Title: Human kinases and polynucleotides encoding the same
Abstract: Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.
Patent Number: 6,861,240 Issued on 03/01/2005 to Walke, et al.
| Inventors:
|
Walke; D. Wade (Spring, TX);
Scoville; John (Houston, TX);
Friddle; Carl Johan (The Woodlands, TX)
|
| Assignee:
|
Lexicon Genetics Incorporated (The Woodlands, TX)
|
| Appl. No.:
|
803278 |
| Filed:
|
March 18, 2004 |
| Current U.S. Class: |
435/194; 530/350; 435/320.1; 435/325; 435/252.3 |
| Intern'l Class: |
C12N 009//12; C12N 015//00; C12N 005//00; C12N 001//20; C07K 001//00 |
| Field of Search: |
435/194,252.3,320.1,325
530/350
|
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|
Primary Examiner: Monshipouri; Maryam
Parent Case Text
The present application is a divisional application of U.S. application
Ser. No. 10/196,927, filed May 20, 2002, now U.S. Pat. No. 6,797,510,
issued Sep. 28, 2004, which claims the benefit of U.S. Provisional
application No. 60/293,248, which was filed on May 24, 2001, both of which
are herein incorporated by reference in their entirety.
Claims
What is claimed is:
1. A substantially isolated protein having the kinase activity of the
protein shown in SEQ ID NO:4, which is encoded by a nucleotide sequence
that hybridizes to SEQ ID NO:3 under highly stringent conditions.
2. An isolated protein comprising the amino acid sequence of SEQ ID NO:4.
Description
1. INTRODUCTION
The present invention relates to the discovery, identification, and
characterization of novel human polynucleotides encoding proteins sharing
sequence similarity with animal kinases. The invention encompasses the
described polynucleotides, host cell expression systems, the encoded
proteins, fusion proteins, polypeptides and peptides, antibodies to the
encoded proteins and peptides, and genetically engineered animals that
either lack or overexpress the disclosed polynucleotides, antagonists and
agonists of the proteins, and other compounds that modulate the expression
or activity of the proteins encoded by the disclosed polynucleotides,
which can be used for diagnosis, drug screening, clinical trial
monitoring, the treatment of diseases and disorders, and cosmetic or
nutriceutical applications.
2. BACKGROUND OF THE INVENTION
Kinases mediate the phosphorylation of a wide variety of proteins and
compounds in the cell. Along with phosphatases, kinases are involved in a
range of regulatory pathways. Given the physiological importance of
kinases, they have been subject to intense scrutiny and are proven drug
targets.
3. SUMMARY OF THE INVENTION
The present invention relates to the discovery, identification, and
characterization of nucleotides that encode novel human proteins, and the
corresponding amino acid sequences of these proteins. The novel human
proteins (NHPs) described for the first time herein share structural
similarity with animal kinases, including, but not limited to,
serine-threonine kinases and G2 protein kinases. Accordingly, the
described NHPs encode novel kinases having homologues and orthologs across
a range of phyla and species.
The novel human polynucleotides described herein encode open reading frames
(ORFs) encoding proteins of 645 and 482 amino acids in length (see,
respectively, SEQ ID NOS:2 and 4).
The invention also encompasses agonists and antagonists of the described
NHPs, including small molecules, large molecules, mutant NHPs, or portions
thereof, that compete with native NHPs, peptides, and antibodies, as well
as nucleotide sequences that:can be used to inhibit the expression of the
described NHPs (e.g., antisense and ribozyme molecules, and open reading
frame or regulatory sequence replacement constructs) or to enhance the
expression of the described NHPs (e.g., expression constructs that place
the described polynucleotide under the control of a strong promoter
system), and transgenic animals that express a NHP sequence, or
"knock-outs" (which can be conditional) that do not express a functional
NHP. Knock-out mice can be produced in several ways, one of which involves
the use of mouse embryonic stem cell ("ES cell") lines that contain gene
trap mutations in a murine homolog of at least one of the described NHPs.
When the unique NHP sequences described in SEQ ID NOS:1-5 are
"knocked-out" they provide a method of identifying phenotypic expression
of the particular gene, as well as a method of assigning function to
previously unknown genes. In addition, animals in which the unique NHP
sequences described in SEQ ID NOS:1-5 are "knocked-out" provide a unique
source in which to elicit antibodies to homologous and orthologous
proteins, which would have been previously viewed by the immune system as
"self" and therefore would have failed to elicit significant antibody
responses.
Additionally, the unique NHP sequences described in SEQ ID NOS:1-5 are
useful for the identification of protein coding sequences, and mapping a
unique gene to a particular chromosome. These sequences identify
biologically verified exon splice junctions, as opposed to splice
junctions that may have been bioinformatically predicted from genomic
sequence alone. The sequences of the present invention are also useful as
additional DNA markers for restriction fragment length polymorphism (RFLP)
analysis, and in forensic biology, particularly given the presence of
nucleotide polymorphisms within the described sequences.
Further, the present invention also relates to processes for identifying
compounds that modulate, i.e., act as agonists or antagonists of, NHP
expression and/or NHP activity that utilize purified preparations of the
described NHPs and/or NHP products, or cells expressing the same. Such
compounds can be used as therapeutic agents for the treatment of any of a
wide variety of symptoms associated with biological disorders or
imbalances.
4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
The Sequence Listing provides the sequence of the novel human ORFs encoding
the described novel human kinase proteins. SEQ ID NO:5 describes a NHP ORF
and flanking sequences.
5. DETAILED DESCRIPTION OF THE INVENTION
The NHPs described for the first time herein are novel proteins that are
expressed in, inter alia, human cell lines and human fetal brain, brain,
pituitary, lung, kidney, lymph node, testis, thyroid, adrenal gland, fetal
kidney, fetal lung and osteosarcoma cells. The described sequences were
compiled from sequences available in GENBANK, and cDNAs generated from
human lymph node, brain, fetal brain, thyroid, and testis mRNAs (Edge
Biosystems, Gaithersburg, Md.) that were identified using primers
generated from human genomic DNA.
The present invention encompasses the nucleotides presented in the Sequence
Listing, host cells expressing such nucleotides, the expression products
of such nucleotides, and: (a) nucleotides that encode mammalian homologs
of the described nucleotides, including the specifically described NHPs,
and the NHP products; (b) nucleotides that encode one or more portions of
a NHP that correspond to functional domains, and the polypeptide products
specified by such nucleotide sequences, including, but not limited to, the
novel regions of any active domain(s); (c) isolated nucleotides that
encode mutant versions, engineered or naturally occurring, of the
described NHPs, in which all or a part of at least one domain is deleted
or altered, and the polypeptide products specified by such nucleotide
sequences, including, but not limited to, soluble proteins and peptides;
(d) nucleotides that encode chimeric fusion proteins containing all or a
portion of a coding region of a NHP, or one of its domains (e.g., a
receptor/ligand binding domain, accessory protein/self-association domain,
etc.) fused to another peptide or polypeptide; or (e) therapeutic or
diagnostic derivatives of the described polynucleotides, such as
oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or gene
therapy constructs, comprising a sequence first disclosed in the Sequence
Listing.
As discussed above, the present invention includes the human DNA-sequences
presented in the Sequence Listing (and vectors comprising the same), and
additionally contemplates any nucleotide sequence encoding a contiguous
NHP open reading frame (ORF) that hybridizes to a complement of a
DNA-sequence presented in the Sequence Listing under highly stringent
conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4,
7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing
in 0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., eds., 1989,
Current Protocols in Molecular Biology, Vol. I, Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., N.Y., at p. 2.10.3) and
encodes a functionally equivalent expression product. Additionally
contemplated are any nucleotide sequences that hybridize to the complement
of a DNA sequence that encodes and expresses an amino acid sequence
presented in the Sequence Listing under moderately stringent conditions,
e.g., washing in 0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel et al.,
1989, supra), yet still encode a functionally equivalent NHP product.
Functional equivalents of a NHP include naturally occurring NHPs present
in other species, and mutant NHPs, whether naturally occurring or
engineered (by site directed mutagenesis, gene shuffling, directed
evolution as described in, for example, U.S. Pat. Nos. 5,837,458 or
5,723,323 both of which are herein incorporated by reference). The
invention also includes degenerate nucleic acid variants of the disclosed
NHP polynucleotide sequences.
Additionally contemplated are polynucleotides encoding NHP ORFs, or their
functional equivalents, encoded by polynucleotide sequences that are about
99, 95, 90, or about 85 percent similar to corresponding regions of SEQ ID
NOS:1 or 3 (as measured by BLAST sequence comparison analysis using, for
example, the GCG sequence analysis package, as described herein, using
default parameters).
The invention also includes nucleic acid molecules, preferably DNA
molecules, that hybridize to, and are therefore the complements of, the
described NHP-encoding polynucleotides. Such hybridization conditions can
be highly stringent or less highly stringent, as described herein. In
instances where the nucleic acid molecules are deoxyoligonucleotides ("DNA
oligos"), such molecules are generally about 16 to about 100 bases long,
or about 20 to about 80 bases long, or about 34 to about 45 bases long, or
any variation or combination of sizes represented therein that incorporate
a contiguous region of sequence first disclosed in the Sequence Listing.
Such oligonucleotides can be used in conjunction with the polymerase chain
reaction (PCR) to screen libraries, isolate clones, and prepare cloning
and sequencing templates, etc.
Alternatively, such NHP oligonucleotides can be used as hybridization
probes for screening libraries, and assessing gene expression patterns
(particularly using a microarray or high-throughput "chip" format).
Additionally, a series of NHP oligonucleotide sequences, or the
complements thereof, can be used to represent all or a portion of the
described NHP sequences. An oligonucleotide or polynucleotide sequence
first disclosed in at least a portion of one or more of the sequences of
SEQ ID NOS:1-5 can be used as a hybridization, probe in conjunction with a
solid support matrix/substrate (resins, beads, membranes, plastics,
polymers, metal or metallized substrates, crystalline or polycrystalline
substrates, etc.). Of particular note are spatially addressable arrays
(i.e., gene chips, microtiter plates, etc.) of oligonucleotides and
polynucleotides, or corresponding oligopeptides and polypeptides, wherein
at least one of the biopolymers present on the spatially addressable array
comprises an oligonucleotide or polynucleotide sequence first disclosed in
at least one of the sequences of SEQ ID NOS:1-5, or an amino acid sequence
encoded thereby. Methods for attaching biopolymers to, or synthesizing
biopolymers on, solid support matrices, and conducting binding studies
thereon, are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637,
5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326,
5,424,186, and 4,689,405, the disclosures of which are herein incorporated
by reference in their entirety.
Addressable arrays comprising sequences first disclosed in SEQ ID NOS:1-5
can be used to identify and characterize the temporal and tissue specific
expression of a gene. These addressable arrays incorporate oligonucleotide
sequences of sufficient length to confer the required specificity, yet be
within the limitations of the production technology. The length of these
probes is usually within a range of between about 8 to about 2000
nucleotides. Preferably the probes consist of 60 nucleotides, and more
preferably 25 nucleotides, from the sequences first disclosed in SEQ ID
NOS:1-5.
For example, a series of NHP oligonucleotide sequences, or the complements
thereof, can be used in chip format to represent all or a portion of the
described sequences. The oligonucleotides, typically between about 16 to
about 40 (or any whole number within the stated range) nucleotides in
length, can partially overlap each other, and/or the sequence may be
represented using oligonucleotides that do not overlap. Accordingly, the
described polynucleotide sequences shall typically comprise at least about
two or three distinct oligonucleotide sequences of at least about 8
nucleotides in length that are each first disclosed in the described
Sequence Listing. Such oligonucleotide sequences can begin at any
nucleotide present within a sequence in the Sequence Listing, and proceed
in either a sense (5'-to-3') orientation vis-a-vis the described sequence
or in an antisense orientation.
Microarray-based analysis allows the discovery of broad patterns of genetic
activity, providing new understanding of gene-functions, and generating
novel and unexpected insight into transcriptional processes and biological
mechanisms. The use of addressable arrays comprising sequences first
disclosed in SEQ ID NOS:1-5 provides detailed information about
transcriptional changes involved in a specific pathway, potentially
leading to the identification of novel components, or gene functions that
manifest themselves as novel phenotypes.
Probes consisting of sequences first disclosed in SEQ ID NOS:1-5 can also
be used in the identification, selection, and validation of novel
molecular targets for drug discovery. The use of these, unique sequences
permits the direct confirmation of drug targets, and recognition of drug
dependent changes in gene expression that are modulated through pathways
distinct from the intended target of the drug. These unique sequences
therefore also have utility in defining and monitoring both drug action
and toxicity.
As an example of utility, the sequences first disclosed in SEQ ID NOS:1-5
can be utilized in microarrays, or other assay formats, to screen
collections of genetic material from patients who have a particular
medical condition. These investigations can also be carried out using the
sequences first disclosed in SEQ ID NOS:1-5 in silico, and by comparing
previously collected genetic databases and the disclosed sequences using
computer software known to those in the art.
Thus the sequences first disclosed in SEQ ID NOS:1-5 can be used to
identify mutations associated with a particular disease, and also in
diagnostic or prognostic assays.
Although the presently described sequences have been specifically described
using nucleotide sequence, it should be appreciated that each of the
sequences can uniquely be described using any of a wide variety of
additional structural attributes, or combinations thereof. For example, a
given sequence can be described by the net composition of the nucleotides
present within a given region of the sequence, in conjunction with the
presence of one or more specific oligonucleotide sequence(s) first
disclosed in SEQ ID NOS:1-5. Alternatively, a restriction map specifying
the relative positions of restriction endonuclease digestion sites, or
various palindromic or other specific oligonucleotide sequences, can be
used to structurally describe a given sequence. Such restriction maps,
which are typically generated by widely available computer programs (e.g.,
the University of Wisconsin GCG sequence analysis package, SEQUENCHER 3.0,
Gene Codes Corp., Ann Arbor, Mich., etc.), can optionally be used in
conjunction with one or more discrete nucleotide sequence(s) present in
the sequence that can be described by the relative position of the
sequence relative to one or more additional sequence(s) or one or more
restriction sites present in the disclosed sequence.
For oligonucleotide probes, highly stringent conditions may refer, e.g., to
washing in 6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C. (for
14-base oligos), 48.degree. C. (for 17-base oligos), 55.degree. C. (for
20-base oligos), and 60.degree. C. (for 23-base oligos). These nucleic
acid molecules may encode or act as NHP antisense molecules, useful, for
example, in NHP gene regulation and/or as antisense primers in
amplification reactions of NHP nucleic acid sequences. With respect to NHP
gene regulation, such techniques can be used to regulate biological
functions. Further, such sequences can be used as part of ribozyme and/or
triple helix sequences that are also useful for NHP gene regulation.
Inhibitory antisense or double stranded oligonucleotides can additionally
comprise at least one modified base moiety that is selected from the group
including, but not limited to, 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,
7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),
wybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,
and 2,6-diaminopurine.
The antisense oligonucleotide can also comprise at least one modified sugar
moiety selected from the group including, but not limited to, arabinose,
2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide will comprise at
least one modified phosphate backbone selected from the group including,
but not limited to, a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or analog
thereof.
In yet another embodiment, the antisense oligonucleotide is an
.alpha.-anomeric oligonucleotide. An .alpha.-anomeric oligonucleotide
forms specific double-stranded hybrids with complementary RNA in which,
contrary to the usual .beta.-units, the strands run parallel to each other
(Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide
is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS
Lett. 215:327-330). Alternatively, double stranded RNA can be used to
disrupt the expression and function of a targeted NHP.
Oligonucleotides of the invention can be synthesized by standard methods
known in the art, e.g., by use of an automated DNA synthesizer (such as
are commercially available from Biosearch, Applied Biosystems, etc.). As
examples, phosphorothioate oligonucleotides can be synthesized by the
method of Stein et al. (1988, Nucl. Acids Res. 16:3209), and
methylphosphonate oligonucleotides can be prepared by use of controlled
pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.
USA 85:7448-7451), etc.
Low stringency conditions are well-known to those of skill in the art, and
will vary predictably depending on the specific organisms from which the
library and the labeled sequences are derived. For guidance regarding such
conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (and
periodic updates thereof), and Ausubel et al., 1989, supra.
Alternatively, suitably labeled NHP nucleotide probes can be used to screen
a human genomic library using appropriately stringent conditions or by
PCR. The identification and characterization of human genomic clones is
helpful for identifying polymorphisms (including, but not limited to,
nucleotide repeats, microsatellite alleles, single nucleotide
polymorphisms, or coding single nucleotide polymorphisms), determining the
genomic structure of a given locus/allele, and designing diagnostic tests.
For example, sequences derived from regions adjacent to the intron/exon
boundaries of the human gene can be used to design primers for use in
amplification assays to detect mutations within the exons, introns, splice
sites (e.g., splice acceptor and/or donor sites), etc., that can be used
in diagnostics and pharmacogenomics.
For example, the present sequences can be used in restriction fragment
length polymorphism (RFLP) analysis to identify specific individuals. In
this technique, an individuals genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique bands
for identification (as generally described in U.S. Pat. No. 5,272,057,
incorporated herein by reference). In addition, the sequences of the
present invention can be used to provide polynucleotide reagents, e.g.,
PCR primers, targeted to specific loci in the human genome, which can
enhance the reliability of DNA-based forensic identifications by, for
example, providing another "identification marker" (i.e., another DNA
sequence that is unique to a particular individual). Actual base sequence
information can be used for identification as an accurate alternative to
patterns formed by restriction enzyme generated fragments.
Further, a NHP gene homolog can be isolated from nucleic acid from an
organism of interest by performing PCR using two degenerate or "wobble"
oligonucleotide primer pools designed on the basis of amino acid sequences
within the NHP products disclosed herein. The template for the reaction
may be total RNA, mRNA, genomic DNA and/or cDNA obtained by reverse
transcription of mRNA prepared from, for example, human or non-human cell
lines or tissue known to express, or suspected of expressing, an allele of
a NHP gene.
The PCR product can be subcloned and sequenced to ensure that the amplified
sequences represent the sequence of the desired NHP gene. The PCR fragment
can then be used to isolate a full length cDNA clone by a variety of
methods. For example, the amplified fragment can be labeled and used to
screen a cDNA library, such as a bacteriophage cDNA library.
Alternatively, the labeled fragment can be used to isolate genomic clones
via the screening of a genomic library.
PCR technology can also be used to isolate full length cDNA sequences. For
example, RNA can be isolated, following standard procedures, from an
appropriate cellular or tissue source (i.e., one known to express, or
suspected of expressing, a NHP gene). A reverse transcription (RT)
reaction can be performed on the RNA using an oligonucleotide primer
specific for the most 5' end of the amplified fragment for the priming of
first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed"
using a standard terminal transferase reaction, the hybrid may be digested
with RNase H, and second strand synthesis may then be primed with a
complementary primer. Thus, cDNA sequences upstream of the amplified
fragment can be isolated. For a review of cloning strategies that can be
used, see, e.g., Sambrook et al., 1989, supra.
A cDNA encoding a mutant NHP sequence can be isolated, for example, by
using PCR. In this case, the first cDNA strand may be synthesized by
hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known
to express, or suspected of expressing, a NHP, in an individual putatively
carrying a mutant NHP allele, and by extending the new strand with reverse
transcriptase. The second strand of the cDNA is then synthesized using an
oligonucleotide that hybridizes specifically to the 5' end of the normal
sequence. Using these two primers, the product is then amplified via PCR,
optionally cloned into a suitable vector, and subjected to DNA sequence
analysis through methods well-known to those of skill in the art. By
comparing the DNA sequence of the mutant NHP allele to that of a
corresponding normal NHP allele, the mutation(s) responsible for the loss
or alteration of function of the mutant NHP gene product can be
ascertained.
Alternatively, a genomic library can be constructed using DNA obtained from
an individual suspected of carrying, or known to carry, a mutant NHP
allele (e.g., a person manifesting a NHP-associated phenotype such as, for
example, behavioral disorders, immune disorders, obesity, high blood
pressure, etc.), or a cDNA library can be constructed using RNA from a
tissue known to express, or suspected of expressing, a mutant NHP allele.
A normal NHP gene, or any suitable fragment thereof, can then be labeled
and used as a probe to identify the corresponding mutant NHP allele in
such libraries. Clones containing mutant NHP sequences can then be
purified and subjected to sequence analysis according to methods
well-known to those skilled in the art.
Additionally, an expression library can be constructed utilizing cDNA
synthesized from, for example, RNA isolated from a tissue known to
express, or suspected of expressing, a mutant NHP allele in an individual
suspected of carrying, or known to carry, such a mutant allele. In this
manner, gene products made by the putatively mutant tissue may be
expressed and screened using standard antibody screening techniques in
conjunction with antibodies raised against a normal NHP product, as
described below (for screening techniques, see, for example, Harlow and
Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
Press, Cold Spring Harbor, N.Y.).
Additionally, screening can be accomplished by screening with labeled NHP
fusion proteins, such as, for example, alkaline phosphatase-NHP or
NHP-alkaline phosphatase fusion proteins. In cases where a NHP mutation
results in an expression product with altered function (e.g., as a result
of a missense or a frameshift mutation), polyclonal antibodies to a NHP
are likely to cross-react with a corresponding mutant NHP expression
product. Library clones detected via their reaction with such labeled
antibodies can be purified and subjected to sequence analysis according to
methods well-known in the art.
An additional application of the described novel human polynucleotide
sequences is their use in the molecular mutagenesis/evolution of proteins
that are at least partially encoded by the described novel sequences
using, for example, polynucleotide shuffling or related methodologies.
Such approaches are described in U.S. Pat. Nos. 5,830,721, 5,837,458,
6,117,679, and 5,723,323, which are herein incorporated by reference in
their entirety.
The invention also encompasses: (a) DNA vectors that contain any of the
foregoing NHP coding sequences and/or their complements (i.e., antisense);
(b) DNA expression vectors that contain any of the foregoing NHP coding
sequences operatively associated with a regulatory element that directs
the expression of the coding sequences (for example, baculovirus as
described in U.S. Pat. No. 5,869,336, herein incorporated by reference);
(c) genetically engineered host cells that contain any of the foregoing
NHP coding sequences operatively associated with a regulatory element that
directs the expression of the coding sequences in the host cell; and (d)
genetically engineered host cells that express an endogenous NHP sequence
under the control of an exogenously introduced regulatory element (i.e.,
gene activation). As used herein, regulatory elements include, but are not
limited to, inducible and non-inducible promoters, enhancers, operators,
and other elements known to those skilled in the art that drive and
regulate expression. Such regulatory elements include, but are not limited
to, the cytomegalovirus (hCMV) immediate early gene, regulatable, viral
elements (particularly retroviral LTR promoters), the early or late
promoters of SV40 or adenovirus, the lac system, the trp system, the TAC
system, the TRC system, the major operator and promoter regions of phage
lambda, the control regions of fd coat protein, the promoter for
3-phosphoglycerate kinase (PGK), the promoters of acid phosphatase, and
the promoters of the yeast .alpha.-mating factors.
Where, as in the present instance, some of the described NHP peptides or
polypeptides are thought to be cytoplasmic or nuclear proteins (although
processed forms or fragments can be secreted or membrane associated),
expression systems can be engineered that produce soluble derivatives of a
NHP (corresponding to a NHP extracellular and/or intracellular domains, or
truncated polypeptides lacking one or more hydrophobic domains) and/or NHP
fusion protein products (especially NHP-Ig fusion proteins, i.e., fusions
of a NHP domain to an IgFc), NHP antibodies, and anti-idiotypic antibodies
(including Fab fragments) that can be used in therapeutic applications.
Preferably, the above expression systems are engineered to allow the
desired peptide or polypeptide to be recovered from the culture media.
The present invention also encompasses antibodies and anti-idiotypic
antibodies (including Fab fragments), antagonists and agonists of a NHP,
as well as compounds or nucleotide constructs that inhibit expression of a
NHP sequence (transcription factor inhibitors, antisense and ribozyme
molecules, or open reading frame sequence or regulatory sequence
replacement constructs), or promote the expression of a NHP (e.g.,
expression constructs in which NHP coding sequences are operatively
associated with expression control elements such as promoters,
promoter/enhancers, etc.)
The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences,
antibodies, antagonists and agonists can be useful for the detection of
mutant NHPs, or inappropriately expressed NHPs, for the diagnosis of
disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucleotide
sequences, host cell expression systems, antibodies, antagonists, agonists
and genetically engineered cells and animals can be used for screening for
drugs (or high throughput screening of combinatorial libraries) effective
in the treatment of the symptomatic or phenotypic manifestations of
perturbing the normal function of a NHP in the body. The use of engineered
host cells and/or animals can offer an advantage in that such systems
allow not only for the identification of compounds that bind to the
endogenous receptor/ligand of a NHP, but can also identify compounds that
trigger NHP-mediated activities or pathways.
Finally, the NHP products can be used as therapeutics. For example, soluble
derivatives such as NHP peptides/domains corresponding to NHPs, NHP fusion
protein products (especially NHP-Ig fusion proteins, i.e., fusions of a
NHP, or a domain of a NHP, to an IgFc), NHP antibodies and anti-idiotypic
antibodies (including Fab fragments), antagonists or agonists (including
compounds that modulate or act on downstream targets in a NHP-mediated
pathway) can be used to directly treat diseases or disorders. For
instance, the administration of an effective amount of a soluble NHP, a
NHP-IgFc fusion protein, or an anti-idiotypic antibody (or its Fab) that
mimics the NHP, could activate or effectively antagonize the endogenous
NHP or a protein interactive therewith. Nucleotide constructs encoding
such NHP products can be used to genetically engineer host cells to
express such products in vivo; these genetically engineered cells function
as "bioreactors" in the body delivering a continuous supply of a NHP, a
NHP peptide, or a NHP fusion protein, to the body. Nucleotide constructs
encoding functional NHPs, mutant NHPs, as well as antisense and ribozyme
molecules, can also be used in "gene therapy" approaches for the
modulation of NHP expression. Thus, the invention also encompasses
pharmaceutical formulations and methods for treating biological disorders.
Various aspects of the invention are described in greater detail in the
subsections below.
5.1 The NHP Sequences
The cDNA sequences and corresponding deduced amino acid sequences of the
described NHPs are presented in the Sequence Listing.
Expression analysis has provided evidence that the described NHPs can be
expressed in a relatively narrow range of human tissues. In addition to
serine-threonine kinases, the described NHPs also share significant
similarity to a range of additional kinase families, including kinases
associated with signal transduction, from a variety of phyla and species.
An A/G polymorphism was identified at the region represented by nucleotide
position number 350 of, for example, SEQ ID NO:1 or SEQ ID NO:3, which can
result in an asp or gly being present at corresponding amino acid (aa)
position 117 of, for example, SEQ ID NO:2 or SEQ ID NO:4; and a T/A
polymorphism was identified at the region represented by nucleotide
position number 1463 of, for example, SEQ ID NO:1, which can result in a
val or glu being present at corresponding aa position 488 of, for example,
SEQ ID NO:2. The present invention contemplates sequences comprising any
and all combinations and permutations of the above polymorphisms. As these
polymorphisms are coding single nucleotide polymorphisms, they are
particularly useful in forensic analysis.
The gene encoding the described NHPs is apparently encoded on human
chromosome 3 (see GENBANK accession no. AC010210). Accordingly, the
described sequences are also useful for mapping and identifying the coding
regions of the human genome, and for defining exon splice junctions.
Given the physiological importance of protein kinases, they have been
subject to intense scrutiny, as exemplified and discussed in U.S. Pat.
Nos. 5,756,289 and 5,817,479, and 6,340,583, herein incorporated by
reference in their entirety, which additionally describe a variety of uses
and applications for the described NHPs.
NHP gene products can also be expressed in transgenic animals. Animals of
any species, including, but not limited to, worms, mice, rats, rabbits,
guinea pigs, pigs, micro-pigs, birds, goats, and non-human primates, e.g.,
baboons, monkeys, and chimpanzees, may be used to generate NHP transgenic
animals.
Any technique known in the art may be used to introduce a NHP transgene
into animals to produce the founder lines of transgenic animals. Such
techniques include, but are not limited to, pronuclear microinjection
(Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus-mediated
gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl.
Acad. Sci. USA 82:6148-6152); gene targeting in embryonic stem cells
(Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo,
1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer
(Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such
techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.
115:171-229, which is incorporated by reference herein in its entirety.
The present invention provides for transgenic animals that carry a NHP
transgene in all their cells, as well as animals that carry a transgene in
some, but not all their cells, i.e., mosaic animals or somatic cell
transgenic animals. A transgene may be integrated as a single transgene,
or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. A
transgene may also be selectively introduced into and activated in a
particular cell-type by following, for example, the teaching of Lasko et
al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatory
sequences required for such a cell-type specific activation will depend
upon the particular cell-type of interest, and will be apparent to those
of skill in the art.
When it is desired that a NHP transgene be integrated into the chromosomal
site of the endogenous NHP gene, gene targeting is preferred. Briefly,
when such a technique is to be utilized, vectors containing some
nucleotide sequences homologous to the endogenous NHP gene are designed
for the purpose of integrating, via homologous recombination with
chromosomal sequences, into and disrupting the function of the nucleotide
sequence of the endogenous NHP gene (i.e., "knockout" animals).
The transgene can also be selectively introduced into a particular
cell-type, thus inactivating the endogenous NHP gene in only that
cell-type, by following, for example, the teaching of Gu et al., 1994,
Science 265:103-106. The regulatory sequences required for such a
cell-type specific inactivation will depend upon the particular cell-type
of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the
recombinant NHP gene may be assayed utilizing standard techniques. Initial
screening may be accomplished by Southern blot analysis or PCR techniques
to analyze animal tissues to assay whether integration of the transgene
has taken place. The level of mRNA expression of the transgene in the
tissues of the transgenic animals may also be assessed using techniques
that include, but are not limited to, Northern blot analysis of tissue
samples obtained from the animal, in situ hybridization analysis, and
RT-PCR. Samples of NHP gene-expressing tissue may also be evaluated
immunocytochemically using antibodies specific for the NHP transgene
product.
The present invention also provides for "knock-in" animals. Knock-in
animals are those in which a polynucleotide sequence (i.e., a gene or a
cDNA) that the animal does not naturally have in its genome is inserted-in
such a way that it is expressed. Examples include, but are not limited to,
a human gene or cDNA used to replace its murine ortholog in the mouse, a
murine cDNA used to replace the murine gene in the mouse, and a human gene
or cDNA or murine cDNA that is tagged with a reporter construct used to
replace the murine ortholog or gene in the mouse. Such replacements can
occur at the locus of the murine ortholog or gene, or at another specific
site. Such knock-in animals are useful for the in vivo study, testing and
validation of, intra alia, human drug targets, as well as for compounds
that are directed at the same, and therapeutic proteins.
5.2 NHPS and NHP Polypeptides
NHPs, NHP polypeptides, NHP peptide fragments, mutated, truncated, or
deleted forms of the NHPS, and/or NHP fusion proteins can be prepared for
a variety of uses. These uses include, but are not limited to, the
generation of antibodies, as reagents in diagnostic assays, for the
identification of other cellular gene products related to a NHP, and as
reagents in assays for screening for compounds that can be used as
pharmaceutical reagents useful in the therapeutic treatment of mental,
biological, or medical disorders and diseases. Given the similarity
information and expression data, the described NHPs can be targeted (by
drugs, oligos, antibodies, etc.) in order to treat disease, or to
therapeutically augment the efficacy of therapeutic agents.
The Sequence Listing discloses the amino acid sequences encoded by the
described NHP-encoding polynucleotides. The NHPs display initiator
methionines that are present in DNA sequence contexts consistent with
eucaryotic translation initiation sites. The NHPs do not display
signal-like sequences, which indicates that they may not be membrane
associated, and are possibly cytoplasmic or nuclear proteins, although
they may also be secreted proteins.
The NHP amino acid sequences of the invention include the amino acid
sequences presented in the Sequence Listing, as well as analogues and
derivatives thereof. Further, corresponding NHP homologues from other
species are encompassed by the invention. In fact, any NHP protein encoded
by the NHP nucleotide sequences described herein are within the scope of
the invention, as are any novel polynucleotide sequences encoding all or
any novel portion of an amino acid sequence presented in the Sequence
Listing. The degenerate nature of the genetic code is well-known, and,
accordingly, each amino acid presented in the Sequence Listing is
generically representative of the well-known nucleic acid "triplet" codon,
or in many cases codons, that can encode the amino acid. As such, as
contemplated herein, the amino acid sequences presented in the Sequence
Listing, when taken together with the genetic code (see, for example,
Table 4-1 at page 109 of "Molecular Cell Biology", 1986, J. Darnell et
al., eds., Scientific American Books, New York, N.Y., herein incorporated
by reference), are generically representative of all the various
permutations and combinations of nucleic acid sequences that can encode
such amino acid sequences.
The invention also encompasses proteins that are functionally equivalent to
the NHPs encoded by the presently described nucleotide sequences, as
judged by any of a number of criteria, including, but not limited to, the
ability to bind and modify a NHP substrate, the ability to effect an
identical or complementary downstream pathway, or a change in cellular
metabolism (e.g., proteolytic activity, ion flux, phosphorylation, etc.).
Such functionally equivalent NHP proteins include, but are not limited to,
additions or substitutions of amino acid residues within the amino acid
sequence encoded by the NHP nucleotide sequences described herein, but
that result in a silent change, thus producing a functionally equivalent
expression product. Amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues involved.
For example, nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
polar neutral amino acids include glycine, serine, threonine, cysteine,
tyrosine, asparagine, and glutamine; positively charged (basic) amino
acids include arginine, lysine, and histidine; and negatively charged
(acidic) amino acids include aspartic acid and glutamic acid.
A variety of host-expression vector systems can be used to express the NHP
nucleotide sequences of the invention. Where the NHP peptide or
polypeptide can exist, or has been engineered to exist, as a soluble or
secreted molecule, the soluble NHP peptide or polypeptide can be recovered
from the culture media. Such expression systems also encompass engineered
host cells that express a NHP, or functional equivalent, in situ.
Purification or enrichment of a NHP from such expression systems can be
accomplished using appropriate detergents and lipid micelles and methods
well-known to those skilled in the art. However, such engineered host
cells themselves may be used in situations where it is important not only
to retain the structural and functional characteristics of a NHP, but to
assess biological activity, e.g., in certain drug screening assays.
The expression systems that may be used for purposes of the invention
include, but are not limited to, microorganisms such as bacteria (e.g., E.
coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing NHP nucleotide sequences;
yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast
expression vectors containing NHP nucleotide sequences; insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus) containing NHP nucleotide sequences; plant cell systems
infected with recombinant virus expression vectors (e.g., cauliflower
mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid) containing NHP
nucleotide sequences; or mammalian ce
