Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE :fNVENTION
CYCLIN-DEPErfDENT PROTEIN KINASE
CROSS-REFERENCE TO RELATED APPLICATIONS
Pro~risional Application U.S. Serial Number 60/037,855 filed
February 7, 1997.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates to an isolated nucleic acid
molecule (polynucleotide) which encodes a novel human cyclin-
dependent kinas~e (CDK:) comprising a novel cyclin binding domain
signature sequence and lacking several heretofore conserved amino acid
residues involved in regulation of the cdk/cyclin complex. The present
invention also relates to associated human CDK proteins and human
CDK mutant proteins.
BACKGROUND OF THE INVENTION
Cell growth and division in eukaryotic organisms is
mediated through the cell cycle. The cell cycle consists of two major
events separated by two central gap phases. DNA synthesis and
replication occwc during the S phase while mitosis occurs during the M
phase. A first gap phase, called G1, which occurs between the M phase
and the S phase:, allowf~ for accumulation of enzymes and other
compounds necessary to drive DNA synthesis and genome replication.
A second gap phase, called G2, occurs between the S phase and the M
phase, allowing for controls to check for proper DNA replication prior to
committing to cell division.
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protein kinases (CDKs). Activation of a CDK requires binding to a cyclin
regulatory subunit, and in the case of CDKl - CDK6, phosophorylation of
threonine 160/161 (Thr160/161). These CDKs contain a cyclin binding site
near the amino terminal portion of the protein. The activated
CDK/cyclin complex phosphorylates proteins involved in various stages
of the cell cycle.
The family of cyclin proteins may generally be classified as
either G1 cyclins or mitotic cyclins, depending on peak expression levels.
A CDK may bind a subset of cyclins. For example, CDK4 is known to
bind cyclin D1 or cyclin D3 whereas CDK2 is known to bind cyclin A,
cyclin B1, cyclin B2, cyclin B3 and cyclin E. The vertebrate cyclins show
homology within a region of approximately 100 amino acids, referred to
as the cyclin box. This region is responsible for CDK binding and activity
(Kobayashi, et al., 1992, Molec. Biol. Cell. 3: 1279-1294; Lees, et al., 1993,
Molec. Cell. Biol., 1993, 13: 1194-1201). It is this region of the cyclin
protein which interacts with the cyclin binding domain of a respective
CDK protein.
Complete activation of a known CDK/cyclin complex
requires phosphorlyation by a CDK-Activating Kinase (CAK). The
vertebrate CAK has been identified as a CDK/cyclin complex, more
specifically CDK7/cyclinH (Fisher and Morgan, 1994, Cell 78: 713-724).
The CAK enzyme comprises a threonine 170 residue (in human CDK7)
which has been shown to be required for optimal activity (Poon, et al.,
1994, J. Cell Sci. 107: 2789-2799; Fisher and Morgan, 1994, Cell 78: 713-
724).
Inhibition of CDK/cyclin complexes are thought to occur via
phosphorylation at threonine 14 (Thrl4) and/or tyrosine 15 (Tyrl5) of the
CDK subunit. The Weel kinase has been suggested as either a Thrl4
kinase or as a Thrl4 and TyrlS kinase. Additionally, CDC25 is thought
to be a dual kinase targeting both Thrl4 and/or Tyrl5 (Morgan, 1995,
Nature 374: 131-134).
It would be advantageous to identify a gene encoding an
additional CDK protein. A nucleic acid molecule expressing a CDK
protein would be extremely useful in screening for compounds acting as
a modulator of the cell cycle. Such a compound or compounds will be
useful in controlling cell growth associated with cancer or immune cell
proliferation. Additionally, the recombinant form of protein expressed
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from such a novel gene would be useful for an in vitro assay to determine
specificity toward substrate proteins, inhibitors and cyclin activators.
Additionally, an isolated and purified (~DK10 cDNA which encodes
CDK-10 or an active mutant thereof zvill also be useful for the
recombinant production of large quantities of respec;l~ive protein. The
ability to praducE~ large quantities of the protein ~~ould be useful for the
production of a therapeutic agent comprising the (~DK10 protein or a
mutant Such as i;he exemplified mutant disclosed herein. A therapeutic
agent comprised of C.'.DliIO protein wotdd be useful in the treatment of
cell cycle and/or ~,'.;rJK.7.(? .related diseases or conditions wh.i.ch are
CDK10
r esponsive. The present invention addresses and meets this need.
SUMMARY OF 'CHE INVENTION
The present invention relates to an isolated nucleic acid
molecule (polynu.cleotide) which encodes a novel human cyclin-
dependent kinasn. This CDK comprises a novel cyclin binding domain
signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1),
lacks Thrl4 and/or Tyrl.S, and also lacks the T-loop domain containing
the conserved Th.r160/161 residue.
The presenlt invention relates to biologically active
fragments or mutants o:f a novel isolated nucleic acid molecule which
encodes mRNA f:xpressing a novel human cyclin-dependent kinase.
Any such biologically active fragment and/or mutant will encode a
protein or protein fragment comprising a novel cyclin binding domain
signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1),
which lacks Thr:l4 and/or TyrlS as well as a T-loop domain containing
the conserved Trir160/lfil residue. Any such polynucleotide includes but
is not necessarily limited to nucleotide substitutions, deletions,
additions, amino-terminal truncations and carboxy-terminal
truncations such that these mutations encode mRNA which express a
protein or protein fragment of diagnostic, therapeutic or prophylactic
use.
The isolated nucleic acid molecule of the present invention
may include a deoxyribonucleic acid molecule (DNA), such as genomic
DNA and complementary DNA (cDNA), which may be single (coding or
noncoding strand) or double stranded, as well as synthetic DNA, such
as a synthesized, single stranded polynucleotide. The isolated nucleic
3
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acid molecule of the present invention may also include a ribonucleic
acid molecule (RNA).
A preferred aspect of the present invention is disclosed in
SEQ ID N0:11 and Figure 1, a human DNA fragment which encodes the
novel human cyclin-dependent kinase, CDK10.
The present invention also relates to a substantially purified
novel cyclin-dependent kinase which comprises a novel cyclin binding
domain signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SE(~ ID
NO:1), lacks Thrl4 and Tyrl5 which make up the conserved ATP
binding motif of several known CKDs, and also lacks the T-loop domain
containing the conserved Thr160/161 residue.
The present invention also relates to biologically active
fragments and/or mutants of a novel cyclin-dependent kinase which
comprises a novel cyclin binding domain signature sequence, lacks
Thrl4 and/or Tyrl5 which make up the conserved ATP binding motif of
known CKDs, and also lacks the T-loop domain containing the
conserved Thr160/161 residue, including but not necessarily limited to
amino acid substitutions, deletions, additions, amino terminal
truncations and carboxy-terminal truncations such that these
mutations provide for proteins or protein fragments of diagnostic,
therapeutic or prophylactic use.
A preferred aspect of the present invention is disclosed
in SE(1 ID N0:3 and Figure 2, the amino acid sequence of CDK10.
The open reading frame of the CDK10 coding region runs from
nucleotide 210 to nucleotide 1182 of SEQ ID N0:2.
Another preferred aspect of the present invention is
disclosed in SEQ ID N0:11, wherein nucleotide 588 of the wild-type form
(SEQ ID NO: 2) is mutated from "G" to "A".
Another preferred aspect of the present invention is the
mutant protein, (CDK10-D127N), wherein nucleotide 588 of SEQ ID
N0:11 is mutated from "G" to "A", as compared to the wild-type form
(SEQ ID N0:2), which results in a change of Asp127 to Asn127 as
compared to the wild-type amino acid sequence (SEQ ID N0:3), disclosed
as SEfa ID N0:12.
The present invention also relates to methods of expressing
the cyclin-dependent kinases disclosed herein, assays employing these
cyclin-dependent kinases, cells expressing these cyclin-dependent
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kinases, and compounds identified through the use of these cyclin-
dependent kinases, including modulators of the cyclin-dependents
kinase either through direct contact with the cyclin-dependent kinase,
an associated cyclin, or the CKD/cyclin complex. Such modulators
identified in this process are useful as therapeutic agents for controlling
cell growth or inunune cell proliferation commonly associated with
cancer.
DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleotide sequence (SEQ ID N0:2)
which comprises the full length cDNA encoding human CDK10.
Figure 2 shows the amino acid sequence (SEQ ID N0:3)
of human CDKh).
Figure 3 shows the strategy utilized to generate a full-
length DNA frag;m.ent encoding human CDK10.
Figure 4 shows northern blot analysis of human tissue
mRNA hybridized to a 32P-labeled probe from the 3' region of the
DNA fragment encoding human CDK10.
Figyire 5 shows northern blot analysis of human tissue
mRNA hybridized to a 32P-labeled probe from the 3' region of the
DNA fragment encoding human CDK10.
DETAILED DE~~CRIPTION OF THE INVENTION
The present invention relates to an isolated nucleic acid
molecule (polynucleotide) which encodes a novel cyclin-dependent
kinase which co:mprise~e a novel human cyclin binding domain (Pro-
Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1), lacks Thrl4 and/or Tyrl5
which make up the can;served ATP binding motif of known CDKs,
and also lacks the T-loop domain containing the conserved
Thr160/161 residue.
The; present invention also relates to biologically active
fragments and/or mutants of a novel isolated nucleic acid molecule
which encode mRNA expressing a novel human cyclin-dependent
kinase. Such a protein comprises a novel cyclin binding domain
signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1),
lacks Thrl4 and/or Tyr lS, and also lack a T-loop domain containing
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the conserved Thr160/I61 residue. The protein of the present
invention includes but is not limited to nucleotide substitutions,
deletions, additions, amino terminal truncations and carboxy-
terminal truncations such that these mutations encode mRNA
S which express a protein or protein fragment of diagnostic,
therapeutic or prophylactic use.
A preferred aspect of the present invention is disclosed in
Figure 1 and SEf~ ID N0:2, a human cDNA encoding a novel cyclin-
dependent kinase, CDK10, disclosed herein as:
lO GAAAAGGCGC AGTGGGGCCC GGAGCTGTCA CCCCTGACTC GACGCAGCTT CCGTTCTCCT
GGTGACGTCG CCTACAGGAA CCGCCCCAGT GGTCAGCTGC CGCGCTGTTG CTAGGCAACA
GCGTGCGAGC TCAGATCAGC GTGGGGTGGA GGAGAAGTGG AGTTTGGAAG TTCAGGGGCA
CAGGGGCACA GGCCCACGAC TGCAGCGGGA TGGACCAGTA CTGCATCCTG GGCCGCATCG
GGGAGGGCGC CCACGGCATC GTCTTCAAGG CCAAGCACGT GGAGACTGGC GAGATAGTTG
IS CCCTCAAGAA GGTGGCCCTA AGGCGGTTGG AAGACGGCTT CCCTAACCAG GCCCTGCGGG
AGATTAAGGC TCTGCAGGAG ATGGAGGACA ATCAGTATGT GGTACAACTG AAGGCTGTGT
TCCCACACGG TGGAGGCTTT GTGCTGGCCT TTGAGTTCAT GCTGTCGGAT CTGGCCGAGG
TGGTGCGCCA TGCCCAGAGG CCACTAGCCC AGGCACAGGT CAAGAGCTAC CTGCAGATGC
TGCTCAAGGG TGTCGCCTTC TGCCATGCCA ACAACATTGT ACATCGGGAC CTGAAACCTG
ZO CCAACCTGCT CATCAGCGCC TCAGGCCAGC TCAAGATAGC GGACTTTGGC CTGGCTCGAG
TCTTTTCCCC AGACGGCAGC CGCCTCTACA CACACCAGGT GGCCACCAGG TCTGTGGGCT
GCATCATGGG GGAGCTGTTG AATGGGTCCC CCCTTTTCCC GGGCAAGAAC GATATTGAAC
AGCTTTGCTA TGTGCTTCGC ATCTTGGGCA CCCCAAACCC TCAAGTCTGG CCGGAGCTCA
CTGAGCTGCC GGACTACAAC AAGATCTCCT TTAAGGAGCA GGTGCCCATG CCCCTGGAGG
ZS AGGTGCTGCC TGACGTCTCT CCCCAGGCAT TGGATCTGCT GGGTCAATTC CTTCTCTACC
CTCCTCACCA GCGCATCGCA GCTTCCAAGG CTCTCCTCCA TCAGTACTTC TTCACAGCTC
CCCTGCCTGC CCATCCATCT GAGCTGCCGA TTCCTCAGCG TCTAGGGGGA CCTGCCCCCA
AGGCCCATCC AGGGCCCCCC CACATCCATG ACTTCCACGT GGACCGGCCT CTTGAGGAGT
CGCTGTTGAA CCCAGAGCTG ATTCGGCCCT TCATCCTGGA GGGGTGAGAA GTTGGCCCTG
3O GTCCCGTCTG CCTGCTCCTC AGGACCACTC AGTCCACCTG TTCCTCTGCC ACCTGCCTGG
CTTCACCCTC CAAGGCCTCC CCATGGCCAC AGTGGGCCCA CACCACACCC TGCCCCTTAG
CCCTTGCGAG GGTTGGTCTC GAGGCAGAGG TCATGTTCCC AGCCAAGAGT ATGAGAACAT
CCAGTCGAGC AGAGGAGATT CATGGCCTGT GCTCGGTGAG CCTTACCTTC TGTGTGCTAC
TGACGTACCC ATCAGGACAG TGAGCTCTGC TGCCAGTCAA GGCCTGCATA TGCAGAATGA
3S CGATGCCTGC CTTGGTGCTG CTTCCCCGAG TGCTGCCTCC TGGTCAAGGA GAAGTGCAGA
GAGTAAGGTG TCCTTATGTT GGAAACTCAA GTGGAAGGAA GATTTGGTTT GGTTTTATTC
TCAGAGCCAT TAAACACTAG TTCAGTATGT GAGATATAGA TTCTAAAAAC CTCAGGTGGC
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TCTGCCTTAT GTCTG,TTCCT C'.CTTCATTTC TCTCAAGGGA AATGGCTAAG GTGGCATTGT
CTCATGGCTC TCGTTTTTGG CiGTCATGGGG AGGGTAGCAC CAGGCATAGC CACTTTTGCC
CTGAGGGACT CCTGTGTGCT TCACATCACT GAGCACTCAT TTAGAAGTGA GGGAGACAGA
AGTCTAGGCC CAGGGATGGC TCCAGTTGGG GATCCAGCAG GAGACCCTCT GCACATGAGG
S CTGGTTTACC AACATCTACT C'CCTCAGGAT GAGCGTGAGC CAGAAGCAGC TGTGTATTTA
AGGAAACAAG CGTTCCTGGA ~I,TTAATTTAT AAATTTAATA AATCCCAATA TAATCCCAAA
AA,AAAAAAAA AAAAAATTCC TGCGGCCGCA AGGA (SEQ ID N0:2).
The presenl; invention also relates to a substantially
purified novel cyclin-dependent kinase which comprises a novel
cyclin binding domain F>ignature sequence (Pro-Asn-Gln-Ala-Leu
Arg-Glu; SEQ ID N0:1), lacks Thrl4 and/or Tyrl5 as well as the
T-loop domain containing the conserved Thr160/I61 residue. Any
such nucleic acid. may be isolated and characterized from a
mammalian cell, including but not limited to human, human and
1S rodent. A human form :is an especially preferred form, such as the
isolated cDNA exemplified herein as set forth in SEQ ID N0:2 and a
dominant negative mutant form as set forth in SEQ ID N0:12.
The present; invention also relates to biologically active
fragments and/or mutants of a novel cyclin-dependent kinase which
comprises the navel cyclin binding domain (Pro-Asn-Gln-Ala-Leu-
Arg-Glu; SEQ ID N0:1), lacks Thrl4 and/or Tyrl5 which make up the
conserved ATP banding motif of known CDKs, and also lacks the
T-loop domain containing the conserved Thr160/161 residue,
including but nol; necessarily limited to amino acid substitutions,
2S deletions, additions, amino terminal truncations and carboxy-
terminal truncations such that these mutations provide for proteins
or protein fragments of diagnostic, therapeutic or prophylactic use.
Any such nucleic: acid may be isolated and characterized from a
mammalian cell, including but not limited to human, human and
rodent, with a human form being an especially preferred form.
A preferred aspect of the present invention is disclosed in
SEQ ID N0:3 and Figure 2, the amino acid sequence of CDK10. The open
reading frame of the CDK10 coding region runs from nucleotide 210 to
nucleotide 1182 of SEfa l:D N0:2. The amino acid sequence of the novel
3S cyclin-dependent kinase, CDK10, is disclosed herein as:
MDQYCILGRI GEGAHGIVFK AKHVETGEIV ALKKVALRRL EDGFPNQALR
EIKALQEMED NQS.'VVQLK~~V FPHGGGFVLA FEFMLSDLAE VVRHAQRPLA
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~p'~~~ 0 3 SEP 1998
QAQVKSYLQM LLKGVAFCHA NNIVHRDLKP ANLLISASGQ LKIADFGLAR
VFSPDGSRLY THQV'ATRSVCi CIMGELLNGS PLFPGKNDIE QLCYVLRILG
TPNPQVWPEL TELPDYNKIS FKEQVPMPLE EVLPDVSPQA LDLLGQFLLY
PPHQRIAASK ALLHQYFFTA PLPAHPSELP IPQRLGGPAP KAHPGPPHIH
S DFHVDRPLEE SLLN'PELIRF' FILEG (SEQ ID N0:3).
Another preferred aspect of the present invention is
disclosed in SEQ IUD NO:11, wherein nucleotide 588 of the wild-type form
(SEfa ID NO: 2) is mutated from "G" to "A".
Another preferred aspect of the present invention is the
,..,~ 10 mutant protein, (CDK10-I)127N), wherein nucleotide 588 of SEQ ID
NO:11 is mutated from "G" to "A", as compared to the wild-type form
(SEQ ID N0:2), which results in a change of Asp127 to Asn127 as
compared to the wild-type amino acid sequence (SEQ ID N0:3), disclosed
as SEQ ID N0:12.
1S The present invention also relates to methods of expressing
the cyclin-dependent kinases disclosed herein, assays employing these
cyclin-dependent ~:inases, cells expressing these cyclin-dependent
kinases, and compounds identified through the use of these cyclin-
dependent kinases, including modulators of the cyclin-dependents
20 kinase either through direct contact with the cyclin-dependent kinase,
an associated cyclin, or the CKD/cyclin complex. Such modulators
-~..
__:-e identified in this process .are useful as therapeutic agents for
controlling
cell growth or immune cell proliferation associated with human
cancers. Additionally, an isolated and purified CDK10 cDNA which
2S encodes CDK-10 or an active mutant thereof will also be useful for the
recombinant production o:f large quantities of respective protein. The
ability to produce large quantities of the protein would be useful for the
production of a the~rapeutiic agent comprising the CDK10 protein or a
mutant such as the exemplified mutant disclosed herein. A therapeutic
30 agent comprised of CDK10 protein would be useful in the treatment of
cell cycle and/or C~DK10 related diseases or conditions which are CDK10
responsive or possibly a therapeutic agent comprised of a mutant,
including but not limited to CDK10-D127N, which may be useful in the
treatment of cell cycle diseases or conditions which are responsive to the
3S regulatory effects of the mutant kinase.
The isolated nucleic acid molecule of the present invention
may include a deo~xyribormcleic acid molecule (DNA), such as genomic
- R8
d,N~ENDED SHEET
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DNA and complementary DNA (cDNA), which may be single (coding or
noncoding strand) or double stranded, as well as synthetic DNA, such as
a synthesized, siingle stranded polynucleotide. The isolated nucleic acid
molecule of the present invention may also include a ribonucleic acid
molecule (RNA).
It i;; known that there is a substantial amount of
redundancy in t:he various colons which code for specific amino
acids. Therefore., this invention is also directed to those DNA
sequences which contain alternative colons which code for the
eventual translation of 'the identical amino acid. For purposes of this
specification, a e~equence bearing one or more replaced colons will be
defined as a degenerate variation. Also included within the scope of
this invention are mutations either in the DNA sequence or the
translated proteiin which do not substantially alter the ultimate
physical properties of the expressed protein. For example,
substitution of valine for leucine, arginine for lysine, or asparagine
for glutamine m~~y not cause a change in functionality of the
polypeptide. Therefore, this invention is also directed to those DNA
sequences which express RNA comprising alternative colons which
code for the eventual translation of the identical amino acid, as
shown below:
A=Ala=Alanine: colons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codon~s UGC, UGU
D=Asp=Aspartic: acid: c;odons GAC, GAU
E=Glu=Glutamic acid: colons GAA, GAG
F=Phe=Phenylal.anine: colons UUC, UUU
G=Gly=Glycine: colons GGA, GGC, GGG, GGU
H=His =Histidine: colons CAC, CAU
I=Ile =Isoleucin~e: colons AUA, AUC, AUU
K=Lys=Lysine: colons AAA, AAG
L=Leu=Leucine: colons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methiorune: colon AUG
N=Asp=Asparag;ine: co~dons AAC, AAU
P=Pro=Proline: ~codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: colons CAA, CAG
R=Arg=ArgininE~: colons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: colons AGC, AGU, UCA, UCC, UCG, UCU
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T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Therefore, the present invention discloses codon redundancy which
may result in differing DNA molecules expressing an identical
protein. For purposes of this specification, a sequence bearing one or
more replaced codons will be defined as a degenerate variation. Also
included within the scope of this invention are mutations either in
the DNA sequence or the translated protein which do not
substantially alter the ultimate physical properties of the expressed
protein. For example, substitution of valine for leucine, arginine for
lysine, or asparagine for glutamine may not cause a change in
functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be
altered so as to code for a peptide having properties that are different
than those of the naturally occurring peptide. Methods of altering the
DNA sequences include but are not limited to site directed mutagenesis.
Examples of altered properties include but are not limited to changes in
the affinity of an enzyme for a substrate or a receptor for a ligand.
It is known that DNA sequences coding for a peptide may be
altered so as to code for a peptide having properties that are different
than those of the naturally-occurring peptide. Methods of altering the
DNA sequences include, but are not limited to site directed mutagenesis.
Examples of altered properties include but are not limited to changes in
the affinity of an enzyme for a substrate or a receptor for a ligand.
As used herein, a "biologically active equivalent" or
"functional derivative" of a wild type CDK possesses a biological
activity that is substantially similar to the biological activity of the
wild type CDK10 protein. The term "functional derivative" is intended
to include the "fragments," "mutants," "variants," "degenerate
variants," "analogs" and "homologues" or to "chemical derivatives" of
the wild type CDK10 protein. The term "fragment" is meant to refer
to any polypeptide subset of wild type CDK10. The term "mutant" is
meant to refer to a molecule that may be substantially similar to the
wild type form but possesses distinguishing biological
characteristics. Such altered characteristics include but are in no
fd
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way limited to altered enzymatic activity, altered cyclin binding
altered substrate binding, altered substrate affinity and altered
sensitivity to chemical compounds affecting biological activity.
An exemplified mutant i.s CDK10-D127N, wherein a single base
mutation at nucleotide 588 of SEMI ID N0:2 results in a single amino
acid substitution at residue 127, from aspartic acid to asparagine.
This mutation ah~ers kinase activity of CDK10-D127N as compared to
the wild type CD K10 protein. The term "variant" is meant to refer to a
molecule substaritially similar in structure and function to either the
entire wild type ~~rotein or to a fragment thereof. A molecule is
"substantially similar" to a wild type CDK10-like protein if both
molecules have substantially similar structures or if both molecules
possess similar b~iologicail activity. Therefore, if the two molecules
possess substantiially similar activity, they are considered to be
variants even if the structure of one of the molecules is not found in
the other or even if the fwo amino acid sequences are not identical.
The term "analog" refers to a molecule substantially
similar in function to either the entire wild type CDK10-like protein or
to a fragment thereof.
"Substantial homology" or "substantial similarity", when
referring to nucleic acids means that the segments or their
complementary ,strands,, when optimally aligned and compared, are
identical with appropriate nucleotide insertions or deletions, in at least
75% of the nucleotides. Alternatively, substantial homology exists when
the segments will hybridize to a strand or its complement.
The term "substantial homology", when referring to
polypeptides, indicates that the polypeptide or protein in question
exhibits at least about 30% homology with the naturally occurring
protein in question, usually at least about 65% homology.
The nucleic: acids claimed herein may be present in whole
cells or in cell lysates or in a partially purified or substantially purified
form. A nucleic acid i.s considered substantially purified when it is
purified away from environmental contaminants. Thus, a nucleic acid
sequence isolated from cells is considered to be substantially purified
when purified from cellular components by standard methods while a
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chemically synthesized nucleic acid sequence is considered to be
substantially purified when purified from its chemical precursors.
Any of a variety of procedures may be used to clone
CDK10. These methods include, but are not limited to, (1) a RACE
PCR cloning technique (Frohman, et al., 1988, Proc. Natl. Acad.
Sci.85: 8998-9002). 5' and/or 3' RACE may be performed to
generate a full length cDNA sequence. This strategy involves
using gene-specific oligonucleotide primers for PCR amplification
of CDK10 cDNA. These gene-specific primers are designed
through identification of an expressed sequence tag (EST)
nucleotide sequence which has been identified by searching any
number of publicly available nucleic acid and protein databases;
(2) direct functional expression of the CDK10 cDNA following the
construction of an CDK10-containing cDNA library in an
appropriate expression vector system; (3) screening a CDK10-
containing cDNA library constructed in a bacteriophage or
plasmid shuttle vector with a labeled degenerate oligonucleotide
probe designed from the amino acid sequence of the CDK10
protein; (4) screening a CDK10-containing cDNA library
constructed in a bacteriophage or plasmid shuttle vector with a
partial cDNA encoding the CDK10 protein. This partial cDNA is
obtained by the specific PCR amplification of CDK10 DNA
fragments through the design of degenerate oligonucleotide
primers from the amino acid sequence known for other CDK
kinases which are related to the CDK10 protein; (5) screening an
CDK10-containing cDNA library constructed in a bacteriophage or
plasmid shuttle vector with a partial cDNA encoding the CDK10
protein. This strategy may also involve using gene-specific
oligonucleotide primers for PCR amplification of CDK10 cDNA
identified as an EST as described above; or (6) designing 5' and 3'
gene specific oligonucleotides using SEQ ID N0:2 as a template so
that either the full length cDNA may be generated by known
RACE techniques, or a portion of the coding region may be
generated by these same known RACE techniques to generate and
isolate a portion of the coding region to use as a probe to screen one
of numerous types of cDNA and/or genomic libraries in order to
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isolate a full length version of the nucleotide sequence encoding
CDK10.
It is. readily apparent to those skilled in the art that
other types of lix~raries, as well as libraries constructed from other
cells types or spE~cies types, may be useful for isolating a
CDK10-encoding DNA or a CDK10 homologue. Other types of
libraries include, but are not limited to, cDNA libraries derived
from other cells or cell lines other than human cells or tissue
such as murine cells, rodent cells or any other such vertebrate
host which may contain a CDK10-encoding DNA. Additionally a
CDK10 gene ma~~ be isolated by oligonucleotide- or polynucleotide-
based hybridization scrE~ening of a vertebrate genomic library,
including but not limited to a human genomic library, a murine
genomic library and a rodent genomic library, as well as
concomitant human genomic DNA libraries.
It is readily apparent to those skilled in the art that
suitable cDNA libraries may be prepared from cells or cell lines
which have CDK:10 actiwity. The selection of cells or cell lines for
use in preparing a cDNA library to isolate a CDK10 cDNA may be
done by first me~3suring cell associated CDK10 activity using any
known assay for CDK activity.
Preparation of cDNA libraries can be performed by
standard techniques well known in the art. Well known cDNA
library construction techniques can be found for example, in
Sambrook, et al., 1989, .iVlolecukzr Cloning: A Laboratory Manual;
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Complementary DNA libraries may also be obtained from
numerous commercial sources, including but not limited to
Clontech Laboratories, Inc. and Stratagene.
It is, also readily apparent to those skilled in the art
that DNA encoding CDK10 may also be isolated from a suitable
genomic DNA literary. Construction of genomic DNA libraries
can be performed by standard techniques well known in the art.
Well known genomic DNA library construction techniques can be
found in Sambrook, et al., supra.
In order to clone the CDK10 gene by one of the
preferred methods, the amino acid sequence or DNA sequence of
13
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CDK10 or a homologous protein may be necessary. To accomplish
this, the CDK10 or a homologous protein may be purified and
partial amino acid sequence determined by automated
sequenators. It is not necessary to determine the entire amino
acid sequence, but the linear sequence of two regions of 6 to 8
amino acids can be determined for the PCR amplification of a
partial CDK10 DNA fragment. Once suitable amino acid
sequences have been identified, the DNA sequences capable of
encoding them are synthesized. Because the genetic code is
degenerate, more than one codon may be used to encode a
particular amino acid, and therefore, the amino acid sequence
can be encoded by any of a set of similar DNA oligonucleotides.
Only one member of the set will be identical to the CDK10 sequence
but others in the set will be capable of hybridizing to CDK10 DNA
IS even in the presence of DNA oligonucleotides with mismatches.
The mismatched DNA oligonucleotides may still sufficiently
hybridize to the CDK10 DNA to permit identification and isolation
of CDK10 encoding DNA. Alternatively, the nucleotide sequence of
a region of an expressed sequence may be identified by searching
one or more available genomic databases. Gene-specific primers
may be used to perform PCR amplification of a cDNA of interest
from either a cDNA library or a population of cDNAs. As noted
above, the appropriate nucleotide sequence for use in a PCR-based
method may be obtained from SEIa ID N0:2, either for the purpose
of isolating overlapping 5' and 3' RACE products for generation of
a full-length sequence coding for CDK10, or to isolate a portion of
the nucleotide sequence coding for CDK10 for use as a probe to
screen one or more cDNA- or genomic-based libraries to isolate a
full-length sequence encoding CDK10 or CDK10-like proteins.
In an exemplified method, the RACE PCR technique
(Frohman, et al., 1988, Proc. Nactl. Acad. Sci 85: 8998-9002) is used
for cloning a 5'coding region of CDK10 encoding DNA. First
round PCR used adapter-ligated human placenta cDNA template
(from Clontech), gene-specific primer PK22L234,
(5'-TGATGCAGCCCACAGACCTG-3 ; SEQ ID NO: 4) and an
adapter primer AP1
1~
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(5'-CCATCCTAA'TACCxACTCACTATAGGGC-3'; SEQ ID N0:5).
PCR amplif"lcation was performed using the ElongaseTM.
Thermal cycling was completed and a portion of this first PCR
reaction was added to a aecond PCR reaction as DNA template.
This PCR reaction also differed from the first PCR reaction in that
the nested gene specific primer PK22L161
(5'-GCCGTCTGGGGAAAAGA-3'; SEQ ID N0:6) and the nested
adapter primer h~P2
(5'-ACTCACTAT~~1GGGC;TCGAGCGGC-3', SEQ ID N0:7) were
utilized.
An a.pproxirnately 600 by DNA product was identified
from a 1% agarose electrophoresis gel, excised, and purified using
a Qiagen PCR-spun column (Qiaquick T""). This fragment was
used directly for IDNA sequencing using PK22L161 and AP2
primers, and for cloning into pCR2.1 using the Invitrogen TA-
cloning kit.
A DhTA fragment 3' to and overlapping the 600 by 5'
fragment was idE~ntified by searching public nucleic acid and
protein database;. This 3' fragment is an approximately 1.8 Kb
cDNA insert available a;s a NotI-HindIII fragment in a typical
phagemid vector. This cDNA clone is readily identified by
Genbank Accession No. H17727, Image Clone ID No. 50484,
Washington University Clone ID No. ym40a06, and GBD Clone ID
No. 423294. This cDNA was isolated from a library constructed
from human infant brain mRNA. This construct is available
from Research Genetics,. Inc., 2130 Memorial Parkway SW,
Hunstville, AL 3.5801 (h.ttp://www. resgen.com).
A full lengfh CDK10 coding region was assembled in
pLITMUS28 (New England Biolabs) as an expression cassette with
a BamHI site appended ;just 5' to the ATG translational start
codon. A BamHl~:-XbaI fragment bearing CDK10 was recloned
into pcDNA3.1 e:~pression vector (Invitrogen) and a BamHI-NcoI
fragment bearing; CDKIiD was recloned into pBlueBacHis2
baculovirus expression vector (Invitrogen). A similar construct
was generated which contains dominant-negative single base pair
mutation of CDK:10. This mutant was generated from
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pLITMUS28::CDK10 using the Stratagene "Quik Change" kit and
primers 22U-D 127N
(5'-CAACATTGTACATCGGAACCTGAAACCTGCC-3'; SEQ ID
NO: 8) and 22L-D127N
(5'-GGCAGGTTTCAGGTTCC-GATGTACAATGTTG-3'; SEQ ID
NO: 9). Both mutant constructions were subcloned into pcDNA3.1
(as a BamHI-XbaI fragment) and pBlueBacHis2 (as a BamHI-
NcoI fragment), respectively.
The sequence for the 5' upstream sequences, coding
region and 3' untranslated sequences for the human full-length
cDNA encoding CDK10 is shown in SEQ ID N0:2. The deduced
amino acid sequence of CDK10 from the cloned cDNA is shown in
SEQ ID N0:3. Inspection of the determined cDNA sequence reveals
the presence of a single open reading frame that encodes a 325 amino
acid protein. The open reading frame of the CDK10 coding region
runs from nucleotide 210 to nucleotide 1182 of SEQ ID N0:2.
The nucleotide sequence which encodes a preferred
mutant form (Asp127 to Asn127), is disclosed as SEQ ID N0:11.
The amino acid sequence for this preferred mutant
form, CDK10-D127N, is disclosed in SEQ ID N0:12.
A variety of mammalian expression vectors may be
used to express recombinant CDK10 in mammalian cells.
Expression vectors are defined herein as DNA sequences that are
required for the transcription of cloned DNA and the translation of
their mRNAs in an appropriate host. Such vectors can be used to
express eukaryotic DNA in a variety of hosts such as bacteria, blue
green algae, plant cells, insect cells and animal cells. Specifically
designed vectors allow the shuttling of DNA between hosts such as
bacteria-yeast or bacteria-animal cells. An appropriately
constructed expression vector should contain: an origin of
replication for autonomous replication in host cells, selectable
markers, a limited number of useful restriction enzyme sites, a
potential for high copy number, and active promoters. A promoter
is defined as a DNA sequence that directs RNA polymerase to bind
to DNA and initiate RNA synthesis. A strong promoter is one
which causes mRNAs to be initiated at high frequency.
Expression vectors may include, but are not limited to, cloning
Ib
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vectors, modified cloning vectors, specifically designed plasmids
or viruses.
Conirnercially available mammalian expression vectors
which may be suitable for recombinant CDK10 expression, include
but are not limited to, pcDNA3.1 (Invitrogen), pBlueBacHis2
(Invitrogen), pLITMUS~;B, pLITMUS29, pLITMUS38 and
pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp
(Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl
(Stratagene), pS(15 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-
1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt
(ATCC 371.99), p)R,SVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),
pUCTag (ATCC x7460), .and ~,ZD35 (ATCC 37565).
A variety of bacterial expression vectors may be used to
express recombinant CI)K10 in bacterial cells. Commercially
available bacterial exprcasion vectors which may be suitable for
recombinant CDlKlO expression include, but are not limited to pCR2.1
(Invitrogen), pE~~lla (Novagen), lambda gtll (Invitrogen), pcDNAII
(Invitrogen), pK1~223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to
express recombinant CI)K10 in fungal cells. Commercially available
fungal cell expression vectors which may be suitable for recombinant
CDK10 expression include but are not limited to pYES2 (Invitrogen),
Pichicz expression vector (Invitrogen).
A variety of insect cell expression vectors may be used to
express recombinant receptor in insect cells. Commercially available
insect cell expre;>sion vectors which may be suitable for recombinant
expression of CDK10 include but are not limited to pBlueBacIII and
pBlueBacHis2 (Invitrogen).
The expression vector may be introduced into host cells
via any one of a number of techniques including but not limited to
transformation, transfection, lipofection, protoplast fusion, and
electroporation. The expression vector-containing cells are clonally
propagated and individually analyzed to determine whether they
produce CDK10 protein. Identification of CDK10 expressing host cell
clones may be done by several means, including but not limited to
immunological reactivity with anti-CDK10 antibodies.
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Expression of CDK10 DNA may also be performed using in
vitro produced synthetic mRNA or native mRNA. Synthetic mRNA or
mRNA isolated from CDK10 producing cells can be efficiently translated
in various cell-free systems, including but not limited to wheat germ
extracts and reticulocyte extracts, as well as efficiently translated in cell
based systems, including but not limited to microinjection into frog
oocytes, with microinjection into frog oocytes being preferred.
An expression vector containing DNA encoding a CDK10-
like protein may be used for expression of CDK10 in a recombinant host
cell. Recombinant host cells may be prokaryotic or eukaryotic, including
but not limited to bacteria such as E. coli, fungal cells such as yeast,
mammalian cells including but not limited to cell lines of human,
bovine, porcine, monkey and rodent origin, and insect cells including
but not limited to Drosophila and silkworm derived cell lines. Cell lines
derived from mammalian species which may be suitable and which are
commercially available, include but are not limited to, L cells L-M(TK-)
(ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85),
293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1
(ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61),
3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2),
C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL
171).
The expression vector may be introduced into host cells via
any one of a number of techniques including but not limited to
transformation, transfection, protoplast fusion, and electroporation.
The expression vector-containing cells are individually analyzed to
determine whether they produce CDK10 protein. Identification of
CDK10 expressing cells may be done by several means, including but not
limited to immunological reactivity with anti-CDK10 antibodies, and the
presence of host cell-associated CDK10 activity.
The cloned CDK10 cDNA obtained through the methods
described above may be recombinantly expressed by molecular cloning
into an expression vector (such as pcDNA3.1, pCR2.l, pBlueBacHis2 and
pLITMUS28) containing a suitable promoter and other appropriate
transcription regulatory elements, and transferred into prokaryotic or
eukaryotic host cells to produce recombinant CDK10. Techniques for
such manipulations can be found described in Sambrook, et al., supra ,
I~
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are discussed at length in the Example section and are well known and
easily available to tile artisan of ordinary skill in the art.
Expression of CDK10 DNA may also be performed using in
vitro produced syntlaetic mRNA. Synthetic mRNA can be efficiently
translated in various cell-fi~ee systems, including but not limited to
wheat germ extracts and reticulocyte extracts, as well as efficiently
translated in cell based systems, including but not limited to
microinjection into :frog ooc;ytes, with microinjection into frog oocytes
being preferred.
To determine i~he CDK10 cDNA sequences) that yields
optimal levels of CDK10 protein, CDK10 cDNA molecules including but
not limited to the following can be constructed: the full-length open
reading frame of the CDKlLO cDNA and various constructs containing
portions of the cDNA encoding only specific domains of the protein or
rearranged domain: of the protein. All constructs can be designed to
contain nane, all or portions of the 5' and/or 3' untranslated region of
CDK10. CDK10 activity and levels of protein expression can be
determined following the introduction, both singly and in combination,
of these constructs :into appropriate host cells. Following determination
of the CDK10 cDN~!~ cassette yielding optimal expression in transient
assays, this CDK10 cDNA construct is transferred to a variety of
expression vectors ;including recombinant viruses), including but not
limited to those for mammalian cells, plant cells, insect cells, oocytes,
bacteria, and yeast cells.
Levels of CDK.10 protein in host cells is quantified by a
variety of techniquE~s including, but not limited to, immunoaffinity
and/or ligand aff'lniity techniques. CDK10-specific affinity beads or
CDK10-specific antibodies .are used to isolate 3~S-methionine labeled or
unlabelled CDK10 F~rotein. Labeled CDK10 protein is analyzed by SDS-
PAGE. Unlabelled CDK10 protein is detected by Western blotting, ELISA
or RIA assays employing C;DK10 specific antibodies.
Following expression of CDK10 in a host cell, CDK10 protein
may be recovered to provide CDK10 in active form. Several CDK10
purification proced»res arE~ available and suitable for use. Recombinant
CDK10 may be purified from cell lysates and extracts, or from
conditioned culture medium, by various combinations of, or individual
application of salt :fractionation, ion exchange chromatography, size
~9
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exclusion chromatography, hydroxylapatite adsorption chromatography
and hydrophobic interaction chromatography.
In addition, recombinant CDK10 can be separated from
other cellular proteins by use of an immuno-affinity column made with
monoclonal or polyclonal antibodies specific for full length CDK10, or
polypeptide fragments of CDK10. Additionally, polyclonal or monoclonal
antibodies may be raised against a synthetic peptide (usually from about
9 to about 25 amino acids in length) from a portion of the protein as
disclosed in SEIa ID N0:3. Monospecific antibodies to CDK10 are
purified from mammalian antisera containing antibodies reactive
against CDK10 or are prepared as monoclonal antibodies reactive with
CDK10 using the technique of Kohler and Milstein (1975, Nature 256:
495-497). Monospecific antibody as used herein is defined as a single
antibody species or multiple antibody species with homogenous binding
characteristics for CDK10. Homogenous binding as used herein refers
to the ability of the antibody species to bind to a specific antigen or
epitope, such as those associated with the CDK10, as described above.
CDK10 specific antibodies are raised by immunizing animals such as
mice, rats, guinea pigs, rabbits, goats, horses and the like, with an
appropriate concentration of CDK10 or CDK10 synthetic peptide either
with or without an immune adjuvant.
Preimmune serum is collected prior to the first
immunization. Each animal receives between about 0.1 ~.g and about
1000 ~g of CDK10 associated with an acceptable immune adjuvant. Such
acceptable adjuvants include, but are not limited to, Freund's complete,
Freund's incomplete, alum-precipitate, water in oil emulsion
containing Corynebacterium pa~rr~um and tRNA. The initial
immunization consists of the CDK10 protein or CDK10 synthetic peptide
in, preferably, Freund's complete adjuvant at multiple sites either
subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled
at regular intervals, preferably weekly, to determine antibody titer. The
animals may or may not receive booster injections following the initial
immunizaiton. Those animals receiving booster injections are
generally given an equal amount of CDK10 in Freund's incomplete
adjuvant by the same route. Booster injections are given at about three
week intervals until maximal titers are obtained. At about 7 days after
each booster immunization or about weekly after a single
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immunization, the animals are bled, the serum collected, and aliquots
are stored at about -20°C.
Monoclonal antibodies (mAb) reactive with CDK10 are
prepared by immutuzing inbred mice, preferably Balb/c, with CDK10.
The mice are immunized b;y the IP or SC route with about 1 ~g to about
100 fig, preferably about l0i ~,g, of CDK10 in about 0.5 ml buffer or saline
incorporated in an equal volume of an acceptable adjuvant, as discussed
above. Freund's complete adjuvant is preferred. The mice receive an
initial immunization on da;y 0 and are rested for about 3 to about 30
weeks. Immunized mice are given one or more booster immunizations
of about 1 to about :100 ~g of CDK10 in a buffer solution such as phosphate
buffered saline by t;he intravenous (IV) route. Lymphocytes, from
antibody positive mice, preferably splenic lymphocytes, are obtained by
removing spleens from immunized mice by standard procedures known
in the art. Hybridoma cellLs are produced by mixing the splenic
lymphocytes with an appropriate fusion partner, preferably myeloma
cells, under conditions whiich will allow the formation of stable
hybridomas. Fusion partners may include, but are not limited to:
mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0
being preferred. The antibody producing cells and myeloma cells are
fused in polyethylene glycol, about 1000 mol. wt., at concentrations from
about 30% to about 50%. Fused hybridoma cells are selected by growth in
hypoxanthine, thymidine and aminopterin supplemented Dulbecco's
Modified Eagles Medium (:DMEM) by procedures known in the art.
Supernatant fluids are collected form growth positive wells on about
days 14, 18, and 21 and are screened for antibody production by an
immunoassay such. as solid phase immunoradioassay (SPIRA) using
CDK10 as the antigen. Th.e culture fluids are also tested in the
Ouchterlony precipitation assay to determine the isotype of the mAb.
Hybridoma cells from antibody positive wells are cloned by a technique
such as the soft ag:~r technique of MacPherson, 1973, Soft Agar
Techniques, in Tis:>ue Culture Methods and Applicactions, Kruse and
Paterson, Eds., Academic Press.
Monoclonal antibodies are produced in vivo by injection of
pristine primed Balb/c mice, approximately 0.5 ml per mouse, with
about 2 x 100 to about 6 x 106 hybridoma cells about 4 days after priming.
Ascites fluid is collected at; approximately 8-12 days after cell transfer
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and the monoclonal antibodies are purified by techniques known in the
art.
In vitro production of anti-CDK10 mAb is carried out by
growing the hydridoma in DMEM containing about 2% fetal calf serum
to obtain sufl'lcient quantities of the specific mAb. The mAb are purified
by techniques known in the art.
Antibody titers of ascites or hybridoma culture fluids are
determined by various serological or immunological assays which
include, but are not limited to, precipitation, pas sive agglutination,
enzyme-linked immunosorbent antibody (ELISA) technique and
radioimmunoassay (RIA) techniques. Similar assays are used to detect
the presence of CDK10 in body fluids or tissue and cell extracts.
It is readily apparent to those skilled in the art that the
above described methods for producing monospecific antibodies may be
utilized to produce antibodies specific for CDK10 polypeptide fragments,
or full-length CDK10 polypeptide.
CDK10 antibody affinity columns are made by adding the
antibodies to Affigel-10 (Biorad), a gel support which is pre-activated
with N-hydroxysuccinimide esters such that the antibodies form
covalent linkages with the agarose gel bead support. The antibodies are
then coupled to the gel via amide bonds with the spacer arm. The
remaining activated esters are then quenched with 1M ethanolamine
HC1 (pH 8). The column is washed with water followed by 0.23 M
glycine HC1 (pH 2.6) to remove any non-conjugated antibody or
extraneous protein. The column is then equilibrated in phosphate
buffered saline (pH 7.3) and the cell culture supernatants or cell extracts
containing CDK10 or CDK10 fragments are slowly passed through the
column. The column is then washed with phosphate buffered saline
until the optical density (A2g0) falls to background, then the protein is
eluted with 0.23 M glycine-HCl (pH 2.6). The purified CDK10 protein is
then dialyzed against phosphate buffered saline.
The novel CDK10 of the present invention is suitable for use
in an assay procedure for the identification of compounds which
modulate CDK10 activity. Modulating CDK10 activity, as described
herein includes the inhibition or activation of the protein and also
includes directly or indirectly affecting the cell cycle regulatory
properties associated with CDK10 activity. Compounds which modulate
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CDK10 activity include agonists, antagonists, inhibitors, activators, and
compounds which directly o~r indirectly affect regulation of the CDK10
activity and/or the C:DK10/c;yclin association.
The CD~K10 protein kinase of the present invention may be
obtained from both native and recombinant sources for use in an assay
procedure to identif~;~ CDK10 modulators. In general, an assay
procedure to identif3~ CDK1~0 modulators will contain the CDKIO-protein
of the present invention, native cyclin protein which will form a
CDK10/cyclin complex, and a test compound or sample which contains a
putative CDK10 modulator. The test compounds or samples may be
tested directly on, for example, purified CDK10 protein whether native or
recombinant, subcellular fractions of CDK10-producing cells whether
native or recombinant, andJor whole cells expressing the CDK10
whether native or recombinant. The test compound or sample may be
added to the CDK10 in the ~aresence or absence of a known CDK10
modulator. The mo~3ulatin~; activity of the test compound or sample may
be determined by, for example, analyzing the ability of the test compound
or sample to bind to CDK10 protein, activate the protein, inhibit CDK10
activity, inhibit or enhance the binding of other compounds to the CDK10
protein, modifying receptor regulation, or modifying an intracellular
activity.
The identification of modulators of CDK10 activity are useful
in treating disease ~;tates involving the cell cycle will be useful in
controlling cell grovvth associated with cancer or immune cell
proliferation. Other compounds may be useful for stimulating or
inhibiting activity of the enzyme. These compounds could be of use in
the treatment of diseases in which activation or inactivation of the
CDK10 protein resmlts in either cellular proliferation, cell death,
nonproliferation, induction of cellular neoplastic transformations or
metastatic tumor gt~owth and hence could be used in the prevention
and/or treatment of various cancers.
The present invention is also directed to methods for
screening for compounds which modulate the expression of DNA or
RNA encoding a CDK protE~in of the present invention or which
modulates the function of a such a CDK protein. Compounds which
modulate these activities may be DNA, RNA, peptides, proteins, or
non-proteinaceous organic molecules. Compounds may modulate by
~3
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increasing or attenuating the expression of DNA or RNA encoding
the CDK protein, or the function of a CDK protein. Compounds that
modulate the expression of DNA or RNA encoding the CDK protein or
the biological function thereof may be detected by a variety of assays.
The assay may be a simple "yes/no" assay to determine whether there
is a change in expression or function. The assay may be made
quantitative by comparing the expression or function of a test sample
with the levels of expression or function in a standard sample. Kits
containing modified CDK10, antibodies to CDK10, or modified CDK10
protein may be prepared by known methods for such uses.
The DNA molecules, RNA molecules, recombinant
protein and antibodies of the present invention may be used to screen
and measure levels of CDK10 DNA, RNA or protein. The
recombinant proteins, DNA molecules, RNA molecules and
antibodies lend themselves to the formulation of kits suitable for the
detection and typing of CDK10. Such a kit would comprise a
compartmentalized carrier suitable to hold in close confinement at
least one container. The carrier would further comprise reagents
such as recombinant CDK10 protein or anti-CDK10 antibodies
suitable for detecting CDK10. The carrier may also contain a means
for detection such as labeled antigen or enzyme substrates or the like.
Pharmaceutically useful compositions comprising
modulators of CDK10 may be formulated according to known
methods such as by the admixture of a pharmaceutically acceptable
carrier. Examples of such carriers and methods of formulation may
be found in Remington's Pharmaceutical Sciences. To form a
pharmaceutically acceptable composition suitable for effective
administration, such compositions will contain an effective amount
of the protein, DNA, RNA, or modified CDK10.
Therapeutic or diagnostic compositions of the invention
are administered to an individual in amounts sufficient to treat or
diagnose disorders. The effective amount may vary according to a
variety of factors such as the individual's condition, weight, sex and
age. Other factors include the mode of administration.
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The ph;armace~utical compositions may be provided to the
individual by a variety of routes such as subcutaneous, topical, oral
and intramuscular"
The term "chemical derivative" describes a molecule that
contains additional chemical moieties which are not normally a part
of the base molecule. Such moieties may improve the solubility, half
life, absorption, etc. of the base molecule. Alternatively the moieties
may attenuate undesirable side effects of the base molecule or
decrease the toxicit3~ of the base molecule. Examples of such moieties
are described in a variety o~f texts, such as Remington's
Pharmaceutical Sciences.
Compounds identified according to the methods disclosed
herein may be used alone a.t appropriate dosages. Alternatively, co-
administration or sf~quenti.~l administration of other agents may be
desirable.
The present invention also has the objective of providing
suitable topical, oral, systemic and parenteral pharmaceutical
formulations for use in the novel methods of treatment of the present
invention. The compositions containing compounds identified
according to this invention as the active ingredient can be administered
in a wide variety of therapeutic dosage forms in conventional vehicles for
administration. Fo:r example, the compounds can be administered in
such oral dosage forms as tablets, capsules (each including timed
release and sustained release formulations), pills, powders, granules,
elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by
injection. Likewise, they may also be administered in intravenous (both
bolus and infusion), intraperitoneal, subcutaneous, topical with or
without occlusion, or intramuscular form, all using forms well known
to those of ordinary skill in the pharmaceutical arts.
Advani;ageousay, compounds of the present invention
may be administered in a single daily dose, or the total daily dosage
may be administered in divided doses of two, three or four times
daily. Furthermore:, compounds for the present invention can be
administered in int:ranasal form via topical use of suitable intranasal
vehicles, or via transdermal routes, using those forms of
transdermal skin p~~tches well known to those of ordinary skill in
that art. To be adnunistered in the form of a transdermal delivery
CA 02280206 1999-08-04
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system, the dosage administration will, of course, be continuous
rather than intermittent throughout the dosage regimen.
For combination treatment with more than one active
agent, where the active agents are in separate dosage formulations,
the active agents can be administered concurrently, or they each can
be administered at separately staggered times.
The dosage regimen utilizing the compounds of the present
invention is selected in accordance with a variety of factors including
type, species, age, weight, sex and medical condition of the patient; the
severity of the condition to be treated; the route of administration; the
renal and hepatic function of the patient; and the particular compound
thereof employed. A physician or veterinarian of ordinary skill can
readily determine and prescribe the effective amount of the drug
required to prevent, counter or arrest the progress of the condition.
Optimal precision in achieving concentrations of drug within the range
that yields efficacy without toxicity requires a regimen based on the
kinetics of the drug's availability to target sites. This involves a
consideration of the distribution, equilibrium, and elimination of a drug
Isolated and purified CDK10 is also be useful for the
recombinant production of large quantities of CDK10 protein. The ability
to produce large quantities of the protein would be useful for the
production of a therapeutic agent comprising the CDK10 protein. A
therapeutic agent comprised of CDK10 protein would be useful in the
treatment of cell cycle and/or CDK10 related diseases or conditions
which are CDK10 responsive.
By computer analysis of a genomic database, molecular
cloning and DNA sequencing a novel member of the human CDK
gene family has been identified. This new cDNA fragment encodes a
novel cyclin-dependent kinase which comprises a novel cyclin
binding domain signature sequence, lacks Thrl4 and/or Tyrl5 within
the conserved ATP binding motif of known CDKs, and also lacks the
T-loop domain containing the conserved Thr160/161 residue.
Northern hybridization experiments with RNA from
various cell and tissues indicates that CDK10 is expressed in various
human tissue, including brain, testis, pituitary gland and adrenal
gland derived cells or tissues.
~b
CA 02280206 1999-08-04
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The following examples are provided as illustrative of the
present invention without, however, limiting the same thereto.
EXAMPLE 1
Isolation and Characterization of DNA Fragments Encoding CDK
A 3' portion of the CDK10 coding region was detected among
the Merck-Washington University EST's as 5' EST H17727. EST "H17727"
resembled several (~DK anal MAPK genes. The pH17727 plasmid
construct comprising the 3' coding region and 3' untranslated region of
CDK10 is contained within a NotI-HindIII fragment of approximately
1.8 Kb, in a typical phagetnid vector. The 5' portion of this fragment
overlaps the 3' end of the 600 bp. This cDNA clone is publicly available by
Genbank Accession. No. H:L7727, Image Clone ID No. 50484, Washington
IS University Clone II) No. yrn40a06, and GBD Clone ID No. 423294. This
cDNA was isolated. from a. library constructed from human infant brain
mRNA. This consi;ruct is available from Research Genetics, Inc., 2130
Memorial Parkway SW, Hunstville, AL 35801 (http://www. resgen.com).
The 5' portion of the gene was isolated by performing
5' RACE (Frohman, et al., 1988, Proc. Natl. Acad. Sci.85: 8998-9002)
using MarathonT"'-ready human placenta cDNA available from
Clontech (Protocol #PT1156-1, Catalog #K1802-1). Adapter-ligated double
stranded cDNA generated from human placenta mRNA was used as a
template for PCR amplification using a gene specific primer PK22L234
(5'-TGATGCAGCCCACAGACCTG-3'; SEQ ID NO: 4) and an adapter
primer AP1 (5'-C(~ATCC'rAATACGACTCACTATAGGGC-3' SEA ID
N0:5). PCR ampli~ficatior~ was performed using the ElongaseTM long-
PCR enzyme mix (stored i:n 20mM Tris-HCl (pH 8.0 at 25°C), O.lmM
EDTA, 1mM DTT, stabili~:ers and 50%(v/v) glycerol) and PCR reaction
buffer obtained from Gibco-BRL. The buffer comprised 300mM Tris-S04
(pH 9.1 at 25°C), 90mM (TfH4)2504 and 1.5 mM MgS04. Two microliters
of Marathon placenta cDNA template and 10 pmoles each of PK22L234
and AP1 were added to the reaction mix and brought to a total volume of
20m1 with sterile iwater. 'thermal cycling was ( 1) 94°C/30sec,
68°C/6min
for 5 cycles; {2) 94°'C/30sec, 64°C/30sec, 68°C/4min for
5 cycles; and, (3)
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94°C/30sec, 62°C/30sec and 68°C/4min for 30 cycles. One
microliter from
a 1/20 dilution of this first PCR reaction was added to a second PCR
reaction as DNA template. This PCR reaction also differed from the first
PCR reaction in that nested primers PK22L161
(5'-GCCGTCTGGGGA.AAAGA-3'; SEQ ID N0:6) and AP2
(5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID N0:7) were used.
An approximately 600 by PCR product was identified from a 1% agarose
electrophoresis gel, excised, and purified using a Qiagen PCR-spun
column. This fragment was used directly for DNA sequencing using
PK22L161 and AP2 oligonucleotide primers.
The MarathonT""-ready human placenta cDNA available
from Clontech is enhanced by ligation of a double-stranded, 5' overhang
adapter to the double stranded cDNA template. The 3' end of the adapter
is blocked by an amine group to prevent extension during PCR
amplification. It is within the non-extended 3' region that the AP1 oligo
will hybridize. Therefore, AP1 does not hybridize and extend any of the
original cDNA template molecules, instead beginning extension and
amplification in the second round of PCR.
EXAMPLE 2
Construction of a Full Length DNA Fragment Encoding CDK10
The 3' portion of a DNA fragment which encodes CDK10 is
contained within a DNA plasmid vector, pH17727. This insert contains a
5' XhoI site unique to the insert and a NcoI site in the 3' unstranslated
region unique to the insert. This XhoI-NcoI fragment was isolated and
subcloned into XhoI-NcoI digested pLITMUS28 plasmid DNA (New
England Biolabs), resulting in pLITMUS28:H17727.
The 600 by PCR fragments obtained from 5' RACE were
cloned into pCR2.1 (Invitrogen) using the Invitrogen TA-cloning kit as
described by the manufacturer. A PmlI restriction site is located at
approximately the midpoint of the 600 by PCR product. The PmII site
was used to construct a wild type form of the 600 by 5' fragment from 2
independent 5' RACE PCR clones, pPK22bo4 and pPK22do4. The
PmlI-BamHI restriction fragment of pPK22bo4 (which contains a
ag
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mutation 3' to the PmlI site) was replaced with the with the
PmlI-BamHI fragment of clone pPK22do4 (which contains a mutation 5'
to the PmlI site ). The resulting clone, pPK22bo4/do4, overlaps the 5'
portion of pH1772? through the unique XhoI restriction site. An SpeI-
BamHI-NdeI restricltion site cluster was appended just 5' to the ATG
translational start codon by PCR-amplifying the insert from clone
pPK22bo4/do4 using primers PK22L661
(5'-GCCGTCTGGGGAA.AACxA-3'; SEQ ID NO: fi) and PK22U210
(5'-GGACTAGTGG~~TCCA'rATGGACCAGTACTGCATCCT-3'; SEQ ID
NO:10). The resulting PCR fragment was digested with Spe1 and XhoI
and ligated into BamHI-XhoI digested pLITMUS28:H17727, resulting in
pLITMUS28:CDK10 (Figure 3).
EXAMPLE 3
Construction of CDF~10 Mammalian Expression Vector
A Baml I-Xbal: fragment from pLITMUS28:CDK10
comprising the CKD10 coding region was subcloned into the
mammalian expression vector, pcDNA3.1 (Invitrogen), which was
previously digested with BamHI and XbaI. The resulting construct,
pcDNA3.1:CDK10, contains a portion of the CMV promoter and a T7
primer site upstream of the CDK10 ATG translational start codon as
well as the BGH polyA region downstream of the translational
termination codon. Of course, other components to allow growth in
E. coli and mammalian cells are present in this vector.
EXAMPLE 4
Construction of CDK10 Ba<:ulovirus Transfer Vector
A BamIHI-Ncol fragment from pLITMUS28:CDK10
containing the CKD10 coding region was cloned into the baculovirus
expression vector, pBlueBacHis2 (Invitrogen), which was previously
digested with BamP~I and NcoI. The resulting construct, pBBH:CDK10,
may be used to express recombinant CDK10 from insect cells by
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following the manufacturer's instructions (e.g., see Invitrogen Cat. No.
V375-20 for pBlueBacHis2 A, B, and C).
EXAMPLE 5
Construction of DNA Fragment Encoding a CDK10 Dominant-Negative
Mutant
The pLITMUS:CDK10 construct (see Example 2) was
mutated to generate a "dominant-negative" single base pair mutation.
This mutation was generated from pLITMUS28:CDK10 using the
Stratagene "Quik Change" kit and primers 22U-D127N:
(5'-CAACATTGTACATCGGAACCTGAAACCTGCC-3'; SEQ ID N0:8),
and 22L-D127N: (5'-GGCAGGTTTCAGGTTCCGATGTACAATGTTG-3';
SE(a ID NO: 9), according to the manufacturer's instructions. The
dominant-negative mutation changes the codon GAC (at nucleotides 588-
590 of SEQ ID N0:2) to AAC (at nucleotides 588-590 of SE(a ID NO:11),
thus deletion essential amino acid Asp127 to Asn127 (see SEQ ID N0:12),
which inactivates kinase activity (see Example 7 and van den Heuvel &
Harlow, 1993, Science 262:2050-2054). A CDK10-D 127N construction was
subcloned into pcDNA3.1 (as a BamHI-Xbal fragment), resulting in
pcDNA3.1:CDK10-d127N. A CDK10-D127N construction was also
subcloned into pBlueBacHis2 (as a BamHI-Nco1 fragment), resulting in
pBBH:CDK10-d127N.
EXAMPLE 6
Tissue Distribution Of CDK10 Expression
Human multiple tissue Northern Blot #7760-1, Human
Brain Northern Blot II #7755-1, Human Brain Northern Blot III #7750-1,
and "Human multiple tissue Northern Dot Blot were purchased from
Clontech. The probe was made by PCR amplifying the NotI-HindIII
insert from pH17727 using the "Universal"
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{5'-CCCAGTCACGA,CGTT(~TAAA.ACG-3 ; SEQ ID N0:13) and
"Reverse" (5'-AGCG~GATAACA,ATTTCACACAGG-3': SEQ ID N0:14)
primers from Gibco :BRL. Twenty-five ng of the probe was labeled with
32p using a Pharmacia "Ready-to-go" random priming kit and
hybridized to the four Northern blots at high stringency according to
Clontech instructions.
Figure 4 and Figure 5 show Northern data indicating the
presence of CDK10 i~ranscri.pts in a variety of adult human tissue (Figure
4) as well as in specific regions of the adult and fetal human brain
(Figure 5). This data shows increased expression levels in the testis as
well as in pituitary and adrenal glands. Expression in various regions
of the brain was relatively constant, with increased expression seen in
the frontal and temporal lolbes and the cerebral cortex.
EXAMPLE 7
Effect of CDK:D127hT on Cell Growth
Human osteosarcoma cell line Saos2 (ATCC HBT-85) was
grown in DMEM hi;~h glucose medium + glutamine +10% fetal calf
serum (in concentrations as recommended by Gibco-BRL). Two
replicates of the exl>eriment were performed sequentially. Cells were
split 1:6 into 10 cm culture dishes two days prior to transfection.
Transfection was pE~rformed using the CaP04 method according to Chen
and Okayama ( 1987, Mol. ocnd Cell. Biol. 7:~ 2745-2752). Ten ug of each
plasmid DNA {pcDI~TA3.1, pcDNA3:CDK10, pcDNA3:CDK10-D127N) was
transfected into ~6a~% confl.uent cells in each 10 cm dish. Cells were
rinsed 2x with Dulb~ecco's PBS (Gibco-BRL) and 10 mL fresh medium
was added. After two days, cells were trypsinized and plated in 12 well
dishes in fresh medium + 500ug/mL geneticin (Gibco-BRL). At 11 and 16
days after plating, colony counts were made to determine how many
transfected cells were capable of growth and colony formation (Table 1).
This data indicates that expression of the kinase inactive "dominant-
negative" form of C17K10 (i.e., CDK10-D127N) impairs colony formation
by analogy to the data presented in van den Heuvel and Harlow (1993,
Science 262: 2050-2054).
31
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TABLE 1
Day 11 (Colonies) Day 16 (# Colonies)
DNA construct Ren A Reu B Ren A Ren B
no DNA 2 1 0 0
cDNA3.1 23 42 16 40
cDNA3.1:CDK10 3 2 5 9 23 49
cDNA3.1:CDK-D127N 5 I 2 I
EXAMPLE 8
Specific Effect of the Dominant Mutant CDK:D127N on Expression of Cell
Cycle Genes
HeLa cervical carcinoma cells were treated for 48 hours
with a control adenovirus deleted for the E1 and E3 genes or the same
adenovirus which comprised the construct encoding CDK10-D127N.
Western blots were performed with a rabbit antibody raised to the
C-terminal 25 amino acids of the CDK10 protein (amino acid 301 - amino
acid 325 of SEQ ID NO: 3). The cell line transfected with Ad/CDK10-
D127N expressed CDK10-DI27N at a 50-fold higher level than
endogenous, wild type CDK10. The two infected cell populations were
subjected to mRNA isolations and probes were prepared for gene
expression DNA chip studies essentially as described by Lockhart, et al.
(1996, Nature Biotechnology 14:1675-1680). Among the genes which were
suppressed at the mRNA level by CDK10-D127N are summarized in
Table 2.
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TABLE 2
GENE Ad/CDK10- Ad- Control
D127N
CDC2~ib 8.3 19.1
CDK7 3.1 8.5
CKSl 2Ei.3 82.5
CKS2 17L.6 98.3
Cyclin lfi.5 41.5
B
Cyclin 4.5 11.5
D 1
poly- 186.5 664.5
Ubiquitin
I Quantified arbitrary expression units measured from the fluorescence
image of the oli~;onucleotide array.
These data indicate a cell cycle block by the dominant-negative mutant
gene, CDK10-D 1271\f, which shows the importance of the CDK10 protein to
the cell cycle. Cell cycle analysis (using a fluorescence-activated cell
sorter, or FAGS) of cells treated for 48 hours with the two viruses indicate
that cells are not blocked in any particular phase of the cell cycle.
The dai;a reported in the above Example sections show the
importance of CDK10 in the cell cycle. Therefore, a therapeutic agent
comprising the CDK:10 protein would be useful in the treatment of cell
cycle and/or CDK10 related diseases or conditions which are CDK10
responsive as well as showing a potential use for a dominant-negative
mutant such as CD1~10-D1:?7N, whach may be useful in the treatment of
cell cycle diseases oo conditions which are responsive to the mtuant
proteins ability to rc;gulate a phase or phases of the cell cycle.
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SEQUENCE LISTIDIG
(1) GENERAL INFORMATION:
(i) APPLICANT: Gerhold, David L.
(ii) TITLE OF INVENTION: CYCLIN-DEPENDENT PROTEIN KINASE
(iii) NUMBER OF SEQUENCES: 14
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co., Inc.
(B) STREET: P.O. Box 2000, RY60-30
(C) CITY: Rahway
(D) STATE: NJ
(E) COUNTRY: US
(F) ZIP: 07065-0907
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTV~ARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hand, J. Mark
(B) REGISTRATION NUMBER: 36,545
(C) REFERENCE/DOCKET NUMBER: 19885Y
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 732/594-3905
(B) TELEFAX: 732/594-4720
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Pro Asn Gln Ala Leu Arg Glu
1 5
3H
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(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2074 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:2:
GAAAAGGCGCAGTGGGGC~CGGAGCTGTCACCCCTGACTCGACGCAGCTTCCGTTCTCCT 60
GGTGACGTCGCCTACAGGAACCGCCCCAGTGGTCAGCTGCCGCGCTGTTGCTAGGCAACA 120
GCGTGCGAGCTCAGATCAGCGTGGGGTGGAGGAGAAGTGGAGTTTGGAAGTTCAGGGGCA 180
CAGGGGCACAGGCCCACGACTGCAGCGGGATGGACCAGTACTGCATCCTGGGCCGCATCG 240
GGGAGGGCGCCCACGGCATCGTCTTCAAGGCCAAGCACGTGGAGACTGGCGAGATAGTTG 300
CCCTCAAGAAGG'rGGCCCTAAGGCGGTTGGAAGACGGCTTCCCTAACCAGGCCCTGCGGG 360
AGATTAAGGCTC'rGCAGGAGATGGAGGACAATCAGTATGTGGTACAACTGAAGGCTGTGT 420
TCCCACACGGTGGAGGCTTTGTGCTGGCCTTTGAGTTCATGCTGTCGGATCTGGCCGAGG 480
TGGTGCGCCATGCCCAGAGGCCACTAGCCCAGGCACAGGTCAAGAGCTACCTGCAGATGC 540
TGCTCAAGGGTGTCGCCTTCTGCCATGCCAACAACATTGTACATCGGGACCTGAAACCTG 600
CCAACCTGCTCATCAGCGCCTCAGGCCAGCTCAAGATAGCGGACTTTGGCCTGGCTCGAG 660
TCTTTTCCCCAGACGGCAGCCGCCTCTACACACACCAGGTGGCCACCAGGTCTGTGGGCT 720
GCATCATGGGGGAGCTGTTGAATGGGTCCCCCCTTTTCCCGGGCAAGAACGATATTGAAC 780
AGCTTTGCTATGTGCTTCGCATCTTGGGCACCCCAAACCCTCAAGTCTGGCCGGAGCTCA 84~)
CTGAGCTGCCGGACTACAACAAGF,TCTCCTTTAAGGAGCAGGTGCCCATGCCCCTGGAGG 900
AGGTGCTGCCTGACGTCTCTCCCC'.AGGCATTGGATCTGCTGGGTCAATTCCTTCTCTACC 960
CTCCTCACCAGCGCATCGCAGCTTCCAAGGCTCTCCTCCATCAGTACTTCTTCACAGCTC 1020
CCCTGCCTGCCCATCCATCTGAGCTGCCGATTCCTCAGCGTCTAGGGGGACCTGCCCCCA 1080
AGGCCCATCCAGGGCCCC'CCCACATCCATGACTTCCACGTGGACCGGCCTCTTGAGGAGT 1140
CGCTGTTGAACCCAGAGC'TGATTC:GGCCCTTCATCCTGGAGGGGTGAGAAGTTGGCCCTG 1200
GTCCCGTCTGCCTGCTCC'TCAGGACCACTCAGTCCACCTGTTCCTCTGCCACCTGCCTGG 1260
CTTCACCCTCCAAGGCCTCCCCATGGCCACAGTGGGCCCACACCACACCCTGCCCCTTAG 1320
CCCTTGCGAGGGTTGGTC'TCGAGC>CAGAGGTCATGTTCCCAGCCAAGAGTATGAGAACAT 1380
3~ .
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CCAGTCGAGCAGAGGAGATTCATGGCCTGTGCTCGGTGAGCCTTACCTTC TGTGTGCTAC1440
TGACGTACCCATCAGGACAGTGAGCTCTGCTGCCAGTCAAGGCCTGCATA TGCAGAATGA1500
CGATGCCTGCCTTGGTGCTGCTTCCCCGAGTGCTGCCTCCTGGTCAAGGA GAAGTGCAGA1560
GAGTAAGGTGTCCTTATGTTGGAAACTCAAGTGGAAGGAAGATTTGGTTT GGTTTTATTC1620
TCAGAGCCATTAAACACTAGTTCAGTATGTGAGATATAGATTCTAAAAAC CTCAGGTGGC1680
TCTGCCTTATGTCTGTTCCTCCTTCATTTCTCTCAAGGGAAATGGCTAAG GTGGCATTGT1740
CTCATGGCTCTCGTTTTTGGGGTCATGGGGAGGGTAGCACCAGGCATAGC CACTTTTGCC1800
CTGAGGGACTCCTGTGTGCTTCACATCACTGAGCACTCATTTAGAAGTGA GGGAGACAGA1860
AGTCTAGGCCCAGGGATGGCTCCAGTTGGGGATCCAGCAGGAGACCCTCT GCACATGAGG1920
CTGGTTTACCAACATCTACTCCCTCAGGATGAGCGTGAGCCAGAAGCAGC TGTGTATTTA1980
AGGAAACAAGCGTTCCTGGAATTAATTTATAAATTTAATAAATCCCAATA TAATCCCAAA2040
AAAAAAAAAAAAAAAATTCCTGCGGCCGCAAGGA 2074
(2) INFORMATION
FOR SEQ
ID N0:3:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:325 amino
acids
(B) TYPE:
amino
acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:
linear
(ii) MOLECULE
TYPE:
protein
(xi)SEQUENCE DESCRIPTION: N0:3:
SEQ
ID
MetAsp GlnTyr CysIleLeu GlyArgIle GlyGluGly AlaHisGly
1 5 ~ 10 15
IleVal PheLys AlaLysHis ValGluThr GlyGluIle ValAlaLeu
20 25 30
LysLys ValAla LeuArgArg LeuGluAsp GlyPhePro AsnGlnAla
35 40 45
LeuArg GluIle LysAlaLeu GlnGluMet GluAspAsn GlnTyrVal
50 55 60
ValGln LeuLys AlaValPhe ProHisGly GlyGlyPhe ValLeuAla
65 70 75 80
PheGlu PheMet LeuSerAsp LeuAlaGlu ValValArg HisAlaGln
85 90 95
ArgPro LeuAla GlnAlaGln ValLysSer TyrLeuGln MetLeuLeu
100 105 110
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Lys Gly Val Al.a Phe Cys His Ala Asn Asn Ile Val His Arg Asp Leu
115 120 125
Lys Pro Ala As;n Leu Leu Ile Ser Ala Ser Gly Gln Leu Lys Ile Ala
130 135 140
Asp Phe Gly Leu Ala Arg Val Phe Ser Pro Asp Gly Ser Arg Leu Tyr
145 CL50 155 160
Thr His Gln Val Ala '7~hr Arg Ser Va1 Gly Cys Ile Met Gly Glu Leu
165 170 175
Leu Asn Gly Ser Pro Leu Phe Pro Gly Lys Asn Asp Ile Glu Gln Leu
1E~0 185 190
Cys Tyr Val Le~u Arg 7Cle Leu Gly Thr Pro Asn Pro Gln Val Trp Pro
195 200 205
Glu Leu Thr Gl.u Leu I?ro Asp Tyr Asn Lys Ile 5er Phe Lys Glu Gln
210 215 220
Val Pro Met Pro Leu Glu Glu Val Leu Pro Asp Val Ser Pro Gln Ala
225 230 235 240
Leu Asp Leu Leu Gly C3ln Phe Leu Leu Tyr Pro Pro His Gln Arg Ile
24~i 250 255
Ala Ala Ser L~~s Ala heu Leu His Gln 'hyr Phe Phe Thr Ala Pro Leu
2f>0 265 270
Pro Ala His Pro Ser Glu Leu Pro Ile Pro Gln Arg Leu Gly Gly Pro
275 280 285
Ala Pro Lys A:La His 3?ro Gly Pro Pro His Ile His Asp Phe His Val
290 295 300
Asp Arg Pro Le:u Glu Glu Ser Leu Leu Asn Pro Glu Leu Ile Arg Pro
305 :310 315 320
Phe Ile Leu G:Lu Gly
325
(2) INFORMATION FOR SEQ I17 N0:4:
( i ) SEQUEPdCE c~HARACT:ERISTICS
(A) LENG'PH: 20 abase pairs
(B) TYPE: nuc:Leic acid
( C ) STRA1JDEDNES;S : s ing 1 a
(D) TOPOhOGY: linear
(ii) MOLECULE 'TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
TGATGCAGCC CACAGACCTG 20
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: S:
CCATCCTAAT ACGACTCACT ATAGGGC 27
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
GCCGTCTGGG GAAAAGA 17
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
ACTCACTATA GGGCTCGAGC GGC 23
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(2) INFORMATION FOR SEQ I17 N0:8:
( i ) SEQUENCE C:HARACTIsRISTICS
(A) LENG".!'H: 31 base pairs
(B) TYPE.: nucleic acid
(C) STRALJDEDNESS: single
(D) TOPOhOGY: linear
(ii) MOLECULE 7:'YPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi ) SEQUENCE I)ESCRIP'PION: SEQ ID NO: 3
CAACATTGTA CATCGGA)~CC TGAi~ACCTGC C 31
( 2 ) INFORMATLON FOIL SEQ I17 NO : 9
(i) SEQUENCE CHARACTERISTICS:
(A) LENG'a'H: 31 base pairs
(B) TYPE'; nucleic acid
( C ) STRAIdDEDNESS : s ing 1 a
(D) TOPOhOGY: linear
(ii) MOLECULE ~('YPE: other nucleir_ acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE I)ESCRIP'rION: SEQ ID N0:9:
GGCAGGTTTC AC;GTTCCGAT GTACAATGTT G 31
(2) INFORMATION FOR SEQ I1J N0:10:
( i ) SEQUENCE I~HARACT:ERISTICS
(A) LENG'~H: 36 '.base pairs
(B) TYPE: nuc:Leic acid
( C ) STRA1JDEDNES;S : s ing 1 a
(D) TOPOhOGY: linear
(ii) MOLECULE 'TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQtJENCE :JESCRIP'TION: SEQ ID N0:10:
GGACTAGTGG ATCCATA'1'GG ACCAGTACTG CATCCT 36
3 °/
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(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2074 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:11:
GAAAAGGCGCAGTGGGGCCCGGAGCTGTCACCCCTGACTCGACGCAGCTTCCGTTCTCCT60
GGTGACGTCGCCTACAGGAACCGCCCCAGTGGTCAGCTGCCGCGCTGTTGCTAGGCAACA120
GCGTGCGAGCTCAGATCAGCGTGGGGTGGAGGAGAAGTGGAGTTTGGAAGTTCAGGGGCA180
CAGGGGCACAGGCCCACGACTGCAGCGGGATGGACCAGTACTGCATCCTGGGCCGCATCG240
GGGAGGGCGCCCACGGCATCGTCTTCAAGGCCAAGCACGTGGAGACTGGCGAGATAGTTG300
CCCTCAAGAAGGTGGCCCTAAGGCGGTTGGAAGACGGCTTCCCTAACCAGGCCCTGCGGG360
AGATTAAGGCTCTGCAGGAGATGGAGGACAATCAGTATGTGGTACAACTGAAGGCTGTGT420
TCCCACACGGTGGAGGCTTTGTGCTGGCCTTTGAGTTCATGCTGTCGGATCTGGCCGAGG480
TGGTGCGCCATGCCCAGAGGCCACTAGCCCAGGCACAGGTCAAGAGCTACCTGCAGATGC540
TGCTCAAGGGTGTCGCCTTCTGCCATGCCAACAACATTGTACATCGGAACCTGAAACCTG600
CCAACCTGCTCATCAGCGCCTCAGGCCAGCTCAAGATAGCGGACTTTGGCCTGGCTCGAG660
TCTTTTCCCCAGACGGCAGCCGCCTCTACACACACCAGGTGGCCACCAGGTCTGTGGGCT720
GCATCATGGGGGAGCTGTTGAATGGGTCCCCCCTTTTCCCGGGCAAGAACGATATTGAAC780
AGCTTTGCTATGTGCTTCGCATCTTGGGCACCCCAAACCCTCAAGTCTGGCCGGAGCTCA840
CTGAGCTGCCGGACTACAACAAGATCTCCTTTAAGGAGCAGGTGCCCATGCCCCTGGAGG900
AGGTGCTGCCTGACGTCTCTCCCCAGGCATTGGATCTGCTGGGTCAATTCCTTCTCTACC960
CTCCTCACCAGCGCATCGCAGCTTCCAAGGCTCTCCTCCATCAGTACTTCTTCACAGCTC1020
CCCTGCCTGCCCATCCATCTGAGCTGCCGATTCCTCAGCGTCTAGGGGGACCTGCCCCCA1080
AGGCCCATCCAGGGCCCCCCCACATCCATGACTTCCACGTGGACCGGCCTCTTGAGGAGT1140
CGCTGTTGAACCCAGAGCTGATTCGGCCCTTCATCCTGGAGGGGTGAGAAGTTGGCCCTG1200
GTCCCGTCTGCCTGCTCCTCAGGACCACTCAGTCCACCTGTTCCTCTGCCACCTGCCTGG1260
CTTCACCCTCCAAGGCCTCCCCATGGCCACAGTGGGCCCACACCACACCCTGCCCCTTAG1320
CCCTTGCGAGGGTTGGTCTCGAGGCAGAGGTCATGTTCCCAGCCAAGAGTATGAGAACAT1380
Nn
CA 02280206 1999-08-04
WO 98/35015 PCT/US98/02337
CCAGTCGAGCAGAGGAG.ATTCATGGCCTGTGCTCGGTGAG CCTTACCTTCTGTGTGCTAC 1440
TGACGTACCCATCAGGACAGTGAGCTCTGCTGCCAGTCAA GGCCTGCATATGCAGAATGA 1500
CGATGCCTGCCTTGGTGCTGCTTCCCCGAGTGCTGCCTCC TGGTCAAGGAGAAGTGCAGA 1560
GAGTAAGGTGTCCTTATGTTGGAAACTCAAGTGGAAGGAA GATTTGGTTTGGTTTTATTC 1620
TCAGAGCCATTAAACAC'rAGTTCAGTATGTGAGATATAGA TTCTAAAAACCTCAGGTGGC 1680
TCTGCCTTATGTCTGTTCCTCCTTCATTTCTCTCAAGGGA AATGGCTAAGGTGGCATTGT 1740
CTCATGGCTCTCGTTTT'rGGGGTCATGGGGAGGGTAGCAC CAGGCATAGCCACTTTTGCC 1800
CTGAGGGACTC(:TGTGTGCTTCACATCACTGAGCACTCAT TTAGAAGTGAGGGAGACAGA 1860
AGTCTAGGCCCAGGGATGGCTCCAGTTGGGGATCCAGCAG GAGACCCTCTGCACATGAGG 1920
CTGGTTTACCAACATCT.ACTCCCTCAGGATGAGCGTGAGC CAGAAGCAGCTGTGTATTTA 1980
AGGAAACAAGCGTTCCTGGAATTAATTTATAAATTTAATA AATCCCAATATAATCCCAAA 2040
AAAA.AAAAAAAAAAAAT'rCCTGCGGCCGCAAGGA 2074
(2) INFORMATION
FOR SEQ
ID N0:12:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:325 amino
acids
(B) TYPE:
amino
acid
(C) STRAP1DEDNESS:
single
(D) TOPOLOGY:
linear
(ii) MOLECULE
'TYPE:
protein
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Asp Gln Tyr Cys Ile Leu Gly Arg Ile Gly Glu Gly Ala His Gly
1 5 ~ 10 15
Ile Val Phe Lys Ala Lys His Val Glu Thr Gly Glu Ile Val Ala Leu
20 25 30
Lys Lys Val Ala Leu Arg Arg Leu Glu Asp Gly Phe Pro Asn Gln Ala
35 40 45
Leu Arg Glu Ile Lys Ala Leu Gln Glu Met Glu Asp Asn Gln Tyr Val
50 55 60
Val Gln Leu Lys Ala Val Phe Pro His Gly Gly Gly Phe Val Leu Ala
65 70 75 80
Phe Glu Phe Met Leu Ser Asp Leu Ala Glu Val Val Arg His Ala Gln
85 90 95
Arg Pro Leu Ala Gln Ala Gln Val Lys Ser Tyr Leu Gln Met Leu Leu
100 105 110
CA 02280206 1999-08-04
WO 98/35015 PCTIITS98/02337
Lys Gly Val Ala Phe Cys His Ala Asn Asn Ile Val His Arg Asn Leu
115 120 125
Lys Pro Ala Asn Leu Leu Ile Ser Ala Ser Gly Gln Leu Lys Ile Ala
130 235 140
Asp Phe Gly Leu Ala Arg Val Phe Ser Pro Asp Gly Ser Arg Leu Tyr
145 150 155 160
Thr His Gln Val Ala Thr Arg Ser Val Gly Cys Ile Met Gly Glu Leu
165 170 175
Leu Asn Gly Ser Pro Leu Phe Pro Gly Lys Asn Asp Ile Glu Gln Leu
180 185 190
Cys Tyr Val Leu Arg Ile Leu Gly Thr Pro Asn Pro Gln Val Trp Pro
195 200 205
Glu Leu Thr Glu Leu Pro Asp Tyr Asn Lys Ile Ser Phe Lys Glu Gln
210 215 220
Val Pro Met Pro Leu Glu Glu Val Leu Pro Asp Val Ser Pro Gln Ala
225 230 235 240
Leu Asp Leu Leu Gly Gln Phe Leu Leu Tyr Pro Pro His Gln Arg Ile
245 250 255
Ala Ala Ser Lys Ala Leu Leu His Gln Tyr Phe Phe Thr Ala Pro Leu
260 265 270
Pro Ala His Pro Ser Glu Leu Pro Ile Pro Gln Arg Leu Gly Gly Pro
275 280 285
Ala Pro Lys Ala His Pro Gly Pro Pro His Ile His Asp Phe His Val
290 295 300
Asp Arg Pro Leu Glu Glu Ser Leu Leu Asn Pro Glu Leu Ile Arg Pre
305 310 315 320
Phe Ile Leu Glu Gly
325
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LEIQGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
4~
CA 02280206 1999-08-04
WO 98/35015 PCT/US98/02337
(xi ) SEQUENCE DESCRIP~.L'ION: SEQ ID N0: 13
CCCAGTCACG ACGTTGT~sAA AC:G 23
(2) INFORMATION FOF; SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE; nucleic acid
(C) STRArdDEDNESS: single
(D) TOPOhOGY: linear
(ii) MOLECULE 7:'YPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE I)ESCRIP'.t'ION: SEQ ID N0:14:
AGCGGATAAC AATTTCAC:AC AC=G 23
u3
