U.S. patent application number 10/948842 was filed with the patent office on 2005-04-28 for novel human kinases and polynucleotides encoding the same.
Invention is credited to Friddle, Carl Johan, Hilbun, Erin, Mathur, Brian, Turner, C. Alexander JR..
Application Number | 20050089907 10/948842 |
Document ID | / |
Family ID | 22954261 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089907 |
Kind Code |
A1 |
Friddle, Carl Johan ; et
al. |
April 28, 2005 |
Novel 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.
Inventors: |
Friddle, Carl Johan; (The
Woodlands, TX) ; Hilbun, Erin; (Houston, TX) ;
Mathur, Brian; (The Woodlands, TX) ; Turner, C.
Alexander JR.; (The Woodlands, TX) |
Correspondence
Address: |
Lance K. Ishimoto
LEXICON GENETICS INCORPORATED
8800 Technology Forest Place
The Woodlands
TX
77381
US
|
Family ID: |
22954261 |
Appl. No.: |
10/948842 |
Filed: |
September 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10948842 |
Sep 23, 2004 |
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10434034 |
May 8, 2003 |
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6815188 |
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10434034 |
May 8, 2003 |
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09992481 |
Nov 19, 2001 |
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6593125 |
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60252011 |
Nov 20, 2000 |
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Current U.S.
Class: |
435/6.14 ;
435/194; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 9/1205 20130101 |
Class at
Publication: |
435/006 ;
435/194; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/12 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. An antibody that specifically recognizes the amino acid sequence
of SEQ ID NO:2 or SEQ ID NO:4.
7. A method for producing a polypeptide comprising SEQ ID NO:2 or
SEQ ID NO:4, comprising culturing a host cell comprising a
recombinant expression vector comprising a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4
under conditions sufficient for the production of said polypeptide,
and recovering said polypeptide from the host cell culture.
Description
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/252,011, which was filed on Nov. 20,
2000 and is herein incorporated by reference in its entirety.
1. INTRODUCTION
[0002] 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 genes, antagonists and agonists of the
proteins, and other compounds that modulate the expression or
activity of the proteins encoded by the disclosed genes, 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
[0003] Kinases mediate the phosphorylation of a wide variety of
proteins and compounds in the cell. In conjunction 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
[0004] 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, receptor tyrosine kinases (SEQ ID
NOS:1-2 show particular similarity to NEK family kinases, and SEQ
ID NOS:3-5 are particularly similar to calcium and calmodulin
dependent kinases as well as sequences encoding PK 80), and
serine-threonine kinases. The described NHPs encode novel kinases
having homologues and orthologs across a range of phyla and
species.
[0005] The novel human polynucleotides described herein encode open
reading frames (ORFs) encoding proteins of 692 and 817 amino acids
in length (see respectively SEQ ID NOS:2 and 4).
[0006] 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 NHP,
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 cells
("ES cells") 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 that would have been previously viewed by the
immune system as "self" and therefore would have failed to elicit
significant antibody responses. To these ends, gene trapped
knockout ES cells have been generated in murine homologs of the
described NHPs.
[0007] Additionally, the unique NHP sequences described in SEQ ID
NOS:1-5 are useful for the identification of protein coding
sequence and mapping a unique gene to a particular chromosome (the
gene encoding SEQ ID NOS:1-2 is apparently encoded on human
chromosome 17, see GENBANK accession no. AC010761, and the gene
encoding SEQ ID. NOS:3-5 is apparently encoded on human chromosome
3, see GENBANK accession no. AC068979). 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.
[0008] 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
[0009] 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
[0010] The NHPs described for the first time herein are novel
proteins that are expressed in, inter alia, human cell lines and
pituitary, thymus, spleen, lymph node, bone marrow, trachea,
kidney, prostate, testis, thyroid, adrenal gland, pancreas,
salivary gland, stomach, small intestine, skeletal muscle, heart,
uterus, placenta, adipose, skin, bladder, rectum, pericardium,
ovary, fetal kidney, fetal lung, gall bladder, tongue, aorta, 6-,
9-, and 12-week embryos, adenocarcinoma, osteosarcoma, and
embryonic carcinoma cells (SEQ ID NOS:1-2). SEQ ID NOS:3-5 were
predominantly expressed in fetal brain, brain, spinal cord, thymus,
lymph node, trachea, lung, prostate, testis, thyroid, adrenal
gland, stomach, small intestine, skeletal muscle, uterus, placenta,
mammary gland, skin, bladder, pericardium, hypothalamus, fetal
kidney, fetal lung, tongue, aorta, 6-, 9-, and 12-week embryos, and
embryonic carcinoma cells.
[0011] The described sequences were compiled from sequences
available in GENBANK, and cDNAs generated from pituitary, lymph
node, mammary gland, brain, adrenal gland, fetus, and testis mRNAs
(Edge Biosystems, Gaithersburg, Md.).
[0012] 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 genes, including
the specifically described NHPs, and the NHP products; (b)
nucleotides that encode one or more portions of an 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 in which all or a portion of the
signal sequence is deleted; (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.
[0013] 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., New
York, 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, and/or directed evolution, as described in, for
example, U.S. Pat. No. 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.
[0014] 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 a NHP (as measured by BLAST sequence
comparison analysis using, for example, the GCG sequence analysis
package, as described herein, using default parameters).
[0015] 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.
[0016] 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 the
described 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.
[0017] 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 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.
[0018] For example, a series of the described 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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 and/or prognostic assays.
[0023] 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
the 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.
[0024] 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), or. 60.degree. C. (for
23-base oligos). These nucleic acid molecules may encode or act as
NHP gene antisense molecules, useful, for example, in NHP gene
regulation (and/or as antisense primers in amplification reactions
of NHP gene 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.
[0025] 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-carboxymethylaminomethyluraci- l, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0026] 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.
[0027] 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.
[0028] 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'-O-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.
[0029] 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 (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.
[0030] 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 (and
periodic updates thereof), Cold Spring Harbor Press, N.Y.; and
Ausubel et al., 1989, supra.
[0031] 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.
[0032] For example, the present sequences can be used in
restriction fragment length polymorphism (RFLP) analysis to
identify specific individuals. In this technique, an individual's
genomic DNA is digested with one or more restriction enzymes, and
probed on a Southern blot to yield unique bands for identification
(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.
[0033] 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,
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.
[0034] 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.
[0035] 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.
[0036] 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 mutant NHP allele, 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.
[0037] 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, 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 sequence, 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.
[0038] 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).
[0039] 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.
[0040] 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.
[0041] 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 and 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.
[0042] 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.
[0043] 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.).
[0044] 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.
[0045] 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 soluble NHP, or 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.
[0046] Various aspects of the invention are described in greater
detail in the subsections below.
5.1 The NHP Sequences
[0047] The cDNA sequences and corresponding deduced amino acid
sequences of the described NHPs are presented in the Sequence
Listing.
[0048] Expression analysis has provided evidence that the described
NHPs can be expressed in a range of human tissues, as described in
greater detail herein above. In addition to serine-threonine
kinases, the described NHPs also share significant similarity to
several additional kinase families, including kinases associated
with signal transduction, from a variety of phyla and species.
Several polymorphisms were identified in the described NHPs. These
include a T/C polymorphism in the sequence region represented by
nucleotide position 1170 of SEQ ID NO:1, both of which result in
the same amino acid being present at the corresponding amino acid
(aa) position of SEQ ID NO:2; a T/C polymorphism in the sequence
region represented by nucleotide position 1321 of SEQ ID NO:1, both
of which result in the same amino acid being present at the
corresponding aa position of SEQ ID NO:2; a C/G polymorphism in the
sequence region represented by nucleotide position 94 of SEQ ID
NO:3, which can result in either a leu or val being present at
corresponding aa position 32 of SEQ ID NO:4; an A/G polymorphism at
nucleotide position 112 of SEQ ID NO:3, which can result in either
a lys or glu being present at corresponding aa position 38 of SEQ
ID NO:4; and an A/T polymorphism at nucleotide position 133 of SEQ
ID NO:3, which can result in either a thr or ser being present at
corresponding aa position 45 of SEQ ID NO:4. The above
polymorphisms can be present either singly, or in any combination
or permutation within a given sequence.
[0049] 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 and 5,837,458, which are
herein incorporated by reference in their entirety.
[0050] 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.
[0051] 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.
[0052] The present invention provides for transgenic animals that
carry the NHP transgene in all their cells, as well as animals that
carry the transgene in some, but not all their cells, i.e., mosaic
animals or somatic cell transgenic animals. The transgene may be
integrated as a single transgene or in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The 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.
[0053] 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).
[0054] 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.
[0055] 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.
5.2 NHPS and NHP Polypeptides
[0056] 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, 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 disease. 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.
[0057] 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 consensus signal sequences, which indicates
that they may be cytoplasmic or possibly nuclear proteins, however,
the homology data and presence of hydrophobic domains indicates
that the NHPs are probably membrane associated, or possibly
secreted.
[0058] 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 above 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.
[0059] 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, or the ability to effect an identical or
complementary downstream pathway, or a change in cellular
metabolism (e.g., proteolytic activity, ion flux, tyrosine
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 above, 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.
[0060] 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 the NHP, but to assess biological activity,
e.g., in drug screening assays.
[0061] 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 cell systems
(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing NHP nucleotide sequences and promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter).
[0062] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the NHP
product being expressed. For example, when a large quantity of such
a protein is to be produced for the generation of pharmaceutical
compositions of or containing NHP, or for raising antibodies to a
NHP, vectors that direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include, but are not limited to, the E. coli expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NHP
coding sequence may be ligated individually into the vector in
frame with the lacZ coding region so that a fusion protein is
produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res.
13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. The PGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target
expression product can be released from the GST moiety.
[0063] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) can be used as a vector to express
foreign polynucleotide sequences. The virus grows in Spodoptera
frugiperda cells. A NHP coding sequence can be cloned individually
into non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter). Successful insertion of NHP coding
sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking
the proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted sequence is expressed (e.g., see Smith
et al., 1983, J. Virol. 46:584; Smith, U.S. Pat. No.
4,215,051).
[0064] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the NHP nucleotide sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric sequence may then be inserted in the adenovirus genome by
in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing a. NHP
product in infected hosts (e.g., see Logan and Shenk, 1984, Proc.
Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may
also be required for efficient translation of inserted NHP
nucleotide sequences. These signals include the ATG initiation
codon and adjacent sequences. In cases where an entire NHP gene or
cDNA, including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of a NHP coding sequence is inserted,
exogenous translational control signals, including, perhaps, the
ATG initiation codon, may be provided. Furthermore, the initiation
codon should be in phase with the reading frame of the desired
coding sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bitter et al.; 1987, Methods in Enzymol.
153:516-544).
[0065] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the expression product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and expression products. Appropriate cell lines or host
systems can be chosen to ensure the desired modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells that possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the expression product may be used. Such
mammalian host cells include, but are not limited to, CHO, VERO,
BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, human cell
lines.
[0066] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express the NHP sequences described above can be
engineered. Rather than using expression vectors that contain viral
origins of replication, host cells can be transformed with DNA
controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci, which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines that express the NHP product. Such engineered cell lines may
be particularly useful in screening and evaluation of compounds
that affect the endogenous activity of the NHP product.
[0067] A number of selection systems may be used, including, but
not limited to, the herpes simplex virus thymidine kinase (Wigler
et al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase. (Szybalska and Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes,
which can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad.
Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo,
which confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre et al., 1984, Gene
30:147).
[0068] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, an exemplary system allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA
88:8972-8976). In this system, the sequence of interest is
subcloned into a vaccinia recombination plasmid such that the
sequence's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0069] Also encompassed by the present invention are fusion
proteins that direct the NHP to a target organ and/or facilitate
transport across the membrane into the cytosol. Conjugation of NHPs
to antibody molecules or their Fab fragments could be used to
target cells bearing a particular epitope. Attaching the
appropriate signal sequence to the NHP would also transport the NHP
to the desired location within the cell. Alternatively, targeting
of NHP or its nucleic acid sequence might be achieved using
liposome or lipid complex based delivery systems. Such technologies
are described in "Liposomes: A Practical Approach", New, R.R.C.,
ed., Oxford University Press, New York, and in U.S. Pat. Nos.
4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respective
disclosures, which are herein incorporated by reference in their
entirety. Additionally embodied are novel protein constructs
engineered in such a way that they facilitate transport of the NHP
to the target site or desired organ, where they cross the cell
membrane and/or the nucleus where the NHP can exert its functional
activity. This goal may be achieved by coupling of the NHP to a
cytokine or other ligand that provides targeting specificity,
and/or to a protein transducing domain (see generally U.S.
Provisional Patent Application Ser. Nos. 60/111,701 and 60/056,713,
both of which are herein incorporated by reference, for examples of
such transducing sequences) to facilitate passage across cellular
membranes, and can optionally be engineered to include nuclear
localization.
5.3 Antibodies to NHP Products
[0070] Antibodies that specifically recognize one or more epitopes
of a NHP, or epitopes of conserved variants of a NHP, or peptide
fragments of a NHP, are also encompassed by the invention. Such
antibodies include, but are not limited to, polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies,
single chain antibodies, Fab fragments, F(ab').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the
above.
[0071] The antibodies of the invention can be used, for example, in
the detection of NHP in a biological sample and may, therefore, be
utilized as part of a diagnostic or prognostic technique whereby
patients may be tested for abnormal amounts of NHP. Such antibodies
may also be utilized in conjunction with, for example, compound
screening schemes for the evaluation of the effect of test
compounds on expression and/or activity of a NHP expression
product. Additionally, such antibodies can be used in conjunction
gene therapy to, for example, evaluate the normal and/or engineered
NHP-expressing cells prior to their introduction into the patient.
Such antibodies may additionally be used as a method for the
inhibition of abnormal NHP activity. Thus, such antibodies may,
therefore, be utilized as part of treatment methods.
[0072] For the production of antibodies, various host animals may
be immunized by injection with the NHP, a NHP peptide (e.g., one
corresponding to a functional domain of a NHP), truncated NHP
polypeptides (NHP in which one or more domains have been deleted),
functional equivalents of the NHP, or mutated variants of the NHP.
Such host animals may include, but are not limited to, pigs,
rabbits, mice, goats, and rats, to name but a few. Various
adjuvants may be used to increase the immunological response,
depending on the host species, including, but not limited to,
Freund's adjuvant (complete and incomplete), mineral salts such as
aluminum hydroxide or aluminum phosphate, chitosan, surface active
substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
Alternatively, the immune response could be enhanced by combination
and or coupling with molecules such as keyhole limpet hemocyanin,
tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin or
fragments thereof. Polyclonal antibodies are heterogeneous
populations of antibody molecules derived from the sera of the
immunized animals.
[0073] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be obtained by any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique (Kohler and Milstein, 1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class, including IgG, IgM,
IgE, IgA, and IgD, and any subclass thereof. The hybridoma
producing the mAbs of this invention may be cultivated in vitro or
in vivo. Production of high titers of mAbs in vivo makes this the
presently preferred method of production.
[0074] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. USA, 81:6851-6855; Neuberger et al., 1984, Nature,
312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing
the genes from a mouse antibody molecule of appropriate antigen
specificity together with genes from a human antibody molecule of
appropriate biological activity can be used. A chimeric antibody is
a molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region. Such
technologies are described in U.S. Pat. Nos. 6,075,181 and
5,877,397 and their respective disclosures, which are herein
incorporated by reference in their entirety. Also encompassed by
the present invention is the use of fully humanized monoclonal
antibodies, as described in U.S. Pat. No. 6,150,584 and respective
disclosures, which are herein incorporated by reference in their
entirety.
[0075] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426'; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be
adapted to produce single chain antibodies against NHP expression
products. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0076] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: F(ab').sub.2 fragments, which can be
produced by pepsin digestion of the antibody molecule; and Fab
fragments, which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0077] Antibodies to a NHP can, in turn, be utilized to generate
anti-idiotype antibodies that "mimic" a given NHP, using techniques
well-known to those skilled in the art (see, e.g., Greenspan and
Bona, 1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.
147:2429-2438). For example, antibodies that bind to a NHP domain
and competitively inhibit the binding of NHP to its cognate
receptor/ligand can be used to generate anti-idiotypes that "mimic"
the NHP and, therefore, bind, activate, or neutralize a NHP, NHP
receptor, or NHP ligand. Such anti-idiotypic antibodies, or Fab
fragments of such anti-idiotypes, can be used in therapeutic
regimens involving a NHP mediated pathway.
[0078] Additionally given the high degree of relatedness of
mammalian NHPs, the presently described knock-out mice (having
never seen a NHP, and thus never been tolerized to a NHP) have a
unique utility, as they can be advantageously applied to the
generation of antibodies against the disclosed mammalian NHPs
(i.e., a NHP will be immunogenic in NHP knock-out animals).
[0079] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein, will become
apparent to those skilled in the art from the foregoing
description. Such modifications are intended to fall within the
scope of the appended claims. All cited publications, patents, and
patent applications are herein incorporated by reference in their
entirety.
Sequence CWU 1
1
5 1 2079 DNA homo sapiens 1 atggagaagt acgagcggat ccgagtggtg
gggagaggtg ccttcgggat tgtgcacctg 60 tgcctgcgaa aggctgacca
gaagctggtg atcatcaagc agattccagt ggaacagatg 120 accaaggaag
agcggcaggc agcccagaat gagtgccagg tcctcaagct gctcaaccac 180
cccaatgtca ttgagtacta cgagaacttc ctggaagaca aagcccttat gatcgccatg
240 gaatatgcac caggcggcac tctggctgag ttcatccaaa agcgctgtaa
ttccctgctg 300 gaggaggaga ccatcctgca cttcttcgtg cagatcctgc
ttgcactgca tcatgtgcac 360 acccacctca tcctgcaccg agacctcaag
acccagaaca tcctgcttga caaacaccgc 420 atggtcgtca agatcggtga
tttcggcatc tccaagatcc ttagcagcaa gagcaaggcc 480 tacacggtgg
tgggtacccc atgctatatc tcccctgagc tgtgtgaggg caagccctac 540
aaccagaaga gtgacatctg ggccctgggc tgtgtcctct acgagctggc cagcctcaag
600 agggctttcg aggctgcgaa cttgccagca ctggtgctga agatcatgag
tggcaccttt 660 gcacctatct ctgaccggta cagccctgag cttcgccagc
tggtcctgag tctactcagc 720 ctggagcctg cccagcggcc accactcagc
cacatcatgg cacagcccct ctgcatccgt 780 gccctcctca acctccacac
cgacgtgggc agtgtccgca tgcggagggc agagaagtcc 840 gtggccccca
gcaacacagg gagcaggacc accagtgtcc gctgcagagg tatcccccgg 900
ggacctgtga ggccagccat cccaccacca ctgtcgtcag tgtatgcctg gggtggtggg
960 ctgggcaccc ccctgcggct gccaatgctc aacacagagg tggtccaggt
ggcagctggg 1020 cgcacgcaga aagccggcgt cacgcgctct gggcgtctca
tcctgtggga ggccccaccc 1080 ctaggtgcag gcggaggcag tctccttcct
ggggcagtgg agcagccaca gccccagttc 1140 atctcgcgtt tcctggaggg
ccagtcgggy gtgaccatca agcacgtggc ctgtggggac 1200 ttcttcactg
cctgcctgac tgacagaggc atcatcatga cattcggcag cggcagcaat 1260
gggtgcctag gccatggcag cctcactgac atcagccagc ccaccattgt ggaggctttg
1320 ytgggctatg aaatggtgca ggtggcctgt ggggcctctc acgtgctggc
cctgtccact 1380 gagcgagaac tatttgcctg gggccgtgga gacagcggca
gactggggct aggcaccagg 1440 gagtcccaca gctgccccca gcaggtgccc
atgcccccag gacaggaagc tcagcgagtt 1500 gtatgtggta tcgattcctc
catgatcctc actgtgcctg gccaagccct agcctgtggg 1560 agcaacaggt
tcaacaagct gggcctggac cacctctccc tgggggagga gcctgtcccc 1620
caccagcaag tggaggaggc cctgagcttc acactactag gctctgcacc cctggaccag
1680 gagcctctgc tgagtataga cctgggcact gctcactcag ctgctgtgac
tgcctcgggt 1740 gattgctaca cttttggcag caatcagcac ggacagttgg
gcaccaatac tcgccgaggc 1800 agtcgggcac cctgtaaggt ccaaggcctt
gagggcatca agatggcaat ggtagcctgt 1860 ggggatgcct tcactgtagc
tattggggca gagagcgaag tgtactcttg gggcaaaggg 1920 gcgcgaggtc
gattgggaag gagggatgag gatgccggac tccctcggcc agtgcagttg 1980
gatgagacac acccttacac ggtgacttcc gtgtcctgtt gccatggaaa caccctcctg
2040 gctgttcgat cggtcacaga tgagccggtc cccccctga 2079 2 692 PRT homo
sapiens 2 Met Glu Lys Tyr Glu Arg Ile Arg Val Val Gly Arg Gly Ala
Phe Gly 1 5 10 15 Ile Val His Leu Cys Leu Arg Lys Ala Asp Gln Lys
Leu Val Ile Ile 20 25 30 Lys Gln Ile Pro Val Glu Gln Met Thr Lys
Glu Glu Arg Gln Ala Ala 35 40 45 Gln Asn Glu Cys Gln Val Leu Lys
Leu Leu Asn His Pro Asn Val Ile 50 55 60 Glu Tyr Tyr Glu Asn Phe
Leu Glu Asp Lys Ala Leu Met Ile Ala Met 65 70 75 80 Glu Tyr Ala Pro
Gly Gly Thr Leu Ala Glu Phe Ile Gln Lys Arg Cys 85 90 95 Asn Ser
Leu Leu Glu Glu Glu Thr Ile Leu His Phe Phe Val Gln Ile 100 105 110
Leu Leu Ala Leu His His Val His Thr His Leu Ile Leu His Arg Asp 115
120 125 Leu Lys Thr Gln Asn Ile Leu Leu Asp Lys His Arg Met Val Val
Lys 130 135 140 Ile Gly Asp Phe Gly Ile Ser Lys Ile Leu Ser Ser Lys
Ser Lys Ala 145 150 155 160 Tyr Thr Val Val Gly Thr Pro Cys Tyr Ile
Ser Pro Glu Leu Cys Glu 165 170 175 Gly Lys Pro Tyr Asn Gln Lys Ser
Asp Ile Trp Ala Leu Gly Cys Val 180 185 190 Leu Tyr Glu Leu Ala Ser
Leu Lys Arg Ala Phe Glu Ala Ala Asn Leu 195 200 205 Pro Ala Leu Val
Leu Lys Ile Met Ser Gly Thr Phe Ala Pro Ile Ser 210 215 220 Asp Arg
Tyr Ser Pro Glu Leu Arg Gln Leu Val Leu Ser Leu Leu Ser 225 230 235
240 Leu Glu Pro Ala Gln Arg Pro Pro Leu Ser His Ile Met Ala Gln Pro
245 250 255 Leu Cys Ile Arg Ala Leu Leu Asn Leu His Thr Asp Val Gly
Ser Val 260 265 270 Arg Met Arg Arg Ala Glu Lys Ser Val Ala Pro Ser
Asn Thr Gly Ser 275 280 285 Arg Thr Thr Ser Val Arg Cys Arg Gly Ile
Pro Arg Gly Pro Val Arg 290 295 300 Pro Ala Ile Pro Pro Pro Leu Ser
Ser Val Tyr Ala Trp Gly Gly Gly 305 310 315 320 Leu Gly Thr Pro Leu
Arg Leu Pro Met Leu Asn Thr Glu Val Val Gln 325 330 335 Val Ala Ala
Gly Arg Thr Gln Lys Ala Gly Val Thr Arg Ser Gly Arg 340 345 350 Leu
Ile Leu Trp Glu Ala Pro Pro Leu Gly Ala Gly Gly Gly Ser Leu 355 360
365 Leu Pro Gly Ala Val Glu Gln Pro Gln Pro Gln Phe Ile Ser Arg Phe
370 375 380 Leu Glu Gly Gln Ser Gly Val Thr Ile Lys His Val Ala Cys
Gly Asp 385 390 395 400 Phe Phe Thr Ala Cys Leu Thr Asp Arg Gly Ile
Ile Met Thr Phe Gly 405 410 415 Ser Gly Ser Asn Gly Cys Leu Gly His
Gly Ser Leu Thr Asp Ile Ser 420 425 430 Gln Pro Thr Ile Val Glu Ala
Leu Leu Gly Tyr Glu Met Val Gln Val 435 440 445 Ala Cys Gly Ala Ser
His Val Leu Ala Leu Ser Thr Glu Arg Glu Leu 450 455 460 Phe Ala Trp
Gly Arg Gly Asp Ser Gly Arg Leu Gly Leu Gly Thr Arg 465 470 475 480
Glu Ser His Ser Cys Pro Gln Gln Val Pro Met Pro Pro Gly Gln Glu 485
490 495 Ala Gln Arg Val Val Cys Gly Ile Asp Ser Ser Met Ile Leu Thr
Val 500 505 510 Pro Gly Gln Ala Leu Ala Cys Gly Ser Asn Arg Phe Asn
Lys Leu Gly 515 520 525 Leu Asp His Leu Ser Leu Gly Glu Glu Pro Val
Pro His Gln Gln Val 530 535 540 Glu Glu Ala Leu Ser Phe Thr Leu Leu
Gly Ser Ala Pro Leu Asp Gln 545 550 555 560 Glu Pro Leu Leu Ser Ile
Asp Leu Gly Thr Ala His Ser Ala Ala Val 565 570 575 Thr Ala Ser Gly
Asp Cys Tyr Thr Phe Gly Ser Asn Gln His Gly Gln 580 585 590 Leu Gly
Thr Asn Thr Arg Arg Gly Ser Arg Ala Pro Cys Lys Val Gln 595 600 605
Gly Leu Glu Gly Ile Lys Met Ala Met Val Ala Cys Gly Asp Ala Phe 610
615 620 Thr Val Ala Ile Gly Ala Glu Ser Glu Val Tyr Ser Trp Gly Lys
Gly 625 630 635 640 Ala Arg Gly Arg Leu Gly Arg Arg Asp Glu Asp Ala
Gly Leu Pro Arg 645 650 655 Pro Val Gln Leu Asp Glu Thr His Pro Tyr
Thr Val Thr Ser Val Ser 660 665 670 Cys Cys His Gly Asn Thr Leu Leu
Ala Val Arg Ser Val Thr Asp Glu 675 680 685 Pro Val Pro Pro 690 3
2454 DNA homo sapiens 3 atgcccgccg ccactccagc cccgcagccg ccgccgcccc
cggcccggcc agccccagcc 60 tgcccggcgc ggcctgcccc gggacagcaa
ggcctatgtg accattctct aaaatattta 120 agctcgagaa tcacagagcg
gaagctgcaa ggctcctggc tgcctgccag ccgagggaat 180 ctggagaaac
cattcctggg gccgcgtggc cccgtcgtgc ccttgttctg ccctcggaat 240
ggccttcact cagcacatcc tgagaacagc cctctgaagc ccagggtcgt gaccgtagtg
300 aagctgggtg ggcagcgccc ccgaaagatc actctgctcc tcaacaggcg
atcagtgcag 360 acgttcgagc agctcttagc tgacatctca gaagccttgg
gctctcccag atggaagaat 420 gaccgtgtga ggaaactgtt taacctcaag
ggcagggaaa tcaggagcgt ctctgatttc 480 ttcagggaag gggatgcttt
catagctatg ggcaaagaac cactgacact gaagagcatt 540 caggtggctg
tagaagaact gtaccccaac aaagcccggg ccctgacact ggcccagcac 600
agccgtgccc cttctccaag gctgaggagc aggctgttta gcaaggctct gaaaggagac
660 caccgctgtg gggagaccga gacccccaag agctgcagcg aagttgcagg
atgcaaggca 720 gccatgaggc accaggggaa gatccccgag gagctttcac
tagatgacag agcgaggacc 780 cagaagaagt gggggagggg gaaatgggag
ccagaaccca gtagcaagcc ccccagggaa 840 gccactctgg aagagaggca
cgcaagggga gagaagcatc ttggggtgga gattgaaaag 900 acctcgggtg
aaattatcag atgcgagaag tgcaagagag agagggagct tcagcagagc 960
ctggagcgtg agaggctttc tctggggacc agtgagctgg atatggggaa gggcccaatg
1020 tatgatgtgg agaagctggt gaggaccaga agctgcagga ggtctcccga
ggcaaatcct 1080 gcaagtgggg aggaagggtg gaagggtgac agccacagga
gcagccccag gaatcccact 1140 caagagctga ggagacccag caagagcatg
gacaagaaag aggacagagg cccagaggat 1200 caagaaagcc atgctcaggg
agcagccaag gccaagaagg accttgtgga agttcttcct 1260 gtcacagagg
aggggctgag ggaggtgaag aaggacacca ggcccatgag caggagcaaa 1320
catggtggct ggctcctgag agagcaccag gcgggctttg agaagctccg caggacccga
1380 ggagaagaga aggaggcaga gaaggagaaa aagccatgta tgtctggagg
cagaaggatg 1440 actctcagag atgaccaacc tgcaaagcta gaaaaggagc
ccaagacgag gccagaagag 1500 aacaagccag agcggcccag cggtcggaag
ccacggccca tgggcatcat tgccgccaat 1560 gtggaaaagc attatgagac
tggccgggtc attggggatg ggaactttgc tgtcgtgaag 1620 gagtgcagac
accgcgagac caggcaggcc tatgcgatga agatcattga caagtccaga 1680
ctcaagggca aggaggacat ggtggacagt gagatcttga tcatccagag cctctctcac
1740 cccaacatcg tgaaattgca tgaagtctac gaaacagaca tggaaatcta
cctgatcctg 1800 gagtacgtgc agggaggaga cctttttgac gccatcatag
aaagtgtgaa gttcccggag 1860 cccgatgctg ccctcatgat catggactta
tgcaaagccc tcgtccacat gcacgacaag 1920 agcattgtcc accgggacct
caagccggaa aaccttttgg ttcagcgaaa tgaggacaaa 1980 tctactacct
tgaaattggc tgattttgga cttgcaaagc atgtggtgag acctatattt 2040
actgtgtgtg ggaccccaac ttacgtagct cccgaaattc tttctgagaa aggttatgga
2100 ctggaggtgg acatgtgggc tgctggcgtg atcctctata tcctgctgtg
tggctttccc 2160 ccattccgca gccctgagag ggaccaggac gagctcttta
acatcatcca gctgggccac 2220 tttgagttcc tcccccctta ctgggacaat
atctctgatg ctgctaaaga tctggtgagc 2280 cggttgctgg tggtagaccc
caaaaagcgc tacacagctc atcaggttct tcagcacccc 2340 tggatcgaaa
cagctggcaa gaccaataca gtgaaacgac agaagcaggt gtcccccagc 2400
agcgatggtc acttccggag ccagcacaag agggttgtgg agcaggtatc atag 2454 4
817 PRT homo sapiens 4 Met Pro Ala Ala Thr Pro Ala Pro Gln Pro Pro
Pro Pro Pro Ala Arg 1 5 10 15 Pro Ala Pro Ala Cys Pro Ala Arg Pro
Ala Pro Gly Gln Gln Gly Leu 20 25 30 Cys Asp His Ser Leu Lys Tyr
Leu Ser Ser Arg Ile Thr Glu Arg Lys 35 40 45 Leu Gln Gly Ser Trp
Leu Pro Ala Ser Arg Gly Asn Leu Glu Lys Pro 50 55 60 Phe Leu Gly
Pro Arg Gly Pro Val Val Pro Leu Phe Cys Pro Arg Asn 65 70 75 80 Gly
Leu His Ser Ala His Pro Glu Asn Ser Pro Leu Lys Pro Arg Val 85 90
95 Val Thr Val Val Lys Leu Gly Gly Gln Arg Pro Arg Lys Ile Thr Leu
100 105 110 Leu Leu Asn Arg Arg Ser Val Gln Thr Phe Glu Gln Leu Leu
Ala Asp 115 120 125 Ile Ser Glu Ala Leu Gly Ser Pro Arg Trp Lys Asn
Asp Arg Val Arg 130 135 140 Lys Leu Phe Asn Leu Lys Gly Arg Glu Ile
Arg Ser Val Ser Asp Phe 145 150 155 160 Phe Arg Glu Gly Asp Ala Phe
Ile Ala Met Gly Lys Glu Pro Leu Thr 165 170 175 Leu Lys Ser Ile Gln
Val Ala Val Glu Glu Leu Tyr Pro Asn Lys Ala 180 185 190 Arg Ala Leu
Thr Leu Ala Gln His Ser Arg Ala Pro Ser Pro Arg Leu 195 200 205 Arg
Ser Arg Leu Phe Ser Lys Ala Leu Lys Gly Asp His Arg Cys Gly 210 215
220 Glu Thr Glu Thr Pro Lys Ser Cys Ser Glu Val Ala Gly Cys Lys Ala
225 230 235 240 Ala Met Arg His Gln Gly Lys Ile Pro Glu Glu Leu Ser
Leu Asp Asp 245 250 255 Arg Ala Arg Thr Gln Lys Lys Trp Gly Arg Gly
Lys Trp Glu Pro Glu 260 265 270 Pro Ser Ser Lys Pro Pro Arg Glu Ala
Thr Leu Glu Glu Arg His Ala 275 280 285 Arg Gly Glu Lys His Leu Gly
Val Glu Ile Glu Lys Thr Ser Gly Glu 290 295 300 Ile Ile Arg Cys Glu
Lys Cys Lys Arg Glu Arg Glu Leu Gln Gln Ser 305 310 315 320 Leu Glu
Arg Glu Arg Leu Ser Leu Gly Thr Ser Glu Leu Asp Met Gly 325 330 335
Lys Gly Pro Met Tyr Asp Val Glu Lys Leu Val Arg Thr Arg Ser Cys 340
345 350 Arg Arg Ser Pro Glu Ala Asn Pro Ala Ser Gly Glu Glu Gly Trp
Lys 355 360 365 Gly Asp Ser His Arg Ser Ser Pro Arg Asn Pro Thr Gln
Glu Leu Arg 370 375 380 Arg Pro Ser Lys Ser Met Asp Lys Lys Glu Asp
Arg Gly Pro Glu Asp 385 390 395 400 Gln Glu Ser His Ala Gln Gly Ala
Ala Lys Ala Lys Lys Asp Leu Val 405 410 415 Glu Val Leu Pro Val Thr
Glu Glu Gly Leu Arg Glu Val Lys Lys Asp 420 425 430 Thr Arg Pro Met
Ser Arg Ser Lys His Gly Gly Trp Leu Leu Arg Glu 435 440 445 His Gln
Ala Gly Phe Glu Lys Leu Arg Arg Thr Arg Gly Glu Glu Lys 450 455 460
Glu Ala Glu Lys Glu Lys Lys Pro Cys Met Ser Gly Gly Arg Arg Met 465
470 475 480 Thr Leu Arg Asp Asp Gln Pro Ala Lys Leu Glu Lys Glu Pro
Lys Thr 485 490 495 Arg Pro Glu Glu Asn Lys Pro Glu Arg Pro Ser Gly
Arg Lys Pro Arg 500 505 510 Pro Met Gly Ile Ile Ala Ala Asn Val Glu
Lys His Tyr Glu Thr Gly 515 520 525 Arg Val Ile Gly Asp Gly Asn Phe
Ala Val Val Lys Glu Cys Arg His 530 535 540 Arg Glu Thr Arg Gln Ala
Tyr Ala Met Lys Ile Ile Asp Lys Ser Arg 545 550 555 560 Leu Lys Gly
Lys Glu Asp Met Val Asp Ser Glu Ile Leu Ile Ile Gln 565 570 575 Ser
Leu Ser His Pro Asn Ile Val Lys Leu His Glu Val Tyr Glu Thr 580 585
590 Asp Met Glu Ile Tyr Leu Ile Leu Glu Tyr Val Gln Gly Gly Asp Leu
595 600 605 Phe Asp Ala Ile Ile Glu Ser Val Lys Phe Pro Glu Pro Asp
Ala Ala 610 615 620 Leu Met Ile Met Asp Leu Cys Lys Ala Leu Val His
Met His Asp Lys 625 630 635 640 Ser Ile Val His Arg Asp Leu Lys Pro
Glu Asn Leu Leu Val Gln Arg 645 650 655 Asn Glu Asp Lys Ser Thr Thr
Leu Lys Leu Ala Asp Phe Gly Leu Ala 660 665 670 Lys His Val Val Arg
Pro Ile Phe Thr Val Cys Gly Thr Pro Thr Tyr 675 680 685 Val Ala Pro
Glu Ile Leu Ser Glu Lys Gly Tyr Gly Leu Glu Val Asp 690 695 700 Met
Trp Ala Ala Gly Val Ile Leu Tyr Ile Leu Leu Cys Gly Phe Pro 705 710
715 720 Pro Phe Arg Ser Pro Glu Arg Asp Gln Asp Glu Leu Phe Asn Ile
Ile 725 730 735 Gln Leu Gly His Phe Glu Phe Leu Pro Pro Tyr Trp Asp
Asn Ile Ser 740 745 750 Asp Ala Ala Lys Asp Leu Val Ser Arg Leu Leu
Val Val Asp Pro Lys 755 760 765 Lys Arg Tyr Thr Ala His Gln Val Leu
Gln His Pro Trp Ile Glu Thr 770 775 780 Ala Gly Lys Thr Asn Thr Val
Lys Arg Gln Lys Gln Val Ser Pro Ser 785 790 795 800 Ser Asp Gly His
Phe Arg Ser Gln His Lys Arg Val Val Glu Gln Val 805 810 815 Ser 5
2824 DNA homo sapiens 5 cgggctcgtg gctgctcgtc tcgccccgcc ttcccgcgcc
tgctcgaccg tcgagccgcg 60 tccccgcgct gccacctctg ctccaggctc
tccccgagcc cgccgccgcg ccatgcccgc 120 cgccactcca gccccgcagc
cgccgccgcc cccggcccgg ccagccccag cctgcccggc 180 gcggcctgcc
ccgggacagc aaggcctatg tgaccattct ctaaaatatt taagctcgag 240
aatcacagag cggaagctgc aaggctcctg gctgcctgcc agccgaggga atctggagaa
300 accattcctg gggccgcgtg gccccgtcgt gcccttgttc tgccctcgga
atggccttca 360 ctcagcacat cctgagaaca gccctctgaa gcccagggtc
gtgaccgtag tgaagctggg 420 tgggcagcgc ccccgaaaga tcactctgct
cctcaacagg cgatcagtgc agacgttcga 480 gcagctctta gctgacatct
cagaagcctt gggctctccc agatggaaga atgaccgtgt 540 gaggaaactg
tttaacctca agggcaggga aatcaggagc gtctctgatt tcttcaggga 600
aggggatgct ttcatagcta tgggcaaaga accactgaca ctgaagagca ttcaggtggc
660 tgtagaagaa ctgtacccca acaaagcccg ggccctgaca ctggcccagc
acagccgtgc 720 cccttctcca aggctgagga gcaggctgtt tagcaaggct
ctgaaaggag accaccgctg 780 tggggagacc gagaccccca agagctgcag
cgaagttgca ggatgcaagg cagccatgag 840 gcaccagggg aagatccccg
aggagctttc actagatgac agagcgagga cccagaagaa 900 gtgggggagg
gggaaatggg agccagaacc cagtagcaag ccccccaggg aagccactct 960
ggaagagagg cacgcaaggg gagagaagca tcttggggtg gagattgaaa agacctcggg
1020 tgaaattatc agatgcgaga agtgcaagag agagagggag cttcagcaga
gcctggagcg 1080 tgagaggctt tctctgggga ccagtgagct ggatatgggg
aagggcccaa tgtatgatgt 1140 ggagaagctg gtgaggacca gaagctgcag
gaggtctccc gaggcaaatc ctgcaagtgg 1200 ggaggaaggg tggaagggtg
acagccacag gagcagcccc aggaatccca ctcaagagct 1260 gaggagaccc
agcaagagca tggacaagaa agaggacaga ggcccagagg atcaagaaag 1320
ccatgctcag ggagcagcca aggccaagaa ggaccttgtg gaagttcttc ctgtcacaga
1380 ggaggggctg agggaggtga agaaggacac caggcccatg agcaggagca
aacatggtgg 1440 ctggctcctg agagagcacc aggcgggctt tgagaagctc
cgcaggaccc gaggagaaga 1500 gaaggaggca gagaaggaga aaaagccatg
tatgtctgga ggcagaagga tgactctcag 1560 agatgaccaa cctgcaaagc
tagaaaagga gcccaagacg aggccagaag agaacaagcc 1620 agagcggccc
agcggtcgga agccacggcc catgggcatc attgccgcca atgtggaaaa 1680
gcattatgag actggccggg tcattgggga tgggaacttt gctgtcgtga aggagtgcag
1740 acaccgcgag accaggcagg cctatgcgat gaagatcatt gacaagtcca
gactcaaggg 1800 caaggaggac atggtggaca gtgagatctt gatcatccag
agcctctctc accccaacat 1860 cgtgaaattg catgaagtct acgaaacaga
catggaaatc tacctgatcc tggagtacgt 1920 gcagggagga gacctttttg
acgccatcat agaaagtgtg aagttcccgg agcccgatgc 1980 tgccctcatg
atcatggact tatgcaaagc cctcgtccac atgcacgaca agagcattgt 2040
ccaccgggac ctcaagccgg aaaacctttt ggttcagcga aatgaggaca aatctactac
2100 cttgaaattg gctgattttg gacttgcaaa gcatgtggtg agacctatat
ttactgtgtg 2160 tgggacccca acttacgtag ctcccgaaat tctttctgag
aaaggttatg gactggaggt 2220 ggacatgtgg gctgctggcg tgatcctcta
tatcctgctg tgtggctttc ccccattccg 2280 cagccctgag agggaccagg
acgagctctt taacatcatc cagctgggcc actttgagtt 2340 cctcccccct
tactgggaca atatctctga tgctgctaaa gatctggtga gccggttgct 2400
ggtggtagac cccaaaaagc gctacacagc tcatcaggtt cttcagcacc cctggatcga
2460 aacagctggc aagaccaata cagtgaaacg acagaagcag gtgtccccca
gcagcgatgg 2520 tcacttccgg agccagcaca agagggttgt ggagcaggta
tcatagtcac caccttggga 2580 atctgtccag cccccagttc tgctcaagga
cagagaaaag gatagaagtt tgagagaaaa 2640 acaatgaaag aggcttcttc
acataattgg tgaatcagag ggagagacac tgagtatatt 2700 ttaaagcata
ttaaaaaaat taagtcaatg ttaaatgtca caacatattt ttagatttgt 2760
atatttaaag cctttaatac atttttgggg ggtaagcatt gtcatcagtg aggaattttg
2820 gtaa 2824
* * * * *