC CCA GGA TCC TCA CAG AAC CCC TGG CAG CGG TTG GTC AAA 1l58
Leu Gly Thr Pro Gly Ser Ser Gln Asn Pro Trp Gln Arg Leu Val Lys
360 365 370 375
AGA ATG ACA CGA TTC TTT ACC AAA TTG GAT GCA GAC AAA TCT TAT CAA 1206
Arg Met Thr Arg Phe Phe Thr Lys Leu Asp Ala Asp Lys Ser Tyr Gln
380 385 390
TGC CT'G AAA GAG ACT TGT GAG AL1G TTG GGC TAT CAA TGG AAG AAA AGT l254
Cys Leu Lys Glu Thr Cys Glu Lys Leu Gly Tyr Gln Trp Lye Lys Ser
395 400 405
TGT ATG AAT CAG GTT ACT ATA TCA ACA ACT GAT AGG AGA AAC AAT AAA l302
Cys Met Asn Gln Val Thr Ile Ser Thr Thr Asp Arg Arg Asn Asn Lys
410 415 420
CTC ATT TTC A14A GTG AAT TTG TTA GAA ATG GAT GAT AAA ATA TTG GTT 1350
Leu Ile Phe Lys Val Asn Leu Leu Glu Met Asp Asp Lys Ile Leu Val
425 430 435
GAC TTC CGG CTT TCT AAG GGT GAT GGA TTG GAG TTC AAG AGA CAC TTC 1398
Asp Phe Arg Leu Ser Lye Gly Asp Gly Leu Glu Phe Lys Arg His Phe
440 445 450 455
CTG AAG ATT AAA GGG AAG CTG ATT GAT ATT GTG AGC AGC CAG AAG GTT 1446
Leu Lys Ile Lys Gly Lys Leu Ile Asp Ile Val Ser Ser Gln Lys Val
460 465 470
TGG CTT CCT GCC ACA TGATCGGACC ATCGGCTCTG GGGAATCCTG GTGAATATAG 1501
Trp Leu Pro Ala Thr
SUBSTITUTE SHEET (RULE 26)
CA 02270911 1999-OS-04
WO 99/11795 PCT/US98/18558
-4-
475
TGCTGCTATG TTGACATTAT TCTTCCTAGA GAAGATTATC CTGTCCTGCA1561
AACTGCAAAT
AGTAGTTCCT GAAGTGTTCA CTTCCCTGTT TATCCAF1ACA TCTTCCAATTl621
TATTTTGTTT
GTTCGGCATA CAAATAATAC CTATATCTTA ATTGTAAGCA 1~~AACT2'IGGG16
GrAAACGATGA 81
ATAGAATTCA TTrGATTATT TCTTCATGTG TGTTTAGTAT CZGrAATTTGA1741
AACTCATCTG
GTGGAAACCA AG'tTrCAGGG GACATGAGTT TTCCAGCTIT TATACACACG1801
TATCTCATTT
TTATCAAAAC ATTTTGTTTA ATTCAAAAAG TACATATTCC ATGTTGATTT1861
AATTCTAAGA
TGAACCAATA AAGACATAAT TCTrGTGACT TTTGGACAGT AGATTTATCA1921
GTCTGTGAAG
CGAAGCCAGC TT 1933
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 476 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Val Pro Phe Val Glu Asp Trp Asp Leu Val Gln
Thr Leu Gly
1 5 10 15
Glu Gly Ala Tyr Gly Glu Val Gln Leu Ala Val Aen Arg
Val Thr Glu
20 25 30
Glu Ala Val Ala Val Lys Ile Val Asp Met Lys Arg Ala
Val Asp Cys
35 40 45
Pro Glu Aen Ile Lys Lys Glu Ile Cys Ile Asn Lys Met
Leu Asn His
50 55 60
Glu Asn Val Val Lys Phe Tyr Gly His Arg Arg Glu Gly
Asn Ile Gln
65 70 75 80
Tyr Leu Phe Leu Glu Tyr Cys Ser Gly Gly Glu Leu Phe Asp Arg Ile
85 90 95
Glu Pro Asp Ile Gly Met Pro Glu Pro Asp Ala Gln Arg Phe Phe His
100 105 1l0
Gln Leu Met Ala Gly Val Val Tyr Leu His Gly Ile Gly Ile Thr His
115 120 125
Arg Asp Ile Lys Pro Glu Asn Leu Leu Leu Asp Glu Arg Asp Asn Leu
130 l35 140
Lys Ile Ser Asp Phe Gly Leu Ala Thr Val Phe Arg Tyr Asn Asn Arg
SU6ST1TUTE SHEET (RULE 26)
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WO 99/11795 PCT/US98/18558
-5-
145 l50 l55 16d
Glu Arg Leu Leu Asn Lys Met Cys Gly Thr Leu Pro Tyr Val Ala Pro
165 170 175
Glu Leu Leu Lys Arg Arg Glu Phe His Ala Glu Pro Val Asp Val Trp
180 l85 190
Ser Cys Gly Ile Val Leu Thr Ala Met Leu Ala Gly Glu Leu Pro Trp
195 200 205
Asp Gln.Pro Ser Aep Ser Cye Gln Glu Tyr Ser Aep Trp Lys Glu Lys
210 215 220
Lys Thr Tyr Leu Asn Pro Trp Lys Lys Ile Asp Ser Ala Pro Leu Ala
225 230 235 240
Leu Leu His Lys Ile Leu Val Glu Asn Pro Ser Ala Arg Ile Thr Ile
245 250 Z55
Pro Asp IIe Lys Lys Asp Arg Tzp Tyr Asn Lys Pro Leu Lys Lys Gly
260 265 270
Ala Lys Arg Pro Arg Val Thr Ser Gly Gly Val Ser Glu Ser Pro Ser
Z75 280 285
Gly Phe Ser Lys His Ile Gln Ser Asn Leu Asp Phe Ser Pro Val Asn
290 295 300
Ser Ala Ser Ser Glu Glu Asn Val Lys Tyr Ser Ser Ser Gln Pro Glu
305 3l0 3l5 320
Pro Arg Thr Gly Leu Ser Leu Txp Asp Thr Ser Pro Ser Tyr Ile Asp
325 330 335
Lys Leu Val Gln Gly Ile Ser Phe Ser Gln Pro Thr Cys Pro Asp His
340 345 350
Met Leu Leu Asn Ser Gln Leu Leu Gly Thr Pro Gly Ser Ser Gln Aen
355 360 365
Pro Trp Gln Arg Leu Val Lys Arg Met Thr Arg Phe Phe Thr Lys Leu
370 375 380
Asp Ala Asp Lys Ser Tyr Gln Gars Leu Lys Glu Thr Cys Glu Lys Leu
385 390 395 400
Gly Tyr Gln Trp Lys Lya Ser Cys Met Asn Gln Val Thr Ile Ser Thr
405 4l0 4l5
Thr Asp Arg Arg Asn Asn Lys Leu Ile Phe Lys Val Asn Leu Leu Glu
420 425 430
Met Asp Asp Lys Ile Leu Val Asp Phe Arg Leu Ser Lys Gly Asp Gly
435 440 445
SUBSTITUTE SHEET (RULE 2fi)
CA 02270911 1999-OS-04
WO 99/11795 PCT/US98/18558
-6-
Leu Glu Phe Lys Arg His Phe Leu Lys Ile Lys Gly Lys Leu Ile Asp
450 455 460
Ile Val Ser Ser Gln Lys Val Trp Leu Pro Ala Thr
465 470 475
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
°Mouse Chkl"
(ix) FEATURE:
(A} NAME/KEY: CDS
(B) LOCATION: l..742
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
48
96
GGA GAG TTG CCG TGG GAC CAG CCC AGT GAT AGC TGT CAG GAA TAT CTG
Gly Glu Leu Pro Tzp Asp Gln Pro Ser Asp Ser Cys Gln Glu Tyr Leu
1 5 10 15
ATT GTA AAG AAA AAA AAA ACC TAT CTC AAT CCT TGG AAA AAA ATT GAT
Ile Val Lys Lys Lys Lys Thr Tyr Leu Asn Pro Trp Lys Lye Ile Asp
20 25 30
TCT GCT CCT CTG GCT TTG CTT CAT AAA ATT CTA GTT GAG ACT CCA TCA 144
Ser Ala Pro Leu Ala Leu Leu His Lys Ile Leu Val Glu Thr Pro Ser
35 40 45
TCA AGG ATC ACC ATC CCA GAC ATT AAG AAA GAT AGA TGG TAC AAC AAA 192
Ser Arg Ile Thr Ile Pro Asp Ile Lys Lys Asp Arg Trp Tyr Asn Lys
50 55 60
CCA CTT AAC AGA GGA GCA AAG AGG CCA CGC GCC ACA TCA GGT GGT ATG 240
Pro Leu Aen Arg Gly Ala Lys Arg Pro Arg Ala Thr Ser Gly Gly Met
65 70 75 80
TCA GAG TCT TCT AGT GGA TTC TCT AAG CAC ATT CAT TCC AAT TTG GAC 288
Ser Glu Ser Ser Ser Gly Phe Ser Lys His Ile His Ser Asn Leu Asp
85 90 95
TTT TCT CCA GTA AAT AAT GGT TCC AGT GAA GAA ACC GTG AAG TTC TCT 336
Phe Ser Pro Val Asn Asn Gly Ser Ser Glu Glu Thr Val Lys Phe Ser
l00 l05 110
AGT TCC CAG CCA GAG CCG AGA ACA GGG CTT TCC TTG TGG GAC ACT GGT 384
Ser Ser Gln Pro Glu Pro Arg Thr Gly Leu Ser Leu Trp Asp Thr Gly
SUBSTITUTE SHEET (RULE 26)
CA 02270911 1999-OS-04
WO 99/11795 PCT/US98/18558
l15 l20 l25
CCC TCG AAC GTG GAC AAA CTG GTT CAG GGC ATC AGT TTT TCC CAG CCT 432
Pro Ser Asn Val Asp Lys Leu Val Gln Gly Ile Ser Phe Ser Gln Pro
l30 135 l40
ACG TGT CCT GAG CAT ATG CTT GTA AAC AGT CAG TTA CTC GGT ACC CCT 480
Thr Cys Pro Glu His Met Leu Val Asn Ser Gln Leu Leu Gly Thr Pro
145 150 l55 160
GGA TCT TCA CAG AAC CCC TGG CAG CGC TTG GTC AAA AGA ATG ACG AGG 528
Gly Ser Ser Gln Aen Pro Trp Gln Arg Leu Val Lys Arg Met Thr Arg
l65 170 175
TTC TTT ACT AAA TIG GAT GCG GAC AAG TCT TAC CAA TGC 576
CTG AAA GAG
Phe Phe Thr Lys Leu Asp Ala Asp Lys Ser Tyr Gln Cys
Leu Lys Glu
180 185 190
ACC TTC GAG AAG TTG GGC TAT CAG TGG AAG AAG AGT TGT 624
ATG AAT CAG
Thr Phe Glu Lys Leu Gly Tyr Gln Tzp Lys Lys Ser Cys
Met Asn Gln
195 200 205
GTT ACT GTA TCA ACA ACT GAT AGA AGA AAC AAT AAG TTG 672
ATT TTC AAA
Val Thr Val Ser Thr Thr Asp Arg Arg Asn Asn Lys Leu
Ile Phe Lys
2l0 2l5 220
ATA AAT TTG GTG GAA ATG GAT GAG AAG ATA CTG GTT GAC 720
TTC CGA CTT
Ile Asn Leu Val Glu Met Asp Glu Lys Ile Leu Val Asp
Phe Arg Leu
225 230 235 240
TCT AAA GGC GAC GGC TAC AAT T 742
Ser Lys Gly Asp Gly Tyr Asn
245
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 247 amino acids
(B) TYPB: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Gly Glu Leu Pro Trp Aep Gln Pro Ser Asp Ser Cys Gln Glu Tyr Leu
1 5 10 15
Ile Val Lys Lye Lys Lys Thr Tyr Leu Asn Pro Trp Lys Lys Ile Asp
20 25 30
Ser Ala Pro Leu Ala Leu Leu His Lys Ile Leu Val Glu Thr Pro Ser
35 40 45
Ser Arg Ile Thr Ile Pro Asp Ile Lys Lys Asp Arg Trp Tyr Asn Lys
50 55 60
SUBSTITUTE SHEET (RULE 26)
CA 02270911 1999-OS-04
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_g_
Pro Leu Asn Arg Gly Ala Lys Arg Pro Arg Ala Thr Ser Gly Gly Met
65 70 75 80
Ser Glu Ser Ser Ser Gly Phe Ser Lys His Ile His Ser Asn Leu Asp
85 90 95
Phe Ser Pro Val Asn Asn Gly Ser Ser Glu Glu Thr Val Lys Phe Ser
100 105 110
Ser Ser Gln Pro Glu Pro Arg Thr Gly Leu Ser Leu Trp Asp Thr Gly
l15 l20 125
Pro Ser Asn Val Asp Lys Leu Val Gln Gly Ile Ser Phe Ser Gln Pro
l30 l35 l40
Thr Cys Pro Glu His Met Leu Val Asn Ser Gln Leu Leu Gly Thr Pro
l45 150 l55 160
Gly Ser Ser Gln Asn Pro Trp Gln Arg Leu Val Lys Arg Met Thr Arg
l65 l70 175
Phe Phe Thr Lys Leu Asp Ala Asp Lys Ser Tyr Gln Cys Leu Lys Glu
180 185 l90
Thr Phe Glu Lys Leu Gly Tyr Gln Trp Lys Lys Ser Cys Met Asn Gln
l95 200 205
Val Thr Val Ser Thr Thr Asp Arg Arg Asn Asn Lys Leu Ile Phe Lys
210 215 220
Ile Asn Leu Val Glu Met Asp Glu Lys Ile Leu Val Asp Phe Arg Leu
225 230 235 240
Ser Lys Gly Asp Gly Tyr Asn
245
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
ACGTGGACAA ACTGGTTCAG G 21
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
SUBSTtTUTE SHEET (RULE 26)
CA 02270911 1999-OS-04
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-9-
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CTGATAGCCC AACZTCTCGA A 21
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GGCTCTGGGG AATCCT'GGTG AATATAGTGC TGC 33
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
TCCCCTGJaAA CTZGGTTTCC ACCAGATGAG 30
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNBSS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GTl'GAGACTC CATCATCAAG G 21
SUBSTITUTE SHEET (RULE 26)
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- 10-
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
TCTGGCT'GGG AACTAGrAGAPr C 21
SUBSTITUTE SHEET (RULE 26)
