U.S. patent application number 11/129653 was filed with the patent office on 2006-12-28 for human orthologues of wart.
This patent application is currently assigned to Sugen, Inc.. Invention is credited to Peter Flanagan, Gregory Plowman.
Application Number | 20060292573 11/129653 |
Document ID | / |
Family ID | 22105085 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060292573 |
Kind Code |
A1 |
Plowman; Gregory ; et
al. |
December 28, 2006 |
Human orthologues of WART
Abstract
The present invention relates in part to hWART nucleic acid
molecules. The invention also relates in part to nucleic acid
molecules encoding portions of hWART full-length proteins, nucleic
acid vectors containing hWART nucleic acid molecules, recombinant
cells containing such nucleic acid vectors, polypeptides purified
from such recombinant cells, antibodies to such polypeptides, and
methods of identifying compounds that modulate the function of an
hWART polypeptide. Also disclosed are methods for diagnosing
abnormal cell proliferative conditions in an organism using
hWART-related molecules or compounds.
Inventors: |
Plowman; Gregory; (San
Carlos, CA) ; Flanagan; Peter; (San Francisco,
CA) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Sugen, Inc.
|
Family ID: |
22105085 |
Appl. No.: |
11/129653 |
Filed: |
May 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10284130 |
Oct 31, 2002 |
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11129653 |
May 16, 2005 |
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09233857 |
Jan 20, 1999 |
6495353 |
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10284130 |
Oct 31, 2002 |
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60072023 |
Jan 21, 1998 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 435/7.2; 530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
G01N 33/574 20130101; C12N 9/1205 20130101; C12Q 1/485
20130101 |
Class at
Publication: |
435/006 ;
530/388.22; 435/007.2; 435/069.1; 435/320.1; 435/325;
536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/567 20060101 G01N033/567; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/705 20060101
C07K014/705; C07K 16/28 20060101 C07K016/28 |
Claims
1-9. (canceled)
10. An isolated, enriched or purified hWART polypeptide of SEQ ID
NO:4.
11. The polypeptide of claim 10, wherein said polypeptide is a
fragment of the protein encoded by the full length amino acid
sequence set forth in SEQ ID NO:4.
12. The polypeptide of claim 10, wherein said polypeptide comprises
an amino acid sequence having (a) the full length amino acid
sequence set forth in SEQ ID NO:4; (b) the full length amino acid
sequence of the sequence set forth in SEQ ID NO:4, expect that it
lacks one or more of the following segments of amino acid residues
1-33, 43-139, 342-466, 467-480, 514-518, 974-1048 of SEQ ID NO:4;
(c) the amino acid sequence set forth in SEQ ID NO:4 from amino
acid residues residues 1-33, 43-139, 342-466, 467-480, 514-518,
974-1048 of SEQ ID NO:4; or (d) the full length amino acid sequence
set forth in SEQ ID NO:4 except that it lacks one or more of the
domains selected from the group consisting of an N-terminal domain,
a catalytic domain, and a C-terminal domain.
13-14. (canceled)
15. A method for identifying a substance capable of modulating
hWART activity comprising the steps of: (a) contacting a
polypeptide of SEQ ID NO: 4 with a test substance; and (b)
determining whether said substance alters the activity of said
polypeptide.
16-22. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is related to and claims priority to the
U.S. Provisional Application Ser. No. 60/072,023, filed on Jan. 21,
1998, by Plowman et al., and entitled "HUMAN ORTHOLOGUES OF WART"
(Lyon & Lyon Docket No. 224/006), which is hereby incorporated
by reference herein in its entirety, including any drawings.
FIELD OF THE INVENTION
[0002] The present invention relates in part to protein kinases. In
particular, the invention concerns the identification of protein
kinase proteins which are human orthologues of the drosophila WART
gene (hWART).
BACKGROUND OF THE INVENTION
[0003] The following description is provided to aid in
understanding the invention, but is not admitted to describe or
constitute prior art to the invention.
[0004] Cellular signal transduction is a fundamental mechanism
whereby extracellular stimuli are relayed to the interior of cells
and thereby regulate diverse cellular processes. One of the key
biochemical mechanisms of signal transduction involves the
reversible phosphorylation of proteins. Phosphorylation of
polypeptides regulates the activity of mature proteins by altering
their structure and function. Phosphate most often resides on the
hydroxyl moiety of serine, threonine, or tyrosine amino acids in
proteins.
[0005] Enzymes that mediate phosphorylation of cellular effectors
generally fall into two classes. The first class consists of
protein kinases which transfer a phosphate moiety from nucleotide
triposphates to protein substrates. The second class consists of
protein phosphatases which hydrolyze phosphate moieties from
phosphoryl protein substrates. The converse functions of protein
kinases and protein phosphatases balance and regulate the flow of
signals in signal transduction processes.
[0006] Protein kinases are generally divided into two classes:
receptor and non-receptor type proteins. Protein kinases may also
be divided into three classes based upon the amino acids they act
upon: (1) Some catalyze the addition or hydrolysis of phosphate on
serine or threonine only; (2) some catalyze the addition or
hydrolysis of phosphate on tyrosine only; and (3) some catalyze the
addition or hydrolysis of phosphate on serine, threonine, and
tyrosine.
[0007] Altered protein kinase activity has been associated with
multiple abnormal cellular functions, including increased cell
proliferation. Increased cell proliferation can result from at
least two cellular events: (i) mutation, chromosome translocation,
or gene amplification of proto-oncogenes (Bishop, Cell 64:235-248,
1991), or (ii) inactivation, loss by mutation, chromosomal loss,
mitotic recombination, or gene conversion of tumor suppressor genes
(Lasko et al., Ann Rev Genet 25:281-314).
[0008] A large number of potential tumor suppressor genes have been
isolated from Drosophila melanogaster, a species of fruit fly.
Watson et al., J. Cell Sci. 18:19-33, 1994. Potential tumor
suppressor genes are identified in this organism by first deleting,
obstructing, or mutating a gene, and then detecting
over-proliferative cell growth of specific tissues in dissected
larvae and pupae. Xu et al., Development 121:1053-1063, 1995. This
organism provides an ideal system for identifying potential tumor
suppressor genes as it reproduces rapidly and its genome is readily
manipulated by persons skilled in the art.
[0009] An example of a putative tumor suppressor gene identified in
Drosophila is the wts gene. Loss or inactivation of both copies of
the wts gene results in the growth of tumors on the legs and wings
of the flies. Bryant et al., Development 1993 Supplement: 239-249,
1993. The large size of these tumors suggests that the cells
undergo more divisions than normal. Id. In addition, the rounded
shape of the tumors suggests that the division of the mutant cells
is not preferentially oriented. Id. These observations taken
together with the increased thickness of the cuticles around the
mutant cells suggest that the wts gene regulates cell adhesion,
cell contact inhibition, and/or cell boundary recognition in
Drosophila.
[0010] Several of the genes characterized as potential tumor
suppressors in Drosophila are cloned. In particular, the wts gene
contains a region that bears sequence similarity to the catalytic
regions of mammalian non-receptor serine/threonine protein kinases.
Watson, BioEssays 17:673-676, 1995. However, the human orthologues
of the drosophila wts gene have not been reported.
SUMMARY OF THE INVENTION
[0011] The invention relates in part to novel human orthologues of
the Drosophila wts gene (hWARTs). The Drosophila wts gene encodes a
non-receptor serine/threonine kinase. The properties of the human
orthologues are described herein. The present invention concerns
polypeptides of hWART, nucleic acids encoding such polypeptides,
cells, tissues and animals containing such nucleic acids,
antibodies to the polypeptides, assays utilizing the polypeptides,
and methods relating to all of the foregoing.
[0012] The term "orthologue" as used herein, refers to a gene that
is more closely related, in terms of nucleic acid sequence, to
another gene than a gene which is a homologue. In the context of
this invention, "homologous" indicates that the nucleotide
sequences of two genes and/or the sequences of the gene products
(e.g., amino acid sequences) have significant similarity, and that
the gene products perform a similar cellular function. Thus, two
homologous genes may have sequences which have 50, 60, 70, 80, 90,
or greater percent nucleotide sequence identity. By "closely
related" in the context of this invention, it is meant nucleic acid
sequences that have greater than 90% identity.
[0013] The hWARTS genes encode proteins that are potential drug
targets for controlling aberrant cell proliferation. Unlike their
Drosophila ortholog, the hWARTS genes may not function as tumor
suppressor genes. While their mRNA is absent from most normal cells
they are abundantly expressed in many types of tumor cells.
However, based on the high degree of sequence identity in the
catalytic and non-catalytic regions between the hWART proteins and
the Drosophila wts, it is likely that the hWART genes are involved
in regulating cell adhesion, cell contact inhibition, and/or cell
boundary recognition, and in regulation of signal transduction
pathways related to cell proliferation.
[0014] Thus, in a first aspect, the invention features an isolated,
enriched, or purified nucleic acid molecule encoding an hWART
polypeptide.
[0015] By "isolated" in reference to nucleic acid it is meant a
polymer of 14, 17, 21 or more nucleotides conjugated to each other,
including DNA or RNA that is isolated from a natural source or that
is synthesized. The isolated nucleic acid of the present invention
is unique in the sense that it is not found in a pure or separated
state in nature. Use of the term "isolated" indicates that a
naturally occurring sequence has been removed from its normal
cellular (i.e., chromosomal) environment. Thus, the sequence may be
in a cell-free solution or placed in a different cellular
environment. The term does not imply that the sequence is the only
nucleotide sequence present, but that it is essentially free (about
90-95% pure at. least) of non-nucleotide material naturally
associated with it and thus is meant to be distinguished from
isolated chromosomes.
[0016] By the use of the term "enriched" in reference to nucleic
acid it is meant that the specific DNA or RNA sequence constitutes
a significantly higher fraction (2-5 fold) of the total DNA or RNA
present in the cells or solution of interest than in normal or
diseased cells or in the cells from which the sequence was taken.
This could be caused by a person by preferential reduction in the
amount of other DNA or RNA present, or by a preferential increase
in the amount of the specific DNA or RNA sequence, or by a
combination of the two. However, it should be noted that "enriched"
does not imply that there are no other DNA or RNA sequences
present, just that the relative amount of the sequence of interest
has been significantly increased.
[0017] The term "significant" here is used to indicate that the
level of increase is useful to the person making such an increase,
and generally means an increase relative to other nucleic acids of
about at least 2 fold, more preferably at least 5 to 10 fold or
even more. The term also does not imply that there is no DNA or RNA
from other sources. The other source DNA may, for example, comprise
DNA from a yeast or bacterial genome, or a cloning vector such as
pUC19. This term distinguishes the sequence from naturally
occurring enrichment events, such as viral infection, or tumor type
growths, in which the level of one mRNA may be naturally increased
relative to other species of mRNA. That is, the term is meant to
cover only those situations in which a person has intervened to
elevate the proportion of the desired nucleic acid.
[0018] It is also advantageous for some purposes that a nucleotide
sequence be in purified form. The term "purified" in reference to
nucleic acid does not require absolute purity (such as a
homogeneous preparation); instead, it represents an indication that
the sequence is relatively purer than in the natural environment
(compared to the natural level this level should be at least 2-5
fold greater, e.g., in terms of mg/ml). Individual clones isolated
from a cDNA library may be purified to electrophoretic homogeneity.
The claimed DNA molecules obtained from these clones can be
obtained directly from total DNA or from total RNA. The cDNA clones
are not naturally occurring, but rather are preferably obtained via
manipulation of a partially purified naturally occurring substance
(messenger RNA). The construction of a cDNA library from mRNA
involves the creation of a synthetic substance (cDNA) and pure
individual cDNA clones can be isolated from the synthetic library
by clonal selection of the cells carrying the cDNA library. Thus,
the process which includes the construction of a cDNA library from
mRNA and isolation of distinct cDNA clones yields an approximately
10.sup.6-fold purification of the native message. Thus,
purification of at least one order of magnitude, preferably two or
three orders, and more preferably four or five orders of magnitude
is expressly contemplated. The term is also chosen to distinguish
clones already in existence which may encode hWARTs but which have
not been isolated from other clones in a library of clones. Thus,
the term covers clones encoding hWART which are isolated from other
non-hWART clones.
[0019] The term "nucleic acid molecule" describes a polymer of
deoxyribonucleotides (DNA) or ribonucleotides (RNA) The nucleic
acid molecule may be isolated from a natural source by cDNA cloning
or subtractive hybridization or synthesized manually. The nucleic
acid molecule may be synthesized manually by the triester synthetic
method or by using an automated DNA synthesizer.
[0020] The term "cDNA cloning" refers to hybridizing a small
nucleic acid molecule, a probe, to genomic cDNA. The probe
hybridizes (binds) to complementary sequences of cDNA.
[0021] The term "complementary" describes two nucleotides that can
form multiple favorable interactions with one another. For example,
adenine is complementary to thymine as they can form two hydrogen
bonds. Similarly, guanine and cytosine are complementary since they
can form three hydrogen bonds. Thus if a nucleic acid sequence
contains the following sequence of bases, thymine, adenine, guanine
and cytosine, a "complement" of this nucleic acid molecule would be
a molecule containing adenine in the place of thymine, thymine in
the place of adenine, cytosine in the place of guanine, and guanine
in the place of cytosine. Because the complement can contain a
nucleic acid sequence that forms optimal interactions with the
parent nucleic acid molecule, such a complement can bind with high
affinity to its parent molecule.
[0022] The term "hybridize" refers to a method of interacting a
nucleic acid sequence with a DNA or RNA molecule in solution or on
a solid support, such as nitrocellulose, nylon or some combination
of these materials. If a nucleic acid sequence binds to the DNA or
RNA molecule with high affinity, it is said to "hybridize" to the
DNA or RNA molecule. The strength of the interaction between the
probing sequence and its target can be assessed by varying the
stringency of the hybridization conditions. Under highly stringent
hybridization conditions only highly complementary nucleic acid
sequences hybridize. Preferably, such conditions prevent
hybridization of nucleic acids having one or two mismatches out of
20 contiguous nucleotides.
[0023] Various low or high stringency hybridization conditions may
be used depending upon the specificity and selectivity desired.
Stringency is controlled by varying salt or denaturant
concentrations. Examples of hybridization conditions are shown in
the examples described herein. High stringent conditions may mean,
conditions that are at least as stringent as the following:
hybridization in 50% formamide, 5.times.SSC, 50 mM
NaH.sub.3PO.sub.4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon
sperm DNA, and 5.times.Denhart solution at 42 C overnight; washing
with 2.times.SSC, 0.1% SDS at 45 C; and washing with 0.2.times.SSC,
0.1% SDS at 45 C. Those skilled in the art will recognize how such
conditions can be varied to vary specificity and selectivity.
[0024] cDNAs are molecules that may be reverse-transcribed from
fragments of message RNA from a genomic source. These fragments
form a cDNA library of nucleic acid molecules. cDNA libraries are
constructed from natural sources such as mammalian blood, semen, or
tissue.
[0025] The term "subtractive hybridization" refers to a method
similar to cDNA cloning except that cDNA prepared from mRNA in
unstimulated cells is added to mRNA in stimulated or different
types of cells. cDNA/mRNA can then be precipitated to enrich the
mRNA specific to the stimulation signal or different cell type.
[0026] The term "hWART nucleic acid molecule" as used herein refers
to a nucleic acid molecule that encodes an hWART polypeptide. hWART
nucleic acid molecules can be identified by hybridization
procedures and cloning procedures as described herein.
[0027] An hWART polypeptide can be encoded by a full-length nucleic
acid sequence or any portion of the full-length nucleic acid
sequence. In preferred embodiments, the isolated nucleic acid
comprises, consists essentially of, or consists of a nucleic acid
sequence set forth in SEQ ID NO:1, or SEQ ID NO:2, a nucleic acid
sequence that hybridizes to the nucleic acid sequence set forth in
SEQ ID NO:1, or SEQ ID NO:2, or a functional derivative (as defined
below) of either of the foregoing. The nucleic acid may be isolated
from a natural source by cDNA cloning or subtractive hybridization;
the natural source may be mammalian (human) blood, semen, or tissue
and the nucleic acid may be synthesized by the triester or other
method or by using an automated DNA synthesizer.
[0028] The term "mammalian" refers to such organisms as mice, rats,
rabbits, goats, more preferably monkeys and apes, and most
preferably humans.
[0029] In other preferred embodiments, the nucleic acid molecule of
the invention comprises a nucleotide sequence that (a) encodes a
polypeptide having the full length amino acid sequence set forth in
SEQ ID NO:3 or SEQ ID NO:4; (b) is the complement of the nucleotide
sequence of (a); (c) hybridizes under highly stringent conditions
to the nucleotide molecule of (a) and encodes a naturally occurring
hWART polypeptide; (d) encodes an hWART polypeptide having the full
length amino acid sequence of the sequence set forth in SEQ ID NO:3
or SEQ ID NO:4, except that it lacks one or more of the following
segments of amino acid residues: 12-45, 55-151, 236-377, 404-520,
555-559, 601-702, 691-998, 1011-1086 of SEQ ID NO:3, or 1-33,
43-139, 342-466, 467-480, 514-518, 974-1648 of SEQ ID NO:4; (e) is
the complement of the nucleotide sequence of (d); (f) encodes a
polypeptide having the amino acid sequence set forth in SEQ ID NO:3
or SEQ ID NO:4 from amino acid residues 12-45, 55-151, 236-377,
404-520, 555-559, 601-702, 691-998, 1011-1086 of SEQ ID NO:3, or
1-33, 43-139, 342-466, 467-480, 514-518, 974-1048 of SEQ ID NO:4;
(g) is the complement of the nucleotide sequence of (f); (h)
encodes a polypeptide having the full length amino acid sequence
set forth in SEQ ID NO:3 or SEQ ID NO:4, except that it lacks one
or more of the domains selected from the group consisting of an
N-terminal domain, a catalytic domain, and a C-terminal domain; or
(i) is the complement of the nucleotide sequence of (h). The
nucleic acid molecule of the invention is isolated, enriched, or
purified from, preferably, a mammal, or most preferably from a
human.
[0030] In yet other preferred embodiments, the nucleic acid is an
isolated conserved or unique region, for example those useful for
the design of hybridization probes to facilitate identification and
cloning of additional polypeptides, or for the design of PCR probes
to facilitate cloning of additional polypeptides.
[0031] By "conserved nucleic acid regions", it is meant regions
present on two or more nucleic acids encoding an hWART polypeptide,
to which a particular nucleic acid sequence can hybridize under
lower stringency conditions. Examples of lower stringency
conditions suitable for screening for nucleic acids encoding hWARTs
polypeptides are provided in Abe, et al. J. Biol. Chem. 19:13361,
1992 (hereby incorporated by reference herein in its entirety,
including any drawings). Preferably, conserved regions differ by no
more than 5 out of 20 contiguous nucleotides.
[0032] By "unique nucleic acid region" it is meant a sequence
present in a full length nucleic acid coding for an hWART
polypeptide that is not present in a sequence coding for any other
known naturally occurring polypeptide. Such regions preferably
comprise 14, 17, 21 or more contiguous nucleotides present in the
full length nucleic acid encoding an hWART polypeptide. In
particular, a unique nucleic acid region is preferably of human
origin.
[0033] In yet another aspect, the invention relates to a nucleic
acid vector comprising a nucleic acid molecule encoding an hWART
polypeptide and a promoter element effective to initiate
transcription in a host cell.
[0034] The term "nucleic acid vector" relates to a single or double
stranded circular nucleic acid molecule that can be transfected or
transformed into cells and replicate independently or within the
host cell genome. A circular double stranded nucleic acid molecule
can be cut and thereby linearized upon treatment with restriction
enzymes. An assortment of vectors, restriction enzymes, and the
knowledge of the nucleotide sequences that the restriction enzymes
operate upon are readily available to those skilled in the art. A
nucleic acid molecule of the invention can be inserted into a
vector by cutting the vector with restriction enzymes and ligating
the two pieces together.
[0035] Many techniques are available to those skilled in the art to
facilitate transformation or transfection of the expression
construct into a prokaryotic or eukaryotic organism. The terms
"transformation" and "transfection" refer to methods of inserting
an expression construct into a cellular organism. These methods
involve a variety of techniques, such as treating the cells with
high concentrations of salt, an electric field, or detergent, to
render the host cell outer membrane or wall permeable to nucleic
acid molecules of interest.
[0036] The term "promoter element" describes a nucleotide sequence
that is incorporated into a vector that, once inside an appropriate
cell, can facilitate transcription factor and/or polymerase binding
and subsequent transcription of portions of the vector DNA into
mRNA. The promoter element precedes the 5' end of the. hWART
nucleic acid molecule such that the latter is transcribed into
mRNA. Host cell machinery then translates mRNA into a
polypeptide.
[0037] Those skilled in the art would recognize that a nucleic acid
vector can contain many other nucleic acid elements besides the
promoter element and the hWART nucleic acid molecule. These other
nucleic acid elements include, but are not limited to, origins of
replication, ribosomal binding sites, nucleic acid sequences
encoding drug resistance enzymes or amino acid metabolic enzymes,
and nucleic acid sequences encoding secretion signals, periplasm or
peroxisome localization signals, or signals useful for polypeptide
purification.
[0038] The invention also features a nucleic acid probe for the
detection of a nucleic acid encoding an hWART polypeptide in a
sample.
[0039] The term "nucleic acid probe" refers to a nucleic molecule
that is complementary to and can bind a nucleic acid sequence
encoding the amino acid sequence substantially similar to that set
forth in SEQ ID NO:3, or SEQ ID NO:4.
[0040] In preferred embodiments, the nucleic acid probe hybridizes
to nucleic acid molecules encoding at least 46 contiguous amino
acids of the sequences set forth in SEQ ID NO:3, SEQ ID NO:4, or a
functional derivative thereof. Various low or high stringency
hybridization conditions may be used depending upon the specificity
and selectivity desired. Under highly stringent hybridization
conditions only highly complementary nucleic acid sequences
hybridize. Preferably, such conditions prevent hybridization of
nucleic acids having 1 or 2 mismatches out of 20 contiguous
nucleotides.
[0041] Methods for using the probes include detecting the presence
or amount of hWART RNA in a sample by contacting the sample with a
nucleic acid probe under conditions such that hybridization occurs
and detecting the presence or amount of the probe bound to hWART
RNA. The nucleic acid duplex formed between the probe and a nucleic
acid sequence coding for an hWART polypeptide may be used in the
identification of the sequence of the nucleic acid detected (for
example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p.
275 Academic Press, San Diego (Kricka, ed., 1992) hereby
incorporated by reference herein in its entirety, including any
drawings). Kits for performing such methods may be constructed to
include a container having disposed therein a nucleic acid
probe.
[0042] The invention also features a nucleic acid molecule as set
forth in SEQ ID NO:1 or SEQ ID NO:2 or fragments thereof,
comprising one or more regions that encode an hWART polypeptide or
an hWART domain polypeptide, where the hWART polypeptide or the
hWART domain polypeptide is fused to a non-WART polypeptide. Such
fused polypeptides include, for example, but are not limited to, a
GST-fusion protein.
[0043] The invention also features recombinant nucleic acid,
preferably in a cell or an organism. The recombinant nucleic acid
may contain a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or a
functional derivative thereof and a vector or a promoter effective
to initiate transcription in a host cell. The recombinant nucleic
acid can alternatively contain a transcriptional initiation region
functional in a cell, a sequence complimentary to an RNA sequence
encoding an hWART polypeptide and a transcriptional termination
region functional in a cell.
[0044] Another aspect of the invention relates to a recombinant
cell or tissue comprising a nucleic acid molecule encoding an hWART
polypeptide. The recombinant cell may comprise a nucleic acid
molecule encoding either an hWART polypeptide; an hWART domain
polypeptide; or an hWART polypeptide or hWART domain polypeptide
fused to a non-WART polypeptide.
[0045] The term "recombinant organism" refers to an organism that
has a new combination of genes or nucleic acid molecules. A new
combination of genes or nucleic acid molecules can be introduced to
an organism using a wide array of nucleic acid manipulation
techniques available to those skilled in the art.
[0046] The term "organism" relates to any living being comprised of
a least one cell. An organism can be as simple as one eukaryotic
cell or as complex as a mammal. Therefore, a recombinant organism
can also be a recombinant cell.
[0047] The recombinant cell can be a eukaryotic or prokaryotic
organism.
[0048] The term "eukaryote" refers to an organism comprised of
cells that contain a nucleus. Eukaryotes are differentiated from
"prokaryotes". which do not have a nucleus and lack other cellular
structures found in eukaryotes, such as mitochondria and
endoplasmic reticulum. Prokaryotes include unicellular organisms,
such as bacteria while eukaryotes are represented by yeast,
invertebrates, and vertebrates.
[0049] The recombinant cell can harbor a nucleic acid vector that
is extragenomic. The term "extragenomic" refers to a nucleic acid
vector which does not insert into the cell genome. Many nucleic
acid vectors are designed with their own origins of replication
allowing them to utilize the recombinant cell replication machinery
to copy and propagate the vector nucleic acid sequence. These
vectors are small enough that they are not likely to harbor nucleic
acid sequences homologous to genomic sequences of the recombinant
cell. Thus these vectors replicate independently of the host genome
and do not recombine with or integrate into the genome.
[0050] A recombinant cell can harbor a portion of a nucleic acid
vector in an intragenomic fashion. The term "intragenomic" defines
a nucleic acid construct that is incorporated within the cell
genome. Multiple nucleic acid vectors available to those skilled in
the art contain nucleic acid sequences that are homologous to
nucleic acid sequences in a particular organism's genomic DNA.
These homologous sequences will result in recombination events that
integrate portions of the vector into the genomic DNA. Those
skilled in the art can control which nucleic acid sequences of the
vector are integrated into the cell genome by flanking the portion
to be incorporated into the genome with homologous sequences in the
vector.
[0051] Another aspect of the invention features an isolated,
enriched, or purified hWART polypeptide.
[0052] By "hWART polypeptide" it is meant an amino acid sequence
substantially similar to the sequence shown in SEQ ID NO:3, SEQ ID
NO:4, or fragments thereof. A sequence that is substantially
similar will preferably have at least 90% identity (more preferably
at least 95% and most preferably 99-100%) to the sequence of SEQ ID
NO:3 or SEQ ID NO:4.
[0053] The hWART polypeptides of the present invention preferably
have a substantially similar biological activity to the proteins
encoded by the full length nucleic acid sequence set forth in SEQ
ID NO:1 or SEQ ID NO:2, or to the proteins with amino acid sequence
set forth in SEQ ID NO:3 or SEQ ID NO:4. By "biological activity"
it is meant an activity of the hWART protein in a cell. The
biological activity of the hWART is related to some of the
activities of the cell which include, but are not limited to, cell
proliferation motogenesis, metastasis, tumor escape, cell adhesion,
transformation, or apoptosis.
[0054] By "identity" is meant a property of sequences that measures
their similarity or relationship. Identity is measured by dividing
the number of identical residues in the two sequences by the total
number of residues and multiplying the product by 100. Thus, two
copies of exactly the same sequence have 100% identity, but
sequences that are less highly conserved and have deletions,
additions, or replacements have a lower degree of identity. Those
skilled in the art will recognize that several computer programs
are available for determining sequence identity.
[0055] By "isolated" in reference to a polypeptide is meant a
polymer of 6, 12, 18 or more amino acids conjugated to each other,
including polypeptides that are isolated from a natural source or
that are synthesized. The isolated polypeptides of the present
invention are unique in the sense that they are not found in a pure
or separated state in nature. Use of the term "isolated" indicates
that a naturally occurring sequence has been removed from its
normal cellular environment. Thus, the sequence may be in a
cell-free solution or placed in a different cellular environment.
The term does not imply that the sequence is the only amino acid
chain present, but that it is essentially free (about 90-95% pure
at least) of material naturally associated with it.
[0056] By the use of the term "enriched" in reference to a
polypeptide it is meant that the specific amino acid sequence
constitutes a significantly higher fraction (2-5 fold) of the total
of amino acids present in the cells or solution of interest than in
normal or diseased cells or in the cells from which the sequence
was taken. This could be caused by a person by preferential
reduction in the amount of other amino acids present, or by a
preferential increase in the amount of the specific amino acid
sequence of interest, or by a combination of the two. However, it
should be noted that "enriched" does not imply that there are no
other amino acid sequences present, just that the relative amount
of the sequence of interest has been significantly increased. The
term significant here is used to indicate that the level of
increase is useful to the person making such an increase, and
generally means an increase relative to other amino acids of about
at least 2 fold, more preferably at least 5 to 10 fold or even
more. The term also does not imply that there is no amino acid from
other sources. The other source amino acid may, for example,
comprise amino acid encoded by a yeast or bacterial genome, or a
cloning vector such as pUC19. The term is meant to cover only those
situations in which a person has intervened to elevate the
proportion of the desired nucleic acid.
[0057] It is also advantageous for some purposes that an amino acid
sequence be in purified form. The term "purified" in reference to a
polypeptide does not require absolute purity (such as a homogeneous
preparation); instead, it represents an indication that the
sequence is relatively purer than in the natural environment
(compared to the natural level this level should be at least 2-5
fold greater, e.g., in terms of mg/mL. Purification of at least one
order of magnitude, preferably two or three orders, and more
preferably four or five orders of magnitude is expressly
contemplated. The substance is preferably free of contamination at
a functionally significant level, for example 90%, 95%, or 99%
pure.
[0058] In another aspect, the invention features an isolated,
enriched, or purified hWART polypeptide fragment.
[0059] By "hWART polypeptide fragment" it is meant an amino acid
sequence that is less than the full-length amino acid sequence. The
full-length amino acid sequences of hWART1 and hWART2 are shown in
SEQ ID NO:3 and SEQ ID NO:4. Examples of fragments include hWART
domains, hWART mutants and hWART-specific epitopes.
[0060] By "hWART domain" it is meant a portion of the hWART
polypeptide having homology to amino acid sequences from one or
more known proteins wherein the sequence predicts some common
function, interaction or activity. Well known examples of domains
are the SH2 (Src Homology 2) domain (Sadowski, et al, Mol. Cell.
Biol. 6:4396, 1986; Pawson and Schlessinger, Curr. Biol. 3:434,
1993), the SH3 domain (Mayer, et al, Nature 332:272, 1988; Pawson
and Schlessinger, Curr. Biol. 3:434, 1993), and pleckstrin (PH)
domain (Ponting, TIBS 21:245, 1996; Haslam, et al, Nature 363:309,
1993), all of which are domains that mediate protein:protein
interaction or protein:lipid interaction, and the kinase catalytic
domain (Hanks and Hunter, FASEB J 9:576-595, 1995). Computer
programs designed to detect such homologies are well known in the
art. The relative homology is at least 20%, more preferably at
least 30% and most preferably at least 35%.
[0061] By "hWART mutant" it is meant an hWART polypeptide which
differs from the native sequence in that one or more amino acids
have been changed, added or deleted. Changes in amino acids may be
conservative or non-conservative. By "conservative" it is meant the
substitution of an amino acid for one with similar properties such
as charge, hydrophobicity, structure, etc. Examples of polypeptides
encompassed by this term include, but are not limited to, (1)
chimeric proteins which comprise a portion of an hWART polypeptide
sequence fused to a non-hWART polypeptide sequence, for example, a
polypeptide sequence of hemmaglutinin (HA), (2) hWART proteins
lacking a specific domain, for example the catalytic domain, and
(3) hWART proteins having a point mutation. An hWART mutant will
retain some useful function such as, for example, binding to a
natural binding partner, catalytic activity, or the ability to bind
to an hWART specific antibody (as defined below).
[0062] By "hWART-specific epitope" it is meant a sequence of amino
acids that is both antigenic and unique to an hWART polypeptide. An
hWART-specific epitope can be used to produce hWART-specific
antibodies, as more fully described herein. Particularly preferred
epitopes are shown in the Examples section below.
[0063] By "recombinant hWART polypeptide" it is meant to include a
polypeptide produced by recombinant DNA techniques such that it is
distinct from a naturally occurring polypeptide either in its
location (e.g., present in a different cell or tissue than found in
nature), purity or structure. Generally, such a recombinant
polypeptide will be present in a cell in an amount different from
that normally observed in nature.
[0064] The polypeptide of the invention comprises an amino acid
sequence having (a) the full length amino acid sequence set forth
in SEQ ID NO:3 or SEQ ID NO:4; (b) the full length amino acid
sequence of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4,
except that it lacks one or more of the following segments of amino
acid residues: 12-45, 55-151, 236-377, 404-520, 555-559, 601-702,
691-998, 1011-1086 of SEQ ID NO:3, or 1-33, 43-139, 342-466,
467-480, 514-518, 974-1048 of SEQ ID NO:4; (c) the amino acid
sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 from amino acid
residues 12-45, 55-151, 236-377, 404-520, 555-559, 601-702,
691-998, 1011-1086 of SEQ ID NO:3, or 1-33, 43-139, 342-466,
467-480, 514-518, 974-1048 of SEQ ID NO:4; or (d) the full length
amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except
that it lacks one or more of the domains selected from the group
consisting of an N-terminal domain, a catalytic domain, and a
C-terminal domain.
[0065] In yet another aspect, the invention features an antibody
(e.g., a monoclonal or polyclonal antibody), or antibody fragment,
having specific binding affinity to an hWART polypeptide or hWART
polypeptide fragment.
[0066] By "specific binding affinity" is meant that the antibody
binds to target (hWART) polypeptides with greater affinity than it
binds to other polypeptides under specified conditions. Antibodies
having specific binding affinity to an hWART polypeptide may be
used in methods for detecting the presence and/or amount of an
hWART polypeptide in a sample by contacting the sample with the
antibody under conditions such that an immunocomplex forms and
detecting the presence and/or amount of the antibody conjugated to
the hWART polypeptide. Diagnostic kits for performing such methods
may be constructed to include a first container containing the
antibody and a second container having a conjugate of a binding
partner of the antibody and a label, such as, for example, a
radioisotope. The diagnostic kit may also include notification of
an FDA approved use and instructions therefor.
[0067] The term "polyclonal" refers to antibodies that are
heterogenous populations of antibody molecules derived from the
sera of animals immunized with an antigen or an antigenic
functional derivative thereof. For the production of polyclonal
antibodies, various host animals may be immunized by injection with
the antigen. Various adjuvants may be used to increase the
immunological response, depending on the host species.
[0068] "Monoclonal antibodies" are substantially homogenous
populations of antibodies to a particular antigen. They may be
obtained by any technique which provides for the production of
antibody molecules by continuous cell lines in culture. Monoclonal
antibodies may be obtained by methods known to those skilled in the
art. See, for example, Kohler, et al., Nature 256:495-497 (1975),
and U.S. Pat. No. 4,376,110.
[0069] The term "antibody fragment" refers to a portion of an
antibody, often the hypervariable region and portions of the
surrounding heavy and light chains, that displays specific binding
affinity for a particular molecule. A hypervariable region is a
portion of an antibody that physically binds to the polypeptide
target.
[0070] In another aspect, the invention features a hybridoma which
produces an antibody having specific binding affinity to an hWART
polypeptide. By "hybridoma" is meant an immortalized cell line
which is capable of secreting an antibody, for example an hWART
antibody. In preferred embodiments the hWART antibody comprises a
sequence of amino acids that is able to specifically bind an hWART
polypeptide.
[0071] The invention features a method for identifying human cells
containing an hWART polypeptide, or a related sequence. The method
involves identifying the novel polypeptide in human cells using
techniques that are routine and standard in the art, such as those
described herein for identifying hWART polypeptides (e.g., cloning,
Southern or Northern blot analysis, in situ hybridization, PCR
amplification, etc.).
[0072] The invention also features methods of screening cells for
natural binding partners of hWART polypeptides. By "natural binding
partner" it is meant a protein that interacts with an hWART
polypeptide. Binding partners include ligands, agonists,
antagonists and downstream signaling molecules such as adaptor
proteins and may be identified by techniques well known in the art
such as co-immunoprecipitation or by using, for example, a
two-hybrid screen. (Fields and Song, U.S. Pat. No. 5,283,173,
issued Feb. 1, 1994 and, incorporated be reference herein.) The
present invention also features the purified, isolated or enriched
versions of the polypeptides identified by the methods described
above.
[0073] In another aspect, the invention provides a method for
identifying a substance capable of modulating hWART activity
comprising the steps of (a) contacting an hWART polypeptide with a
test substance; and (b) determining whether the substance alters
the activity of said polypeptide.
[0074] The invention also features another method of identifying
substances capable of modulating the function of an hWART
polypeptide. The method comprises the following steps: (a)
expressing an hWART polypeptide in cells; (b) adding a compound to
the cells; and (c) monitoring a change or an absence of a change in
cell phenotype, cell proliferation, catalytic activity of the hWART
polypeptide, and binding a natural binding partner.
[0075] The term "compound" includes small organic molecules
including, but not limited to, oxindolinones, quinazolines,
tyrphostins, quinoxalines, and those contained within extracts from
natural sources. Examples of such compounds are included in section
XIII, below.
[0076] The term "function" refers to the cellular role of a
serine-threonine protein kinase. The serine-threonine protein
kinase family includes members that regulate many steps in
signaling cascades, including cascades controlling cell growth,
migration, differentiation, gene expression, muscle contraction,
glucose metabolism, cellular protein synthesis, and regulation of
the cell cycle.
[0077] The term "modulates" refers to the ability of a compound to
alter the function of a protein kinase. A modulator preferably
activates the catalytic activity of a protein kinase, more
preferably activates or inhibits the catalytic activity of a
protein kinase depending on the concentration of the compound
exposed to the protein kinase, or most preferably inhibits the
catalytic activity of a protein kinase.
[0078] The term "catalytic activity", in the context of the
invention, defines the ability of a protein kinase to phosphorylate
a substrate. Catalytic activity can be measured, for example, by
determining the amount of a substrate converted to a product as a
function of time. Phosphorylation of a substrate occurs at the
active-site of a protein kinase. The active-site is normally a
cavity in which the substrate binds to the protein kinase and is
phosphorylated.
[0079] The term "substrate" as used herein refers to a molecule
that is phoshorylated by or directly interacts with the protein
kinase. The substrate is preferably a peptide and more preferably a
protein. In relation to the protein kinase RAF, preferred
substrates are MEK and the MEK substrate MAPK.
[0080] The term "activates" refers to increasing the cellular
function of a protein kinase. The protein kinase function is
preferably the interaction with a natural binding partner or
catalytic activity.
[0081] The term "inhibit" refers to decreasing the cellular
function of a protein kinase. The protein kinase function is
preferably the interaction with a natural binding partner or
catalytic activity.
[0082] The term "modulates" also refers to altering the function of
a protein kinase by increasing or decreasing the probability that a
complex forms between a protein kinase and a natural binding
partner. A modulator preferably increases the probability that such
a complex forms between the protein kinase and the natural binding
partner, more preferably increases or decreases the probability
that, a complex forms between the protein kinase and the natural
binding partner depending on the concentration of the compound
exposed to the protein kinase, and most preferably decreases the
probability that a complex forms between the protein kinase and the
natural binding partner.
[0083] The term "complex" refers to an assembly of at least two
molecules bound to one another. Signal transduction complexes often
contain at least two protein molecules bound to one another, either
transiently or in succession. For instance, a receptor protein
tyrosine kinase, GRB2, SOS, and RAF sequentially interact in
response to a mitogenic ligand.
[0084] The term "expressing" as used herein refers to the
production of an hWART polypeptide from a nucleic acid vector
containing an hWART gene within a cell. The nucleic acid vector is
transfected into cells using well known techniques in the art as
described herein.
[0085] The term "adding" as used herein refers to administering a
solution comprising a compound to the medium bathing cells. The
solution comprising the compound can also comprise an agent, such
as dimethyl sulfoxide, which facilitates the uptake of the compound
into the cells.
[0086] The term "monitoring refers to observing the effect of
adding the compound to the cells of the method. The effect can be
manifested in a change in cell phenotype, cell proliferation,
protein kinase catalytic activity, or in the interaction between a
protein kinase and a natural binding partner.
[0087] The term "cell phenotype" refers to the outward appearance
of a cell or tissue or the function of the cell or tissue. Examples
of cell or tissue phenotype are cell size (reduction or
enlargement), cell proliferation (increased or decreased numbers of
cells), cell differentiation (a change or absence of a change in
cell shape), cell survival, apoptosis (cell death), or the
utilization of a metabolic nutrient (e.g., glucose uptake). Changes
or the absence of changes in cell phenotype are readily measured by
techniques known in the art.
[0088] The term "cell proliferation" refers to the rate at which a
group of cells divides. The number of cells growing in a vessel can
be quantitated by a person skilled in the art when that person
visually counts the number of cells in a defined area using a comon
light microscope. Alternatively, cell proliferation rates can be
quantitated by laboratory apparatae that optically measure the
density of cells in an appropriate medium.
[0089] The method can utilize any of the molecules disclosed in the
invention. These molecules include nucleic acid molecules encoding
hWART polypeptides, nucleic acid vectors, recombinant cells,
polypeptides, or antibodies of the invention.
[0090] Substances identified as modulators of hWART activity can be
used to study the effects of hWART modulation in animal models of
cell proliferative disorders. For example, inhibitors of hWART
activity can be tested as treatments for cell proliferative
disorders such as. leukemia or lymphoma using subcutaneous
xenograph models in mice.
[0091] In a preferred embodiment, the invention provides a method
for treating or preventing an abnormal condition by administering a
compound which is a modulator of hWART function in vitro. The
abnormal condition preferably involves abnormality in hWART signal
transduction pathway, and most preferably is cancer. Such compounds
preferably show positive results in one or more in vitro assays for
an activity corresponding to treatment of the disease or disorder
in question (such as the assays described in example 9 below).
Examples of substances that can be screened for favorable activity
are provided in section XIII below.
[0092] The summary of the invention described above is non-limiting
and other features and advantages of the invention will be apparent
from the following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0093] FIG. 1 is a sequence alignment of the hWART1 and hWART2
amino acid sequences.
[0094] FIG. 2 is a sequence alignment of the hWART1 and Drosophila
WART amino acid sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0095] The present invention is based in part upon the isolation
and characterization of nucleic acid molecules encoding novel hWART
proteins. The invention also relates to nucleic acid molecules
encoding portions of hWART polypeptides, nucleic acid molecules
encoding at least one hWART functional portion, nucleic acid
vectors harboring such nucleic acid molecules, recombinant cells
containing such nucleic acid vectors, purified polypeptides encoded
by such nucleic acid molecules, antibodies to such polypeptides,
and methods of identifying compounds that modulate the function of
hWART polypeptides. Also disclosed are methods for diagnosing
abnormal cell proliferative conditions in an organism.
I. The Nucleic Acids of the Invention.
[0096] A. hWART1 Nucleic Acid
[0097] The full-length human Wart1 cDNA is 7,382 bp long and
consists of a 3,390 bp open reading frame (ORF) flanked by 394 and
3,554 bp of 5' and 3' untranslated regions (UTR) respectively. A 41
nucleotide polyA-rich tail follows the 3' UTR. There are two
potential start codons at positions 395 and 431, neither
corresponding to the Kozak consensus for initiating methionines.
Although the second start site aligns to the N-terminal sequence of
the related WART2, we have designated position 395 as the start
site since it is the first start site encountered in this extended
ORF. There are two additional ATGs located 5' to the start codon at
position 395, but they are followed by stop codons after 31 and 10
nucleotides, respectively. The 3,390 nucleotide ORF has the
potential to encode a 1,130 amino acid protein.
[0098] The 5' untranslated region from nucleotide 12-63 displays 10
copies of the tri-nucleotide repeat, GGC. This repeat is very
similar to one found in the human retinoid X receptor beta
(BG:M84820). Such repeats have been reported to undergo expansion
in various human diseases particularly those associated with
neuronal phenotypes. The 3' untranslated region contains an
inverted 289 bp Alu-J subfamily repeat (between nucleotides
6,058-6,346). A polyadenylation signal (AATAAA) is found at
position 7,338 followed by a 20 nucleotide long polyadenylated
stretch.
[0099] Sequence analysis of multiple cDNA clones identified three
polymorphisms in the human Wart1 gene: (1) at nucleotide 978
resulting in an Ala/Gly change; (2) at nucleotide 1,840, silent;
(3) at nucleotides 3,252-3,253 comprising a deletion of two
adenosines that results in a C-terminal truncation of the Wart1_h
gene, disrupting the putative kinase domain. The frame shift
mutation at position 3252 was observed in two independent clones
isolated from the human bone marrow cDNA source. The non-mutated
sequence however, was also confirmed in multiple independent
clones. Conceivably, truncation of the WART1 STK could play a role
in disease progression.
[0100] At least 8 EST fragments match the WART1_h gene over its 3'
untranslated region and only one (GB:Z16134) was found to span part
of the coding region of this gene (nucleotides 2,138-3,977).
[0101] B. hWART2 Nucleic Acid
[0102] The full-length human Wart2 cDNA is 5,276 bp long and
consists of a 3,264 bp open reading frame (ORF) flanked by 394 and
1,612 bp of 5' and 3' untranslated regions (UTR) respectively. A 23
nucleotide polyA-rich tail follows the 3' UTR. This ORF has the
potential to encode a 1,088 amino acid polypeptide. Based on amino
acid sequence homology to the Drosophila and human Wart1 proteins
we beleive that this ORF encodes the human Wart2 protein. There are
5 additional ORF's, none longer than 144 nucleotides, 5' to
nucleotide 375. The ATG at position 375 fits the Kozak consensus
for translational initiation.
[0103] Fourteen EST and one STS fragment match the Wart2_h cDNA
sequence. These ESTs cluster into 5 contigs and match the Wart2_h
coding region at the following positions: N56660 at 712, R75698 at
3,077, H26525 at 3,109, R01798 at 2,751, AA30618 at 163. The latter
is a TIGR EST whose 3' end matches position 5,276 at the 3' end of
the Wart2 gene.
II. The Proteins of the Invention.
[0104] The open reading frame (ORF) of the full-length hWART1
nucleic acid molecule is predicted to encode a protein of 1130
amino acids with a predicted molecular weight of approximately 127
kDa. The ORF of the full-length hWART2 nucleic acid molecule is
predicted to encode a protein of 1088 amino acids with a molecular
weight of approximately 120 kDa. Structural analysis of these
protein sequences predicts that hWART1 and hWART2 are likely to be
intracellular proteins.
[0105] A. hWART1 Protein
[0106] Analysis of the deduced amino acid sequence predicts hWART1
to be an intracellular protein, lacking both a signal sequence and
transmembrane domain. The predicted amino acid sequence contains a
long N-terminal region that is believed to be predominately alpha
helical and hydrophilic followed by a C-terminal domain with all
the motifs characteristic of a serine-threonine kinase. Several
regions of homology exist between the hWART1, hWART2 and Drosophila
homologue. A Smith-Waterman pairwise alignment of hWART1 and hWART2
is shown in FIG. 1, and a similar alignment between hWART1 and
Drosophila WART (SEQ ID NO:13) is shown in FIG. 2. The description
and boundaries of these motifs are described in the following
paragraphs.
[0107] The extreme N-terminal region of the hWART1 protein
extending from 12-45 amino acids, shares 66% identity and 78%
similarity to the corresponding region in hWART2. This domain is
referred to as "BOX A". Box B lies ten amino acids C-terminal to
Box A and extends from amino acids 55-151. Box B shares 56%
identity and 77% similarity to the corresponding region in hWART2.
Drosophila WART protein lacks significant homology to the
N-terminal Box A and B present in the two human proteins. A
Smith-Waterman search of the nonredundant protein database with the
amino acid sequences of Box A and Box B does not reveal any
significant homologies that might suggest a potential function for
these two conserved regions.
[0108] hWART1 contains a proline-rich region, consisting of 26%
prolines, extending from amino acids 236-377. This region is
distantly similar to Volvox extensin proteins (40% amino acid
identity with Volvox cateri extensin GB:x65165 using Smith-Waterman
alignment) and may represent a protein interaction domain as well
as a possible site for interaction with proteins containing SH3
motifs. WART homologues from Drosophila melanogaster (PIR:A56155)
and Caenorhabditis elegans (EMBL:Z8159) have an N-terminal
proline-rich comparable to the one found in hWART1, but this region
is lacking in hWART2. Box C extends from amino acids 404-520 and is
44% identical and 73% similar to hWART2. A small portion of Box C
is also found in a similar position in D. melanogaster WART but is
mostly replaced with a glutamine-rich region. A Smith-Waterman
search of the nonredundant protein database with the amino acid
sequence of Box C did not reveal significant homologies that would
suggest a potential function for this region. The 5' amino acid
motif P.sub.4Y is present between amino acids 555-559 of hWART1 and
is conserved in hWART2 and Drosophila WART. This region may
represent an SH3 or WW domain binding site or may be a site for
tyrosine phosphorylation and SH2 interactions.
[0109] A distinguishing feature of the WART family is the extended
homology flanking both the N- and C-terminal side of their
predicted serine-threonine kinase domain. This extended homology is
present in the human and mouse WART1 and WART2, D. melanogaster
WART, and C. elegans WART. The N-terminal flanking region of the
hWART1 catalytic domain extends from amino acids 601-702 of hWART1
and is 69%, 71%, and 45% identical and 85%, 85%, and 64% similar
with hWART2, D. melanogaster WART, and C. elegans WART,
respectively. The catalytic. domain of WART1 (amino acids 691-998)
is 85%, 75%, and 53% identical.90%, 87%, and 72% similar with
hWART2, D. melanogaster WART, C. elegans WART, respectively. The
region C-terminal to the catalytic domain, extending from amino
acids 1011-1086 in hWART1 is 63%, 53%, and 40% identical and 76%,
73%, and 56% similar with hWART2, D. melanogaster WART, and C.
elegans WART, respectively.
[0110] The extended homology on either side of the catalytic domain
of the WART kinases suggests these regions may actually be a part
of this enzymatic domain. Other Serine-threonine kinases including
Calmodulin-dependent kinases and DUN1 kinases from S. cerevesiae,
are also characterized by an extended kinase domain.
[0111] B. hWART2 Protein
[0112] The 5276 bp human WART2 sequence is predicted to encode a
polypeptide of 1,088 amino acids (SEQ ID NO:4). Analysis of the
deduced amino acid sequence predicts hWART2 to be an intracellular
protein, lacking both a signal sequence and transmembrane domain.
Like hWART1, it contains a long N-terminal region that is
predominately alpha helical and hydrophilic followed by a
C-terminal domain with all the motifs characteristic of a
serine-threonine kinase. Several regions of homology exist between
hWART1, hWART2 and the Drosophila homologue (FIGS. 1 and 2). Box A
extends from amino acids 1-33 and is 66% identical and 78% similar
to the corresponding region in hWART1. Box B lies 21 amino acids
C-terminal to Box A from amino acids 43-139. The hWART2 Box B is
56% identical and 77% similar to the corresponding region in
hWART1. Box C extends from amino acids 342-466 and is 44% identical
and 73% similar to hWART1. A GC nucleotide repeat region encodes
alternating prolines and alanines (PAPA Box) from amino acids
467-480. This motif is also present in the human Cdk-inhibitor
p57KIP2 (GB:U22398), and in the myosin light chain protein from
several species. A recent study examined the human p57KIP2 for
genetic variations in a large number of tumors (Tokino et al.,
"Characterization of the human p57. (KIP2) gene: alternative
splicing, insertion/deletion polymorphisms in VNTR sequences in the
coding region, and mutational analysis." Hum. Genet. 97:625-631,
1996). This study identified 4 types of 12-bp deletions in the
proline/alanine rich region of p57KIP2, GC repeat region of hWART2
may also be subject to variations in size, possibly resulting in
altered gene function. The P.sub.4Y motif lies at amino acids
514-518 in hWART2 and is also found in a similar location in hWART1
and D. melanogaster WART.
[0113] The region immediately N-terminal to the core of the WART2
kinase domain extends from amino acid 564-665 and is 69%, 65%, and
41% identical and 85%, 82%, and 62% similar with human WART1, D.
melanogaster WART, and C. elegans WART, respectively. The catalytic
domain of WART2 (amino acids 666-973) is 85%, 75%, and 53%
identical and 90%, 86%, and 70% similar with hWART1, D.
melanogaster WART, and C. elegans WART, respectively. The region
C-terminal to the catalytic domain extends from amino acids
974-1048 in WART2 is 63%, 50%, and 36% identical and 76%, 72%, and
60% similar with hWART1, D. melanogaster WART, and C. elegans
WART1, respectively.
III. Applications, Biological Significance, and Clinical Utility of
hWARTs
[0114] Experimental studies of the WART homologues from lower
organisms suggest hWART1 may play a role in the regulation of
normal epithelial cell growth. Therefore, compounds that
specifically modulate the function of these proteins would likely
alter the growth or biology of epithelial tumors and would provide
novel potential treatments for human cancer.
IV. A Nucleic Acid Probe for the Detection of hWARTs
[0115] A nucleic acid probe of the present invention may be used to
probe an appropriate chromosomal or cDNA library by usual
hybridization methods to obtain another nucleic acid molecule of
the present invention. A chromosomal DNA or cDNA library may be
prepared from appropriate cells according to recognized methods in
the art (e.g. "Molecular Cloning: A Laboratory Manual", second
edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring
Harbor Laboratory, 1989).
[0116] In the alternative, chemical synthesis is carried out in
order to obtain nucleic acid probes having nucleotide sequences
which correspond to N-terminal and C-terminal portions of the amino
acid sequence of the polypeptide of interest. Thus, the synthesized
nucleic acid probes may be used as primers in a polymerase chain
reaction (PCR). carried out in accordance with recognized PCR
techniques, essentially according to PCR Protocols, "PCR Protocols,
A. Guide to Methods and Applications", edited by Innis et al.,
Academic Press, 1990, utilizing the appropriate chromosomal or cDNA
library to obtain the fragment of the present invention.
[0117] One skilled in the art can readily design such probes based
on the sequence disclosed herein using methods of computer
alignment and sequence analysis known in the art (e.g. "Molecular
Cloning: A Laboratory Manual", second edition, edited by Sambrook,
Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989). The
hybridization probes of the present invention can be labeled by
standard labeling techniques such as with a radiolabel, enzyme
label, fluorescent label, biotin-avidin label, chemiluminescence,
and the like. After hybridization, the probes may be visualized
using known methods.
[0118] The nucleic acid probes of the present invention include RNA
as well as DNA probes and nucleic acids modified in the sugar,
phosphate or even the base portion as long as the probe still
retains the ability to specifically hybridize under conditions as
disclosed herein. Such probes are generated using techniques known
in the art. The nucleic acid probe may be immobilized on a solid
support. Examples of such solid supports include, but are not
limited to, plastics such as polycarbonate, complex carbohydrates
such as agarose and sepharose, acrylic resins, such as
polyacrylamide and latex beads, and nitrocellulose. Techniques for
coupling nucleic acid probes to such solid supports are well known
in the art.
[0119] The test samples suitable for nucleic acid probing methods
of the present invention include, for example, cells or nucleic
acid extracts of cells, or biological fluids. The sample used in
the above-described methods will vary based on the assay format,
the detection method and the nature of the tissues, cells or
extracts to be assayed. Methods for preparing nucleic acid extracts
of cells are well known in the art and can be readily adapted in
order to obtain a sample which is compatible with the method.
utilized.
V. A Probe Based Method And Kit For Detecting hWART
[0120] One method of detecting the presence of hWART in a sample
comprises (a) contacting the sample with one of the above-described
nucleic acid probes, under conditions such that hybridization
occurs, and (b) detecting the presence of the probe bound to a
nucleic acid molecule in the sample. One skilled in the art would
select the nucleic acid probe according to techniques known in the
art as described above. Samples to be tested include but should not
be limited to RNA samples of human tissue.
[0121] A kit for detecting the presence of hWART in a sample
comprises at least one container having disposed therein an
above-described nucleic acid probe. The kit may further comprise
other containers comprising one or more of the following: wash
reagents and reagents capable of detecting the presence of bound
nucleic acid probe. Examples of detection reagents include, but are
not limited to radiolabelled probes, enzymatically labeled probes
(horseradish peroxidase, alkaline phosphatase), and affinity
labeled probes (biotin, avidin, or steptavidin).
[0122] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allow the efficient transfer of
reagents from one compartment to another compartment such that the
samples and reagents are not cross-contaminated and the agents or
solutions of each container can be added in a quantitative fashion
from one compartment to another. Such containers will include a
container which will accept the test sample, a container which
contains the probe or primers used in the assay, containers which
contain wash reagents (such as phosphate buffered saline,
Tris-buffers, and the like), and containers which contain the
reagents used to detect the hybridized probe, bound antibody,
amplified product, or the like. One skilled in the art will readily
recognize that the nucleic acid probes described in the present
invention can readily be incorporated into one of the established
kit formats which are well known in the art.
VI. DNA Constructs Comprising an hWART Nucleic Acid Molecule and
Cells Containing These Constructs
[0123] The present invention also relates to a recombinant DNA
molecule comprising, 5' to 3', a promoter effective to initiate
transcription in a host cell and one of the above-described nucleic
acid molecules. In addition, the present invention relates to a
recombinant DNA molecule comprising a vector and a nucleic acid
molecule described herein. The present invention also relates to a
nucleic acid molecule comprising a transcriptional region
functional in a cell, a sequence complimentary to an RNA sequence
encoding an amino acid sequence corresponding to an hWART
polypeptide, or functional derivative, and a transcriptional
termination region functional in said cell. The above-described
molecules may be isolated and/or purified DNA molecules.
[0124] The present invention also relates to a cell or organism
that contains an hWART nucleic acid molecule, as described herein,
and thereby is capable of expressing a peptide. The polypeptide may
be purified from cells which have been altered to express the
polypeptide. A cell is said to be "altered to express a desired
polypeptide" when the cell, through genetic manipulation, is made
to produce a protein which it normally does not produce or which
the cell normally produces at lower levels. One skilled in the art
can readily adapt procedures for introducing and expressing either
genomic, cDNA, or synthetic sequences into either eukaryotic or
prokaryotic cells.
[0125] A nucleic acid molecule, such as DNA, is said to be "capable
of expressing" a polypeptide if it contains nucleotide sequences
which contain transcriptional and translational regulatory
information and such sequences are is "operably linked" to
nucleotide sequences which encode the polypeptide. An operable
linkage is a linkage in which the regulatory DNA sequences and the
DNA sequence sought to be expressed are connected in such a way as
to permit gene sequence expression. The precise nature of the
regulatory regions needed for gene sequence expression may vary
from organism to organism, but will in general include a promoter
region which, in prokaryotes, contains both the promoter (which
directs the initiation of RNA transcription) as well as the DNA
sequences which, when transcribed into RNA, will signal synthesis
initiation. Such regions will normally include those 5'-non-coding
sequences involved with initiation of transcription and
translation, such as the TATA box, capping sequence, CAAT sequence,
and the like.
[0126] If desired, the non-coding region 3' to the sequence
encoding an hWART gene may be obtained by the above-described
cloning methods. This region may be retained for its
transcriptional termination regulatory sequences, such as
termination and polyadenylation. Thus, by retaining the 3'-region
naturally contiguous to the DNA sequence encoding an hWART gene,
the transcriptional termination signals may be provided. Where the
transcriptional termination signals are not satisfactorily
functional in the expression host cell, then a 3' region functional
in the host cell may be substituted.
[0127] Two DNA sequences (such as a promoter region sequence and an
hWART sequence) are said to be operably linked if the nature of the
linkage between the two DNA sequences does not (1) result in the
introduction of a frame-shift mutation, (2) interfere with the
ability of the promoter region sequence to direct the transcription
of the second sequence, for example an hWART gene sequence, or (3)
interfere with the ability of the second sequence to be transcribed
by the promoter region sequence. Thus, a promoter region would be
operably linked to a DNA sequence if the promoter were capable of
effecting transcription of that DNA sequence. Thus, transcriptional
and translational signals recognized by an appropriate host are
necessary to express an hWART gene.
[0128] The present invention encompasses the expression of an hWART
gene (or a functional derivative thereof) in either prokaryotic or
eukaryotic cells. Prokaryotic hosts are, generally, very efficient
and convenient for the production of recombinant proteins and are,
therefore, one type. of preferred expression system for these
genes. Prokaryotes most frequently are represented by various
strains of E. coli. However, other microbial strains may also be
used, including other bacterial strains.
[0129] In prokaryotic systems, plasmid vectors that contain
replication sites and control sequences derived from a species
compatible with the host may be used. Examples of suitable plasmid
vectors may include pBR322, pUC118, pUC119 and the like; suitable
phage or bacteriophage vectors may include gt10, gt11 and the like;
and suitable virus vectors may include pMAM-neo, pKRC and the like.
Preferably, the selected vector of the present invention has the
capacity to replicate in the selected host cell.
[0130] Recognized prokaryotic hosts include bacteria such as E.
coli and those from genera such as Bacillus, Streptomyces,
Pseudomonas, Salmonella, Serratia, and the like. However, under
such conditions, the polypeptide will not be glycosylated. The
prokaryotic host must be compatible with the replicon and control
sequences in the expression plasmid.
[0131] To express hWART (or a functional derivative thereof) in a
prokaryotic cell, it is necessary to operably link the gene
sequence to a functional prokaryotic promoter. Such promoters may
be either constitutive or, more preferably, regulatable (i.e.,
inducible or derepressible). Examples of constitutive promoters
include the int promoter of bacteriophage 1, the bla promoter of
the -lactamase gene sequence of pBR322, and the CAT promoter of the
chloramphenicol acetyl transferase gene sequence of pPR325, and the
like. Examples of inducible prokaryotic promoters include the major
right and left promoters of bacteriophage 1 (P.sub.L and P.sub.R),
the trp, recA, lacZ, lacI, and gal promoters of E. coli, the
a-amylase (Ulmanen, et at., J. Bacteriol. 162:176-182, 1985) and
the sigma-28-specific promoters of B. subtilis (Gilman, et al.,
Gene sequence 32:11-20, 1984), the promoters of the bacteriophages
of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli,
Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward,
et at., Mol. Gen. Genet. 203:468-478, 1986). Prokaryotic promoters
are reviewed by Glick, J. Ind. Microbiot. 1:277-282, 1987;
Cenatiempo, Biochimie 68:505-516, 1986; and Gottesman, Ann. Rev.
Genet. 18:415-442, 1984.
[0132] Proper expression in a prokaryotic cell also requires the
presence of a ribosome binding site upstream of the gene
sequence-encoding sequence. Such ribosome binding sites are
disclosed (see, for example, Gold, et at., Ann. Rev. Microbiol.
35:365-404, 1981). The selection of control sequences, expression
vectors, transformation methods, and the like, are dependent on the
type of host cell used to express the gene.
[0133] As used herein, "cell", "cell line", and "cell culture" may
be used interchangeably and all such designations include the
progeny of the cells. Thus, the words "transformants" or
"transformed cells" include the primary subject cell and cultures
derived therefrom, without regard to the number of transfers. It is
also understood that all progeny may not be precisely identical in
DNA content, due to deliberate or inadvertent mutations. However,
as defined, mutant progeny have the same functionality as that of
the originally transformed cell.
[0134] Host cells which may be used in the expression systems of
the present invention are not strictly limited, provided that they
are suitable for use in the expression of the hWART peptide of
interest. Suitable hosts may often include eukaryotic cells.
Preferred eukaryotic hosts include, for example, yeast, fungi,
insect cells, and mammalian cells, either in vivo or in tissue
culture. Mammalian cells which may be useful as hosts include HeLa
cells, cells of fibroblast origin such as VERO, 3T3 or CHO-K1, or
cells of lymphoid origin (such as 32D cells) and their derivatives.
Preferred mammalian host cells include SP2/0 and J558L, as well as
neuroblastoma cell lines such as IMR 332 and PC12 which may provide
better capacities for correct post-translational processing.
[0135] In addition, plant cells are also available as hosts, and
control sequences compatible with plant cells are available, such
as the cauliflower mosaic virus 35S and 19S, and nopaline synthase
promoter and polyadenylation signal sequences. Another preferred
host is an insect cell, for example the Drosophila larvae. Using
insect cells as hosts, the Drosophila alcohol dehydrogenase
promoter can be used (Rubin, Science 240:1453-1459, 1988).
Alternatively, baculovirus vectors can be engineered to express
large amounts of hWART in insects cells (Jasny, Science 238:1653,
1987; Miller, et al., In: Genetic Engineering, 1986; Setlow, J. K.,
et al., eds., Plenum, Vol. 8, pp. 277-297).
[0136] Any of a series of yeast gene sequence expression systems
can be utilized which incorporate promoter and termination elements
from the actively expressed gene sequences coding for glycolytic
enzymes; the systems are produced in large quantities when yeast
are grown in mediums rich in glucose. Known glycolytic gene
sequences can also provide very efficient transcriptional control
signals. Yeast provides substantial advantages in that it can also
carry out post-translational peptide modifications. A number of
recombinant DNA strategies exist which utilize strong promoter
sequences and high copy number of plasmids which can be utilized
for production of the desired proteins in yeast. Yeast recognizes
leader sequences on cloned mammalian gene sequence products and
secretes peptides bearing leader sequences (i.e., pre-peptides).
For a mammalian host, several possible vector systems are available
for the expression of hWART.
[0137] A particularly preferred yeast expression system is that
utilizing Schizosaccharmocyces pombe. This system is useful for
studying the activity of members of the Src family (Superti-Furga,
et al., EMBO J. 12:2625, 1993) and other non-receptor-TKs, the
function of which is often regulated by the activity of tyrosine
phosphatases.
[0138] A wide variety of transcriptional and translational
regulatory sequences may be employed, depending upon the nature of
the host. The transcriptional and translational regulatory signals
may be derived from viral sources, such as adenovirus, bovine
papilloma virus, cytomegalovirus, simian virus, or the like, where
the regulatory signals are associated with a particular gene
sequence which has a high level of expression. Alternatively,
promoters from mammalian expression products, such as actin,
collagen, myosin, and the like, may be employed. Transcriptional
initiation regulatory signals may be selected which allow for
repression or activation, so that expression of the gene sequences
can be modulated. Of interest are regulatory signals which are
temperature-sensitive so that by varying the temperature,
expression can be repressed or initiated, or are subject to
chemical (such as metabolite) regulation.
[0139] Expression of hWART in eukaryotic hosts requires the use of
eukaryotic regulatory regions. Such regions will, in general,
include a promoter region sufficient to direct the initiation of
RNA synthesis. Preferred eukaryotic promoters include, for example,
the promoter of the mouse metallothionein I gene sequence (Hamer,
et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of
Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early
promoter (Benoist, et al., Nature (London) 290:304-310, 1981); and
the yeast gal4 gene sequence promoter (Johnston, et al., Proc.
Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver, et al., Proc.
Natl. Acad. Sci. (USA) 81:5951-5955, 1984).
[0140] Translation of eukaryotic mRNA is initiated at the codon
which encodes the first methionine. For this reason, it is
preferable to ensure that the linkage between a eukaryotic promoter
and a DNA sequence which encodes hWART (or a functional derivative
thereof) does not contain any intervening codons which are capable
of encoding a methionine (i.e., AUG). The presence of such codons
results either in the formation of a fusion protein (if the AUG
codon is in the same reading frame as the coding sequence) or a
frame-shift mutation (if the AUG codon is not in the same reading
frame as an hWART coding sequence).
[0141] An hWART nucleic acid molecule and an operably linked
promoter may be introduced into a recipient prokaryotic or
eukaryotic cell either as a nonreplicating DNA (or RNA) molecule,
which may either be a linear molecule or, more preferably, a closed
covalent circular molecule (a plasmid). Since such molecules are
incapable of autonomous replication, the expression of the gene may
occur through the transient expression of the introduced sequence.
Alternatively, permanent or stable expression may occur through the
integration of the introduced DNA sequence into the host
chromosome.
[0142] A vector may be employed which is capable of integrating the
desired gene sequences into the host cell chromosome. Cells which
have stably integrated the introduced DNA into their chromosomes
can be selected by also introducing one or more markers which allow
for selection of host cells which contain the expression vector.
The marker may provide for prototrophy to an auxotrophic host,
biocide resistance, e.g., antibiotics, or heavy metals, such as
copper, or the like. The selectable marker gene sequence can either
be directly linked to the DNA gene sequences to be expressed, or
introduced into the same cell by co-transfection. Additional
elements may also be needed for optimal synthesis of single chain
binding protein mRNA. These elements may include splice signals, as
well as transcription promoters, enhancers, and termination
signals. cDNA expression vectors incorporating such elements
include those described by Okayama, Mol. Cell. Bio. 3:280,
1983.
[0143] The introduced nucleic acid molecule can be incorporated
into a plasmid or viral vector capable of autonomous replication in
the recipient host. Any of a wide variety of vectors may be
employed for this purpose. Factors of importance in selecting a
particular plasmid or viral vector include: the ease with which
recipient cells that contain the vector may be recognized and
selected from those recipient cells which do not contain the
vector; the number of copies of the vector which are desired in a
particular host; and whether it is desirable to be able to
"shuttle" the vector between host cells of different species.
[0144] Preferred prokaryotic vectors include plasmids such as those
capable of replication in E. coil (such as, for example, pBR322,
ColE1, pSC101, pACYC 184, pVX). Such plasmids are, for example,
disclosed by Sambrook (c.f. "Molecular Cloning: A Laboratory
Manual", second edition, edited by Sambrook, Fritsch, &
Maniatis, Cold Spring Harbor Laboratory, 1989). Bacillus plasmids
include pC194, pC221, pT127, and the like. Such plasmids are
disclosed by Gryczan (In: The Molecular Biology of the Bacilli,
Academic Press, NY, 1982, pp. 307-329). Suitable Streptomyces
plasmids include p1J101 (Kendall, et al., J. Bacteriol.
169:4177-4183, 1987), and streptomyces bacteriophages such as fC31
(Chater, et al., In: Sixth International Symposium on
Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary, 1986,
pp. 45-54). Pseudomonas plasmids are reviewed by John, et al., Rev.
Infect. Dis. 8:693-704, 1986, and Izaki, Jpn. J. Bacteriol.
33:729-742, 1978.
[0145] Preferred eukaryotic plasmids include, for example, BPV,
vaccinia, SV40, 2-micron circle, and the like, or their
derivatives. Such plasmids are well known in the art (Botstein, et
al., Miami Wntr. Symp. 19:265-274, 1982); Broach, In: "The
Molecular Biology of the Yeast Saccharomyces: Life Cycle and
Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., p. 445-470 1981; Broach, Cell 28:203-204, 1982; Bollon et
at., J. Clin. Hematol.,Oncol. 10:39-48, 1980; Maniatis, In: Cell
Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence
Expression, Academic Press, NY, pp. 563-608, 1980.
[0146] Once the vector or nucleic acid molecule containing the
construct(s) has been prepared for expression, the DNA construct(s)
may be introduced into an appropriate host cell by any of a variety
of suitable means, i.e., transformation, transfection, conjugation,
protoplast fusion, electroporation, particle gun technology,
calcium phosphate-precipitation, direct microinjection, and the
like. After the introduction of the vector, recipient cells are
grown in a selective medium, which selects for the growth of
vector-containing cells. Expression of the cloned gene molecule (s)
results in the production of hWART or fragments or functional
derivatives thereof. This can take place in the transformed cells
as such, or following the induction of these cells to differentiate
(for example, by administration of bromodeoxyuracil to
neuroblastbma cells or the like). A variety of incubation
conditions for the transformed cells can be used to foster
expression of the polypeptides of the present invention. The most
preferred conditions are those which mimic physiological
conditions.
VII. An Antibody Having Binding Affinity to an hWART Polypeptide
and Hybridomas Producing The Antibody
[0147] The present invention also relates to an antibody having
specific binding affinity to an hWART polypeptide. The polypeptide
may have the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID
NO:4, or a fragment thereof, or at least 41 contiguous amino acids
thereof. Such an antibody may be identified by comparing its
binding affinity to the desired polypeptide, for example an hWART
polypeptide, with its binding affinity to another (nbn-hWART)
polypeptide. Those which bind selectively to the desired
polypeptide would be chosen for use in methods requiring a
distinction between the desired polypeptide and other polypeptides.
Such methods could include, but should not be limited to, the
analysis of altered expression of the desired polypeptide in tissue
containing other polypeptides and assay systems using whole
cells.
[0148] An hWART polypeptide of the present invention can be used to
produce antibodies or hybridomas. One skilled in the art will
recognize that if an antibody is desired, such a peptide would be
generated as described herein and used as an immunogen. The
antibodies of the present invention include monoclonal and
polyclonal antibodies, as well fragments of these antibodies, and
humanized forms. Humanized forms of the antibodies of the present
invention may be generated using one of the procedures known in the
art such as chimerization or CDR grafting. The present invention
also relates to a hybridoma which produces the above-described
monoclonal antibody, or binding fragment thereof. A hybridoma is an
immortalized cell line which is capable of secreting a specific
monoclonal antibody.
[0149] In general, techniques for preparing monoclonal antibodies
and hybridomas are well known in the art (Campbell, "Monoclonal
Antibody Technology: Laboratory Techniques in Biochemistry and
Molecular Biology," Elsevier Science Publishers, Amsterdam, The
Netherlands, 1984; St. Groth et al., J. Immunol. Methods 35:1-21,
1980). Any, animal (mouse, rabbit, and the like) which is known to
produce antibodies can be immunized with the selected polypeptide.
Methods for immunization are well known in the art. Such methods
include subcutaneous or intraperitoneal injection of the
polypeptide. One skilled in the art will recognize that the amount
of polypeptide used for immunization will vary based on the animal
which is immunized, the antigenicity of the polypeptide and the
site of injection.
[0150] The polypeptide may be modified or administered in an
adjuvant in order to increase the peptide antigenicity. Methods of
increasing the antigenicity of a polypeptide are well known in the
art. Such procedures include coupling the antigen with a
heterologous protein (such as globulin or .beta.-galactosidase) or
through the inclusion of an adjuvant during immunization.
[0151] For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, such as SP2/0-Agl4
myeloma cells, and allowed to become monoclonal antibody producing
hybridoma cells. Any one of a number of methods well known in the
art can be used to identify the hybridoma cell which produces an
antibody with the desired characteristics. These include screening
the hybridomas with an ELISA assay, western blot analysis, or
radioimmunoassay (Lutz, et al., Exp. Cell Res. 175:109-124, 1988).
Hybridomas secreting the desired antibodies are cloned and the
class and subclass is determined using procedures known in the art
(Campbell, "Monoclonal Antibody Technology: Laboratory Techniques
in Biochemistry and Molecular Biology", supra, 1984).
[0152] For polyclonal antibodies, antibody containing antisera is
isolated from the immunized animal and is screened for the presence
of antibodies with the desired specificity using one of the
above-described procedures. The above-described antibodies may be
detectably labeled. Antibodies can be detestably labeled through
the use of radioisotopes, affinity labels (such as biotin, avidin,
and the like), enzymatic labels (such as horse radish peroxidase,
alkaline phosphatase, and the like) fluorescent labels (such as
FITC or rhodamine, and the like), paramagnetic atoms, and the like.
Procedures for accomplishing such labeling are well-known in the
art, for example, see (Stemberger, et al., J. Histochem. Cytochem.
18:315, 1970; Bayer, et al., Meth. Enzym. 62:308, 1979; Engval, et
al., Immunot. 109:129, 1972; Goding, J. Immunol. Meth. 13:215,
1976). The labeled antibodies of the present invention can be used
for in vitro, in vivo, and in situ assays to identify cells or
tissues which express a specific peptide.
[0153] The above-described antibodies may also be immobilized on a
solid support. Examples of such solid supports include plastics
such as polycarbonate, complex carbohydrates such as agarose and
sepharose, acrylic resins and such as polyacrylamide and latex
beads. Techniques for coupling antibodies to such solid supports
are well known in the art (Weir et al., "Handbook of Experimental
Immunology" 4th Ed., Blackwell Scientific Publications, Oxford,
England, Chapter 10, 1986; Jacoby, et al., Meth. Enzym. 34,
Academic Press, N.Y., 1974). The immobilized antibodies of the
present invention can be used for in vitro, in vivo, and in situ
assays as well as in immunochromotography.
[0154] Furthermore, one skilled in the art can readily adapt
currently available procedures, as well as the techniques, methods
and kits disclosed above with regard to antibodies, to generate
peptides capable of binding to a specific peptide sequence in order
to generate rationally designed antipeptide peptides, for example
see Hurby et. al., "Application of Synthetic Peptides: Antisense
Peptides", In Synthetic Peptides, A User's Guide, W.H. Freeman, NY,
pp. 289-307, 1992, and Kaspczak, et al., Biochemistry 28:9230-8,
1989.
VIII. An Antibody Based Method And Kit For Detecting hWART
[0155] The present invention encompasses a method of detecting an
hWART polypeptide in a sample comprising incubating a test sample
with one or more of the antibodies of the present invention and
determining whether the antibody binds to the test sample. The
method can include the steps of, for example: (a) contacting the
sample with an above-described antibody, under conditions such that
immunocomplexes form, and (b) detecting the presence of said
antibody bound to the polypeptide. Altered levels, either an
increase or decrease, of hWART in a sample as compared to normal
levels may indicate an abnormality or disorder.
[0156] Conditions for incubating an antibody with a test sample
vary. Incubation conditions depend on the format employed in the
assay, the detection methods employed, and the type and nature of
the antibody used in the assay. One skilled in the art will
recognize that any one of the commonly available immunological
assay formats (such as radioimmunoassays, enzyme-linked
immunosorbent assays, diffusion based Ouchterlony, or rocket
immunofluorescent assays) can readily be adapted to employ the
antibodies of the present invention. Examples of such assays can be
found in Chard, "An Introduction to Radioimmunoassay and Related
Techniques" Elsevier Science Publishers, Amsterdam, The Netherlands
1986; Bullock et al., "Techniques in Immunocytochemistry," Academic
Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985);
Tijssen, "Practice and Theory of Enzyme Immunoassays: Laboratory
Techniques in Biochemistry and Molecular Biology," Elsevier Science
Publishers, Amsterdam, The Netherlands (1985).
[0157] The immunological assay test samples of the present
invention include cells, protein or membrane extracts of cells, or
biological fluids such as blood, serum, plasma, or urine. The test
sample used in the above-described method will vary based on the
assay format, nature of the detection method and the tissues, cells
or extracts used as the sample to be assayed. Methods for preparing
protein extracts or membrane extracts of cells are well known in
the art and can be readily adapted in order to obtain a sample
which is compatible with the system utilized.
[0158] A kit contains all the necessary reagents to carry out the
previously described methods of detection. The kit may comprise:
(i) a first container containing an above-described antibody, and
(ii) a second container containing a conjugate comprising a binding
partner of the antibody and a label. In another preferred
embodiment, the kit further comprises one or more other containers
comprising one or more of the following: wash reagents and reagents
capable of detecting the presence of bound antibodies.
[0159] Examples of detection reagents include, but are not limited
to labeled secondary antibodies, or in the alternative, if the
primary antibody is labeled, the chromophoric, enzymatic, or
antibody binding reagents which are capable of reacting with the
labeled antibody. The compartmentalized kit may be as described
above for nucleic acid probe kits. One skilled in the art will
recognize that the antibodies described in the present invention
can readily be incorporated into one of the established kit formats
which are well known in the art.
IX. Isolation of Natural Binding Partners of hWART
[0160] The present invention also relates to methods of detecting
natural binding partners capable of binding to an hWART
polypeptide. A natural binding partner of hWART may be, for
example, a substrate protein which is dephosphorylated as part of a
signaling cascade. The binding partner(s) may be present within a
complex mixture, for example, serum, body fluids, or cell
extracts.
[0161] In general, methods for identifying natural binding partners
comprise incubating a substance with a first polypeptide, hWART for
the invention described herein, and detecting the presence of a
substance bound to the first polypeptide. Preferred methods include
the two-hybrid system of Fields and Song (supra) and
co-immunoprecipitation wherein the first polypeptide is allowed to
bind to a natural binding partner, then the polypeptide complex is
immunoprecipitated using antibodies specific for the first
polypeptide. The natural binding partner can then be isolated and
identified by techniques well known in the art.
X. Identification of and Uses for Substances Capable of Modulating
hWART Activity
[0162] The present invention also relates to a method of detecting
a substance capable of modulating hWART activity. Such substances
can either enhance activity (agonists) or inhibit activity
(antagonists). Agonists and antagonists can be peptides,
antibodies, products from natural sources such as fungal or plant
extracts or small molecular weight organic compounds. In general,
small molecular weight organic compounds are preferred. Examples of
classes of compounds that can be tested for hWART modulating
activity are, for example but not limited to, thiazoles (see, for
example U.S. application Ser. No. 60/033,522 filed Dec. 19, 1996,
and Ser. No. 08/660,900 filed Jun. 7, 1996), and naphthopyrones
(U.S. Pat. No. 5,602,171, issued Feb. 11, 1997).
[0163] In general the method comprises incubating cells that
produce hWART in the presence of a test substance and detecting
changes in the level of hWART activity or hWART binding partner
activity. A change in activity may be manifested by increased or
decreased binding of an hWART polypeptide to a natural binding
partner or increased or decreased biological response in cells.
Biological responses can include, for example, proliferation,
differentiation, survival, or motility. The substance thus
identified would produce a change in activity indicative of the
agonist or antagonist nature of the substance. Once the substance
is identified it can be isolated using techniques well known in the
art, if not already available in a purified form.
[0164] The present invention also encompasses a method of agonizing
(stimulating) or antagonizing hWART associated activity in a mammal
comprising administering to said mammal an agonist or antagonist to
hWART in an amount sufficient to effect said agonism or antagonism.
Also encompassed in the present application is a method of treating
diseases in a mammal with an agonist or antagonist of hWART-related
activity comprising administering the agonist or antagonist to a
mammal in an amount sufficient to agonize or antagonize hWART
associated function(s). The particular compound can be administered
to a patient either by itself or in a pharmaceutical composition
where it is mixed with suitable carriers or excipient(s). In
treating a patient, a therapeutically effective dose of the
compound is administered. A therapeutically effective dose refers
to that amount of the compound that results in amelioration of
symptoms or a prolongation of survival in a patient.
[0165] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals. Cell culture assays and animal studies can
be used for determining the LD.sub.50 (the dose lethal to 50% of a
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of a population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, which can be
expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit
large therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in
formulating a range of dosages for use in human. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized.
[0166] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays by determining an IC.sub.50 (i.e., the concentration
of the test compound which achieves a half-maximal disruption of
the protein complex, or a half-maximal inhibition of the cellular
level and/or activity of a cellular component, ex. hWART). A dose
can then be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by HPLC. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See e.g. Fingl et al., in "The
Pharmacological Basis of Therapeutics", Ch. 1 p1, 1975).
[0167] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity, or to organ dysfunctions. Conversely, the
attending physician would also know to adjust treatment to higher
levels if the clinical response were not adequate (precluding
toxicity). The magnitude of an administrated dose in the management
of the oncogenic disorder of interest will vary with the severity
of the condition to be treated and to the route of administration.
The severity of the condition may, for example, be evaluated, in
part, by standard prognostic evaluation methods. Further, the dose
and perhaps dose frequency, will also vary according to the age,
body weight, and response of the individual patient. A program
comparable to that discussed above may be used in veterinary
medicine.
[0168] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
"Remington's Pharmaceutical Sciences," 1990, 18th ed., Mack
Publishing Co., Easton, Pa. Suitable routes may include oral,
rectal, transdermal, vaginal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal,
direct intraventricular, intravenous, intraperitoneal, intranasal,
or intraocular injections, just to name a few.
[0169] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0170] Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated. Particular formulations suitable for
parenteral administration of hydrophobic compounds can be found in
U.S. Pat. No. 5,610,173, issued Mar. 11, 1997 and U.S. Provisional
Application Ser. No. 60/039,870, filed Mar. 05, 1997, both of which
are hereby incorporated by reference herein in their entirety,
including any figures and drawings.
[0171] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. Liposomes are
spherical lipid bilayers with aqueous interiors. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external microenvironment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Small organic molecules may be directly
administered intracellularly due to their hydrophobicity.
[0172] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an amount effective to achieve its intended purpose.
Determination of an effective amount is well within the capability
of those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0173] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[0174] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0175] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0176] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable. excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0177] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0178] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
[0179] The present invention also includes a kit containing the the
active ingredients mentioned above. The kit may or may not include
other compounds, such as carriers or excipients, and the active
ingredient may be included in a suitable pharamaceutical
composition. The kit may include a protocol for the use of the
compounds of the invention. Said protocol may be approved by the
Food and Drug Administration or an equivalent agency.
XI. Transgenic Animals
[0180] Also contemplated by the invention are transgenic animals
useful for the study of hWART activity in complex in vivo systems.
A "transgenic animal" is an animal having cells that contain DNA
which has been artificially inserted into a cell, which DNA becomes
part of the genome of the animal which develops from that cell.
Preferred transgenic animals are primates, mice, rats, cows, pigs,
horses, goats, sheep, dogs and cats. The transgenic DNA may encode
a human hWART polypeptide. Native expression in an animal may
alternatively be reduced by providing an amount of antisense RNA or
DNA effective to reduce expression of the target gene.
[0181] A variety of methods are available for the production of
transgenic animals associated with this invention. DNA sequences
encoding hWART can be injected into the pronucleus of a fertilized
egg before fusion of the male and female pronuclei, or injected
into the nucleus of an embryonic cell (e.g., the nucleus of a
two-cell embryo) following the initiation of cell division
(Brinster, et al., Proc. Nat. Acad. Sci. USA 82: 4438, 1985).
Embryos can be infected with viruses, especially retroviruses,
modified to carry inorganic-ion receptor nucleotide sequences of
the invention.
[0182] Pluripotent stem cells derived from the inner cell mass of
the embryo and stabilized in culture can be manipulated in culture
to incorporate nucleotide sequences of the invention. A transgenic
animal can be produced from such cells through implantation into a
blastocyst that is implanted into a foster mother and allowed to
come to term. Animals suitable for transgenic experiments can be
obtained from standard commercial sources such as Charles River
(Wilmington, Mass.), Taconic (Germantown, N.Y.), Harlan Sprague
Dawley (Indianapolis, Ind.), etc.
[0183] The procedures for manipulation of the rodent embryo and for
microinjection of DNA into the pronucleus of the zygote are well
known to those of ordinary skill in the art (Hogan, et al., supra).
Microinjection procedures for fish, amphibian eggs and birds are
detailed in Houdebine and Chourrout, Experientia 47:897-905, 1991.
Other procedures for introduction of DNA into tissues of animals
are described in U.S. Pat. No. 4,945,050 (Sandford et. al., Jul.
30, 1990).
[0184] By way of example only, to prepare a transgenic mouse,
female mice are induced to superovulate. After being allowed to
mate, the females are sacrificed by CO.sub.2 asphyxiation or
cervical dislocation and embryos are recovered from excised
oviducts. Surrounding cumulus cells are removed. Pronuclear embryos
are then washed and stored until the time of injection. Randomly
cycling adult female mice are paired with vasectomized males.
Recipient females are mated at the same time as donor females.
Embryos then are transferred surgically. The procedure for
generating transgenic rats is similar to that of mice. See Hammer,
et al., Cell 63:1099-1112, 1990.
[0185] Methods for the culturing of embryonic stem (ES) cells and
the subsequent production of transgenic animals by the introduction
of DNA into ES cells using methods such as electroporation, calcium
phosphate/DNA precipitation and direct injection also are well
known to those of ordinary skill in the art. (See, for example,
Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E.
J. Robertson, ed., IRL Press, 1987). In cases involving random gene
integration, a clone containing the sequence(s) of the invention is
co-transfected with a gene encoding resistance. Alternatively, a
gene encoding neomycin resistance is physically linked to the
sequence(s) of the invention. Transfection and isolation of desired
clones are carried out by any one of several methods well known to
those of ordinary skill in the art (E. J. Robertson, supra). DNA
molecules introduced into ES cells can also be integrated into the
chromosome through the process of homologous recombination. (See
Capecchi, Science 244: 1288, 1989.) Methods for positive selection
of the recombination event (i.e., neo resistance) and dual
positive-negative selection (i.e., neo resistance and gancyclovir
resistance) and the subsequent identification of the desired clones
by PCR have been described by Capecchi, supra and Joyner et al.,
Nature 338: 153, 1989, the teachings of which are incorporated by
reference herein. The final phase of the procedure is to inject
targeted ES cells into blastocysts and to transfer the blastocysts
into pseudopregnant females. The resulting chimeric animals are
bred and the offspring are analyzed by Southern blotting to
identify individuals that carry the transgene. Procedures for the
production of non-rodent mammals and other animals have been
discussed by others. (See Houdebine and Chourrout, supra; Pursel,
et al., Science 244:1281, 1989; Simms, et al., Bio/Technology
6:179, 1988.)
[0186] Thus, the invention provides transgenic, nonhuman mammals
containing a transgene encoding an hWART polypeptide or a gene
effecting the expression of an hWART polypeptide. Such transgenic
nonhuman mammals are particularly useful as an in vivo test system
for studying the effects of introducing an hWART polypeptide, or
for regulating the expression of an hWART polypeptide (i.e.,
through the introduction of additional genes, antisense nucleic
acids, or ribozymes).
XII. Gene Therapy
[0187] hWART nucleic acid sequences, both mutated and non-mutated,
will also be useful in gene therapy (reviewed in Miller, Nature
357:455-460, 1992). Miller states that advances have resulted in
practical approaches to human gene therapy that have demonstrated
positive initial results. The basic science of gene therapy is
described in Mulligan, Science 260:926, 1993. As used herein "gene
therapy" is a form of gene transfer and is included within the
definition of gene transfer as used herein and specifically refers
to gene transfer to express a therapeutic product from a cell in
vivo or in vitro. Gene transfer can be performed ex vivo on cells
which are then transplanted into a patient, or can be performed by
direct administration of the nucleic acid or nucleic acid-protein
complex into the patient.
[0188] In one preferred embodiment, an expression vector containing
an hWART coding sequence or an hWART mutant coding sequence, as
described above, is inserted into cells, the cells are grown in
vitro and then infused in large numbers into patients. In another
preferred embodiment, a DNA segment containing a promoter of choice
(for example a strong promoter) is transferred into cells
containing an endogenous hWART in such a manner that the promoter
segment enhances expression of the endogenous hWART gene (for
example, the promoter segment is transferred to the cell such that
it becomes directly linked to the endogenous hWART gene).
[0189] The gene therapy may involve the use of an adenovirus
containing hWART cDNA targeted to an appropriate cell type,
systemic hWART increase by implantation of engineered cells
injection with hWART virus, or injection of naked hWART DNA into
appropriate cells or tissues, for example adipose tissue.
[0190] Expression vectors derived from viruses such as
retroviruses, vaccinia virus, adenovirus, adeno-associated virus,
herpes viruses, other RNA viruses, or bovine papilloma virus, may
be used for delivery of nucleotide sequences (eg., cDNA) encoding
recombinant hWART protein into the targeted cell population (e.g.,
tumor cells or fat cells). Methods which are well known to those
skilled in the art can be used to construct recombinant viral
vectors containing coding sequences. See, for example, the
techniques described in Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989), and
in Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and Wiley Interscience, N.Y. (1989).
Alternatively, recombinant nucleic acid molecules encoding protein
sequences can be used as naked DNA or in reconstituted system e.g.,
liposomes or other lipid systems for delivery to target cells (See
e.g., Felgner et al., Nature. 337:387-8, 1989). Several other
methods for the direct transfer of plasmid DNA into cells exist for
use in human gene therapy and involve targeting the DNA to
receptors on cells by complexing the plasmid DNA to proteins. See,
Miller, supra.
[0191] In its simplest form, gene transfer can be performed by
simply injecting minute amounts of DNA into the nucleus of a cell,
through a process of microinjection. (Capecchi M R, Cell 22:479-88,
1980). Once recombinant genes are introduced into a cell, they can
be recognized by the cell's normal mechanisms for transcription and
translation, and a gene product will be expressed. Other methods
have also been attempted for introducing DNA into larger numbers of
cells. These methods include: transfection, wherein DNA is
precipitated with CaPO.sub.4 and taken into cells by pinocytosis
(Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52, 1987);
electroporation, wherein cells are exposed to large voltage pulses
to introduce holes into the membrane (Chu G., et al., Nucleic Acids
Res., 15:1311-26, 1987); lipofection/liposome fusion, wherein DNA
is packaged into lipophilic vesicles which fuse with a target cell
(Felgner P L., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7,
1987); and particle bombardment using DNA bound to small
projectiles (Yang N S., et al., Proc. Natl. Acad. Sci. 87:9568-72,
1990). Another method for introducing DNA into cells is to couple
the DNA to chemically modified proteins.
[0192] It has also been shown that adenovirus proteins are capable
of destabilizing endosomes and enhancing the uptake of DNA into
cells. The admixture of adenovirus to solutions containing DNA
complexes, or the binding of DNA to polylysine covalently attached
to adenovirus using protein crosslinking agents substantially
improves the uptake and expression of the recombinant gene.
(Curiel, et al., Am. J. Respir. Cell. Mol. Biol., 6:247-52,
1992).
[0193] As used herein "gene transfer" means the process of
introducing a foreign nucleic acid molecule into a cell. Gene
transfer is commonly performed to enable the expression of a
particular product encoded by the gene. The product may include a
protein, polypeptide, antisense DNA or RNA, or enzymatically active
RNA. Gene transfer can be performed in cultured cells or by direct
administration into animals. Generally gene transfer involves the
process of nucleic acid contact with a target cell by non-specific
or receptor mediated interactions, uptake of nucleic acid into the
cell through the membrane or by endocytosis, and release of nucleic
acid into the cytoplasm from the plasma membrane or endosome.
Expression may require, in addition, movement of the nucleic acid
into the nucleus of the cell and binding to appropriate nuclear
factors for transcription.
[0194] In another preferred embodiment, a vector having nucleic
acid sequences encoding an hWART is provided in which the nucleic
acid sequence is expressed only in specific tissue. Methods of
achieving tissue-specific gene expression as set forth in
International Publication No. WO 93/09236, filed Nov. 3, 1992. and
published May 13, 1993.
[0195] In all of the preceding vectors set forth above, a further
aspect of the invention is that the nucleic acid sequence contained
in the vector may include additions, deletions or modifications to
some or all of the sequence of the nucleic acid, as defined
above.
[0196] In another preferred embodiment, an hWART nucleic acid is
used in gene replacement. "Gene replacement" as used herein means
supplying a nucleic acid sequence which is capable of being
expressed in vivo in an animal and thereby providing or augmenting
the function of an endogenous gene which is missing or defective in
the animal. Methods of introducing the nucleic acid into the animal
to be treated are as described above.
[0197] One skilled in the art appreciates that any modifications
made to a complex can be manifested in a modification of any of the
molecules in that complex. Thus, the invention includes any
modifications to nucleic acid molecules, polypeptides, antibodies,
or compounds in a complex. All of these aspects and features are
explained in detail with respect to PYK-2 in PCT publication WO
96/18738, which is incorporated herein by reference in its
entirety, including any drawings. Those skilled in the art will
readily appreciate that such descriptions can be easily adapted to
hWART polypeptides and nucleic acid molecules as well, and is
therefore equally applicable to the present invention.
XIII. Compounds that Modulate the Function of hWART Proteins
[0198] In an effort to discover novel treatments for diseases,
biomedical researchers and chemists have designed, synthesized, and
tested molecules that inhibit the function of protein kinases. Some
small organic molecules form a class of compounds that modulate the
function of protein kinases. Examples of molecules that have been
reported to inhibit the function of protein kinases include, but
are not limited to, bis monocyclic, bicyclic or heterocyclic aryl
compounds (PCT WO 92/20642, published Nov. 26, 1992 by Maguire et
al.), vinylene-azaindole derivatives (PCT WO 94/14808, published
Jul. 7, 1994 by Ballinari et al.),
1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992),
styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted
pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline
derivatives (EP Application No. 0 566 266 A1), seleoindoles and
selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny et
al.), tricyclic pblyhydroxylic compounds (PCT WO 92/21660,
published Dec. 10, 1992 by Dow), and benzylphosphonic acid
compounds (PCT WO 91/15495, published Oct. 17, 1991 by Dow et al).
The compounds that can traverse cell membranes and are resistant to
acid hydrolysis are potentially advantageous therapeutics as they
can become highly bioavailable after being administered orally to
patients. However, many of these protein kinase inhibitors only
weakly inhibit the function of protein kinases. In addition, many
inhibit a variety of protein kinases and will therefore cause
multiple side-effects as therapeutics for diseases.
[0199] Some indolinone compounds, however, form classes of acid
resistant and membrane permeable organic molecules. WO 96122976,
published Aug. 1, 1996 by Ballinari et al. describes hydrosoluble
indolinone compounds that harbor tetralin, naphthalene, quinoline,
and indole substituents fused to the oxindole ring. These bicyclic
substituents are in turn substituted with polar moieties including
hydroxylated alkyl, phosphate, and ether moieties. U.S. patent
application Ser. No. 08/702,232, filed Aug. 23, 1996, entitled
"Indolinone Combinatorial Libraries and Related Products and
Methods for the Treatment of Disease" by Tang et al. (Lyon &
Lyon Docket No. 221/187) and U.S. Ser. No. 08/485,323, filed Jun.
7, 1995, entitled "Benzylidene-Z-Indoline Compounds for the
Treatment of Disease" by Tang et al. (Lyon & Lyon Docket No.
223/298) and International Patent Publication WO 96/22976,
published Aug. 1, 1996 by Ballinari et al., all of which are
incorporated herein by reference in their entirety, including any
drawings, describe indolinone chemical libraries of indolinone
compounds harboring other bicyclic moieties as well as monocyclic
moieties fused to the oxindole ring. Applications Ser. No.
08/702,232, filed Aug. 23, 1996, entitled "Indolinone Combinatorial
Libraries and Related Products and Methods for the Treatment of
Disease" by Tang et al. (Lyon & Lyon Docket No. 221/187), Ser.
No. 08/485,323, filed Jun. 7, 1995, entitled
"Benzylidene-Z-Indoline Compounds for the Treatment of Disease" by
Tang et al. (Lyon & Lyon Docket No. 223/298), and WO 96/22976,
published Aug. 1, 1996 by Ballinari et al. teach methods of
indolinone synthesis, methods of testing the biological activity of
indolinone compounds in cells, and inhibition patterns of
indolinone derivatives.
[0200] Other examples of substances capable of modulating hWART
activity include, but are not limited to, tyrphostins,
quinazolines, quinoxolines, and quinolines.
[0201] The quinazolines, tyrphostins, quinolines, and quinoxblines
referred to above include well known compounds such as those
described in the literature. For example, representative
publications describing quinazoline include Barker et al., EPO
Publication No. 0 520 722 A1; Jones et al., U.S. Pat. No.
4,447,608; Kabbe et al., U.S. Pat. No. 4,757,072; Kaul and
Vougioukas, U.S. Pat. No. 5,316,553; Kreighbaum and Comer, U.S.
Pat. No. 4,343,940; Pegg and Wardleworth, EPO Publication No. 0 562
734 A1; Barker et al., Proc. of Am. Assoc. for Cancer Research
32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979);
Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin
et al., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer
Research 43:1117-1123 (1983); Ferris et al. J. Org. Chem.
44(2):173-178; Fry et al., Science 265:1093-1095 (1994); Jackman et
al., Cancer Research 51:5579-5586 (1981); Jones et al. J. Med.
Chem. 29(6):1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362
(1987); Lemus et al., J. Org. Chem. 54:35.11-3518 (1989); Ley and
Seng, Synthesis 1975:415-522 (1975); Maxwell et al., Magnetic
Resonance in Medicine 17:189-196 (1991); Mini et al., Cancer
Research 45:325-330 (1985); Phillips and Castle, J. Heterocyclic
Chem. 17(19):1489-1596 (1980); Reece et al., Cancer Research
47(11):2996-2999 (1977); Sculier et al., Cancer Immunol. and
Immunother. 23:A65 (1986); Sikora et al., Cancer Letters 23:289-295
(1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all
of which are incorporated herein by reference in their entirety,
including any drawings.
[0202] Quinoxaline is described in Kaul and Vougioukas, U.S. Pat.
No. 5,316,553, incorporated herein by reference in its entirety,
including any drawings.
[0203] Quinolines are described in Dolle et al., J. Med. Chem.
37:2627-2629 (1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994);
Burke et al., J. Med. Chem. 36:425-432 (1993); and Burke et al.
BioOrganic Med. Chem. Letters 2:1771-1774 (1992), all of which are
incorporated by reference in their entirety, including any
drawings.
[0204] Tyrphostins are described in Allen et al., Clin. Exp.
Immunol. 91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529
(1993); Baker et al., J. Cell Sci. 102:543-555 (1992); Bilder et
al., Amer. Physiol. Soc. pp. 6363-6143:C721-C730 (1991); Brunton et
al., Proceedings of Amer. Assoc. Cancer Rsch. 33:558 (1992);
Bryckaert et al., Experimental Cell Research 199:255-261 (1992);
Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J.
Immunol. 151(5):2717-2724 (1993); Gazit et al., J. Med. Chem.
32:2344-2352 (1989); Gazit et al.," J. Med. Chem. 36:3556-3564
(1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994); Kaur et
al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al.,
Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J.
6:3275-3282 (1992); Lyall et al., J. Biol. Chem. 264:14503-14509
(1989); Peterson et al., The Prostate 22:335-345 (1993); Pillemer
et al., Int. J. Cancer 50:80-85 (1992); Posner et al., Molecular
Pharmacology 45:673-683 (1993); Rendu et al., Biol. Pharmacology
44(5):881-888 (1992); Sauro and Thomas, Life Sciences 53:371-376
(1993); Sauro and Thomas, J. Pharm. and Experimental Therapeutics
267(3):119-1125 (1993); Wolbring et al., J. Biol. Chem.
269(36):22470-22472 (1994); and Yoneda et al., Cancer Research
51:4430-4435 (1991); all of which are incorporated herein by
reference in their entirety, including any drawings.
[0205] Other compounds that could be used as modulators include
oxindolinones such as those described in U.S. patent application
Ser. No. 08/702,232 filed Aug. 23, 1996, incorporated herein by
reference in its entirety, including any drawings.
EXAMPLES
[0206] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention. The examples below demonstrate the isolation and
characterization of novel human WART nucleic acids and
polypeptides.
Example 1
Cloning of Murine WART1
[0207] Total RNAs were isolated using the Guanidine Salts/Phenol
extraction protocol of Chomczynski and Sacchi (P. Chomczynski and
N. Sacchi, Anal. Biochem. 162:156, 1987) from murine embryos from
gestational day 12. These RNA were used to generate single-stranded
cDNA using the Superscript Preamplification System (GIBCO BRL,
Gaithersburg, Md.; Gerard, G F et al., Focus 11:66, 1989). A
typical reaction used 10 .mu.g total RNA with 1.5 .mu.g
oligo(dT).sub.12-18 in a reaction volume of 60 .mu.L. The product
was treated with RNaseH and diluted to 100 .mu.L with H.sub.2O. For
subsequent PCR amplification, 1-4 .mu.L of the sscDNA was used in
each reaction.
[0208] Degenerate oligonucleotides targeted for the Epidermal
Growth Factor (EGF) family were synthesized on an Applied
Biosystems 3948 DNA synthesizer using established phosphoramidite
chemistry, precipitated with ethanol and used unpurified for PCR.
The sequence of the degenerate oligonucleotide primers used were
the following: TABLE-US-00001 (SEQ ID NO:5) KITDFG = 5' -
CAYGTNAARATHACNGAYTTYGG - 3' and (SEQ ID NO:6) KCWMID = 5' -
GGRTCDATCATCCAGCAYTT - 3'.
These primers were derived from the sense and antisense strands,
respectively of peptide sequences KITDFG (SEQ ID NO:7) and KCWMID
(SEQ ID NO:8). Degenerate nucleotide residue designations are: N=A,
C, G, or T; R=A or G; Y=C or T; H=A, C or T not G; and D=A, G or T
not C.
[0209] PCR reactions were performed using degenerate primers
applied to the murine day 12 embryo single-stranded cDNA. The
primers were added at a final concentration of 5 .mu.M each to a
mixture containing 10 mM TrisHCL (pH 8.3), 50 mM KCL, 1.5 mM
MgCl.sub.2, 200 .mu.M each deoxynucleoside triphosphate, 0.001%
geletin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and
1-4 .mu.L cDNA. Following 3 min denaturation at 95.degree. C., the
cycling conditions were 94.degree. C. for 30 s, 50.degree. C. for 1
min, and 72.degree. C. for 1 min 45 s for 35 cycles. PCR fragments
migrating between 300-350 bp were isolated from 2% agarose gels
using the GeneClean Kit (Bio101), and T-A cloned into the PCRII
vector (Invitrogen Corp. U.S.A.) according to the manufacturer's
protocol.
[0210] Colonies were selected for mini plasmid DNA-preparations
using Qiagen columns and the plasmid DNA was sequenced using cycle
sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS
(ABI, Foster City, Calif.). Sequencing reaction products were run
on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST
alignment algorithm (Altschul, S. F. et al., J. Mol. Biol.
215:403-10). This analysis lead to the isolation of clone 105-4-10
corresponding to murine WART1.
[0211] Clone 105-4-10 exhibits 65% homology with the predicted
amino acid sequence of the Drosophila serine-threonine kinase WART
(Gene Bank (GB): L39847) using MPsrch tnp (Oxford Molecular Group,
UK) a DNA to protein pairwise search implementation of the
Smith-Waterman algorithm. While the 5' primer recognized a sequence
encoding the predicted kinase homology domain, the 3' primer
hybridized to a sequence whose translation was out of frame with
the peptide it had been designed to amplify. Nonetheless, the
intervening sequence contained the expected kinase motifs.
Example 2
cDNA Cloning and Characterization of Human WART1
[0212] A second PCR strategy was designed to isolate the human
orthologue of the novel mouse clone. Degenerate primers based on
clone 105-4-10 were used to amplify templates derived from a pool
of primary human non-small cell lung carcinomas. Total RNAs from
primary human lung tumors were isolated as in Example 1. The
sequence of the degenerate oligonucleotide primers used were as
follows: TABLE-US-00002 (SEQ ID NO:9) 5774 = 5' -
TCCRAACAGDATNACNCCNACNSWCCA - 3' and (SEQ ID NO:10) 5326 = 5' -
TTYGGNYTNTGYACNGGNTTYMGNTGG - 3'.
These primers were derived from the sense and antisense strands,
respectively of peptide sequences FGLCTGFRW (SEQ ID NO:11) and
WSVGVILFE (SEQ ID NO:12) present in the murine WART1 clone. The
amplification conditions were similar to those described in Example
1 using oligonucleotides KITDFG (SEQ ID NO:7 and KCWMID (SEQ ID
NO:8). Two distinct PCR products were isolated, SuSTK15 (268 bp)
and SuSTK17 (273 bp). These two fragments share 72% DNA identity
and 88% amino acid sequence identity to one another. SuSTK15_h has
been designated as hWART1 cDNA because it is more related to the
murine WART1 cDNA (90% DNA identity; 98% amino acid identity), than
SuSTK17 h (74% DNA sequence identity; 83% amino acid identity).
SuSTK17 h has been designated as hWART2.
Example 3
Isolation of hWART1
[0213] A human bone marrow gt11 cDNA library was probed with the
PCR fragments corresponding to human WART1. Probes were
.sup.32P-labeled by random priming and used at 2.times.10.sup.6
cpm/mL following standard techniques known in the art for library
screening. Prehybridization (3 h) and hybridization (overnight)
were conducted at 42.degree. C. in 5.times.SSC, 5.times.Denhart's
solution, 2.5% dextran sulfate, 50 mM Na.sub.2PO.sub.4 [pH 7.0],
50% formamide with 100 mg/mL denatured salmon sperm DNA. Stringent
washes were performed at 65.degree. C. in 0.1.times. SSC and 0.1%
SDS. DNA sequencing was carried out on both strains using a cycle
sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS
(ABI, Foster City, Calif.). Sequencing reaction products were run
on an ABI Prism 377 DNA Sequencer.
[0214] Three cDNAs were isolated and completely sequenced. Two of
the clones were found to be overlapping clones that encoded a long
C-terminal open reading frame (ORF) but lacked an upstream stop
codon. The third clone was found to contain no significant ORFs but
was later found to encode the 3' untranslated region (UTR) of the
human WART1 cDNA. Rescreening of the bone marrow cDNA library
yielded two more cDNA clones which upon sequencing were found to
contain a long ORF contiguous with the two clones isolated from the
previous screening of the bone marrow cDNA library.
[0215] The full-length human WART1 cDNA is 7,382 bp long and
consists of a 3,390 bp ORF. This ORF is flanked by 394 and 3,554 bp
of 5' and 3' untranslated regions (UTR) respectively. A 41
nucleotide polyA-rich tail follows the 3' UTR. There are two
potential start codons at positions 395 and 431, neither
corresponding to the Kozak consensus for initiating methionines.
Although the second start site aligns to the N-terminal sequence of
the related WART2, we have designated position 395 as the start
site since it is the first start site encountered in this extended
ORF. There are two additional ATGs located 5' to the start codon at
position 395, but they are followed by stop codons after 31 and 10
nucleotides, respectively. The 3,390 bp ORF has the potential to
encode a 1,130 amino acid protein.
[0216] The 5' UTR from nucleotide 12-63 displays 10 copies of the
tri-nucleotide repeat, GGC. This repeat is very similar to one
found in the human retinoid X receptor beta (GB:M84820). Such
repeats have been reported to undergo expansion in various human
diseases particulary those associated with neuronal phenotypes. The
3' UTR contains an inverted 289 bp Alu-J subfamily repeat (between
nucleotides 6,058-6,346). A polyadenylation signal (AATAAA) is
found at position 7,338 followed by a 20 nucleotide long
polyadenylated stretch.
[0217] Sequence analysis of multiple cDNA clones identified three
polymorphisms in the human WART1 gene: (1) at nucleotide 978
resulting in an Ala/Gly change; (2) at nucleotide 1,840, silent;
(3) at nucleotides 3,252-3,253 comprising a deletion of two
adenosines that results in a C-terminal truncation of the hWART1
gene, disrupting the putative kinase domain. The frame shift
mutation at position 3252 was observed in two independent clones
isolated from the human bone marrow cDNA source. The non-mutated
sequence, however, was also confirmed in multiple independent
clones. Conceivably, truncation of the WART1 Serine-threonine
kinase could play a role in disease progression.
Example 4
Isolation of cDNA Encoding the hWART2 Gene
[0218] SuSTK17_h was used as a probe to screen a Agt11 human bone
marrow cDNA library. Multiple cDNA clones were isolated and two
(W2D4 and W2D1.8) were sequenced fully on both strands. Clone W2D4
lies 5' of clone W2D1.8 separated by an internal EcoRI site in the
full-length hWART2 cDNA.
[0219] The full-length 5,276 bp hWART2 cDNA consists of a 3,264 bp
ORF flanked by 394 and 1,612 bp of 5' and 3' UTRs, respectively. A
23 nucleotide polyA-rich tail follows the 3' UTR. This ORF has the
potential to encode a 1,088 amino acid polypeptide. Based on amino
acid sequence homology to the Drosophila and human WART1 proteins
we believe that this ORF encodes the hWART2 protein. There are 5
additional ORF's none longer than 144 nucleotides, 5' to nucleotide
375. The ATG at position 375 fits the Kozak consensus for
translational initiation.
Example 5
Distribution of Human WART1 and WART2 mRNA in Normal Tissues and
Tumor Cell Lines
[0220] Northern blots were obtained from Clontech (Palo Alto,
Calif.) containing 2 .mu.g polyA+ RNA from 16 different adult human
tissues (spleen, thymus, prostate, testis, ovary, small intestine,
colonic mucosa, heart, brain, placenta, lung, liver, skeletal
muscle, kidney, pancreas, and peripheral blood leukocytes), and
four different human fetal tissues (brain, lung, liver, and
kidney), on charge-modified nylon membrane. Additional Northern
blots were prepared by running 20 .mu.g total RNA on formaldehyde
1.2% agarose gel and transferring to nylon membranes.
[0221] Filters were hybridized with random prime
[.sup.32P]dCTP-labeled probes synthesized from the 270 bp inserts
from SuSTK15 (hWART1) or SuSTK17 (hWART2). Hybridization was
performed at 60.degree. C. overnight in 6.times.SSC, 0.1% SDS,
1.times.Denhardt's solution, 100 mg/mL denatured herring sperm DNA
with 1-2.times.10.sup.6 cpm/mL of .sup.32P-labeled DNA probes. The
filters were washed in 0.1.times.SSC/0.1% SDS, 65.degree. C., and
exposed overnight on Kodak XAR-2 film.
[0222] hWART1 RNA expression was not detected in 18 normal samples
tested. Similarly hWART2 expression was undetectable in 15 of the
18 samples, but was seen in three hormonally responsive tissues:
uterus, prostate, and testis.
[0223] Expression of hWART1 and hWART2 was next examined in a panel
of human tumor cell lines representing a diverse sampling of tumor
types. hWART1 showed strong expression in cell lines from non-small
cell lung cancer, ovarian tumors, central nervous system tumors,
renal tumors, and breast tumors. hWART2 expression was consistently
expressed, although usually at lower levels than hWART1 in
virtually all samples tested, except for most of the colon cancer
lines. The robust overexpression of hWART1 and hWART2 in tumor
cells versus normal tissues may provide an attractive target for
oncology drug development. The tissue distribution of hWART1 and
hWART2 mRNA is summarized in Table 1. TABLE-US-00003 TABLE 1
Expression of hWART1 and hWART2 in various tissues. hWART1* hWART2*
Cell type Origin expression expression Brain Normal tissue 0 0
Cerebellum Normal tissue 0 0 Thymus Normal tissue 0 0 Salivary
Gland Normal tissue 0 0 Lung Normal tissue 0 0 Heart Normal tissue
0 0 Liver Normal tissue 0 0 Pancreas Normal tissue 0 0 Kidney
Normal tissue 0 0 Stomach Normal tissue 0 0 Duodenum Normal tissue
0 0 Uterus Normal tissue 0 0 Prostate Normal tissue 0 1 Skel.
Muscle Normal tissue 0 0 Placenta Normal tissue 0 0 Fetal Brain
Normal tissue 0 0 Mammary Gland Normal tissue 0 0 Testis Normal
tissue 0 1 HOP-92 Lung tumor 1 1 EKVX Lung tumor 2 1 NCI-H23 Lung
tumor 4 1 NCI-H226 Lung tumor 3 1 NCI-H322M Lung tumor 4 1 NCI-H460
Lung tumor 1 0 NCI-H522 Lung tumor 1 1 A549 Lung tumor 1 0 HOP-62
Lung tumor 1 0 OVCAR-3 Ovarian tumor 0 0 OVCAR-4 Ovarian tumor 1 1
OVCAR-5 Ovarian tumor 1 1 OVCAR-8 Ovarian tumor 1 1 IGROV1 Ovarian
tumor 2 1 SK-OV-3 Ovarian tumor 4 1 SNB-19 CNS tumor 4 0 SNB-75 CNS
tumor 1 1 U251 CNS tumor 2 1 SF-268 CNS tumor 3 3 SF-295 CNS tumor
1 1 SF-539 CNS tumor 3 1 CCRF-CEM Leukemia 3 0 K-562 Leukemia 4 0
MOLT-4 Leukemia 1 0 HL-60 Leukemia 0 0 RPMI 8226 Leukemia 1 0 SR
Leukemia 1 1 DU-145 Prostate 1 1 PC-3 Prostate 1 0 HT-29 Colon
tumor 0 0 HCC-2998 Colon tumor 0 0 HCT-116 Colon tumor 0 0 SW620
Colon tumor 0 0 Colo 205 Colon tumor 0 0 HTC15 Colon tumor 2 1
KM-12 Colon tumor 0 0 UO-31 Colon tumor 0 1 SN12C Kidney tumor 0 3
A498 Kidney tumor 0 0 CaKil Kidney tumor 2 2 RXF-393 Kidney tumor 2
1 ACHN Kidney tumor 0 0 786-0 Kidney tumor 3 0 TK-10 Kidney tumor 3
4 LOX IMVI Melanoma 3 2 Malme-3M Melanoma 0 1 SK-MEL-2 Melanoma 1 1
SK-MEL-5 Melanoma 0 1 SK-MEL-28 Melanoma 1 1 UACC-62 Melanoma 4 1
UACC-257 Melanoma 1 1 M14 Melanoma 1 1 MCF-7 Breast tumor 3 1
MCF-7/ADR RES Breast tumor 1 1 Hs578T Breast tumor 1 1 MDA-MB-231
Breast tumor 0 1 MDA-MB-435 Breast tumor 0 0 MDA-N Breast tumor 0 1
BT-549 Breast tumor 1 1 T47D Breast tumor 4 1 *No expression is
represented by 0 and maximal expression is represented by 4.
Example 6
hWART1 and hWART2 Expression Vector Construction
[0224] Full length expression constructs were generated for hWART1
and hWART2 from fully sequenced cDNA clones. These intact ORFs were
inserted into pcDNAII (Invitrogen) or pRK5 for transient expression
in mammalian cells. The hWART constructs were also tagged, by PCR
mutagenesis, at their carboxy-terminal ends with the Haemophilus
influenza hemaglutinin (HA) epitope YPYDVPDYAS (SEQ ID NO:14) (U.K.
Pati, Gene 114:285-288, 1992).
[0225] An N-terminal myristolated form of both hWART1 and hWART2
were also generated by addition of a 5' amino tag to both proteins
by PCR mediated mutagenesis using techniques known to those skilled
in the art. These altered fragments were inserted into the same
expression vectors. These expression constructs will allow
targeting of the recombinant WART proteins to the membrane,
potentially enhancing or deregulating their biologic effects.
[0226] Dominant negative forms of hWART1 and hWART2 can be
constructed by a lysine to alanine substitution at the ATP-binding
site in their kinase domains.
Example 7
Generation of hWART1- and hWART2-specific Immunoreagents
[0227] hWART1- and hWART2-specific Immunoreagents were raised in
rabbits against KLH-conjugated synthetic peptides specific to the
two proteins. The peptides were conjugated to a cysteine added to
the C-terminal end of each peptide, using techniques known to those
skilled in the art. Amino acid sequences of the peptide immunogens
and their location within the human WART1 and WART2 sequences are:
TABLE-US-00004 hWART1: ISKPSKEDQPSLPK (SEQ ID NO:15) (aa576-589)
N-terminal to kinase domain. DDQNTGSEIKNRDLVYV (SEQ ID NO:16)
(aa1114-1130) C-terminus. hWART2 PsgKNSRDEEKRESRI (SEQ ID NO:17)
(aa579-594) N-terminal to kinase domain. SDLVDQTEGCQPVYV (SEQ ID
NO:18) (1074-1088) C-terminus.
SEQ ID NO:17 has 2 amino acid differences from the hWART2 sequence,
due to only partial sequence information present at the time of its
synthesis. These changes have no apparent effect on the specificity
of the antisera generated using it as an immunogen.
Example 8
Transient Expression of hWART1 and hWART2 Constructs in Mammalian
Cells
[0228] The hWART1 and hWART2 expression plasmids (10 g DNA/100 mm
plate) containing the wild type or HA-tagged hWART1, wild type or
HA-tagged hWART2 or the myristolated forms of hWART1 and hWART2
were introduced into COS and 293 cells with lipofectamine (Gibco
BRL). After 72 hours, the cells were harvested in 0.5 mL
solubilization buffer (20 mM Hepes pH 7.35, 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 1.5 mM MgCl.sub.2, 1 mM EGTA, 2 mM
phenylmethylsulfonyl fluoride, 1 .mu.g/mL aprotinin). Sample
aliquots were resolved by SDS polyacrylamide gel electrophoresis
(PAGE) on 8% acrylamide/0.5% bis-acrylamide gels and
electrophoretically transferred to nitrocellulose. Non-specific
binding was blocked by preincubating blots in Blotto (phosphate
buffered saline containing 5% w/v non-fat dried milk and 0.2% v/v
nonidet P-40 (Sigma)), and recombinant protein was detected using a
murine Mab to the HA decapeptide tag. Alternatively, recombinant
protein can be detected using various hWART1- or hwART2-specific
antisera.
Example 9
Screening Systems for the Identification of Inhibitors of hWART
Activity
[0229] Assays may be performed in vitro or in vivo and are
described in detail herein or can be obtained by modifying existing
assays, such as the growth assay described in patent application
Ser. No. 08/487,088 (Lyon & Lyon Docket No. 212/276), filed
Jun. 7, 1995.; by Tang et al., and entitled "Novel Pharmaceutical
Compounds", or the assays described in patent application Ser. No.
60/005,167 (Lyon & Lyon Docket No. 215/256), filed Oct. 13,
1995 by Seedorf et al., and entitled "Diagnosis and Treatment of
TKA-1 related disorders", all of which are hereby incorporated
herein by reference in their entirety including any drawings.
Another assay which could be modified to use the genes of the
present invention is described in International Application No. WO
94/23039, published Oct. 13, 1994, hereby incorporated herein by
reference in its entirety including any drawings. Other
possibilities include detecting kinase activity in an
autophosphorylation assay or testing for kinase activity on
standard substrates such as histones, myelin basic protein, gamma
tubulin, or centrosomal proteins. Binding partners may be
identified by putting the N-terminal portion of the protein into a
two-hybrid screen or detecting phosphotyrosine of a dual
specificity kinase (Fields and Song, U.S. Pat. No. 5,283,173,
issued Feb. 1, 1994, incorporated by reference herein, including
any drawings).
[0230] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The molecular complexes and the methods, procedures,
treatments, molecules, specific compounds described herein are
presently representative of preferred embodiments are exemplary and
are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention are
defined by the scope of the claims.
[0231] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0232] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0233] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
[0234] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
For example, if X is described as selected from the group
consisting of bromine, chlorine, and iodine, claims for X being
bromine and claims for X being bromine and chlorine are fully
described.
[0235] In view of the degeneracy of the genetic code, other
combinations of nucleic acids also encode the claimed peptides and
proteins of the invention. For example, all four nucleic acid
sequences GCT, GCC, GCA, and GCG encode the amino acid alanine.
Therefore, if for an amino acid there exists an average of three
codons, a polypeptide of 100 amino acids in length will, on
average, be encoded by 3.sup.100, or 5.times.10.sup.47, nucleic
acid sequences. It is understood by those skilled in the art that,
with, Thus, a nucleic acid sequence can be modified to form a
second nucleic acid sequence, encoding the same polypeptide as
encoded by the first second nucleic acid sequences, using routine
procedures and without undue experimentation. Thus, all possible
nucleic acids that encode the claimed peptides and proteins are
also fully described herein, as if all were written out in full
taking into account the codon usage, especially that preferred in
humans.
[0236] Furthermore, changes in the amino acid sequences of
polypeptides, or in the corresponding nucleic acid sequence
encoding such polypeptide, may be designed or selected to take
place in an area of the sequence where the significant activity of
the polypeptide remains unchanged. For example, an amino acid
change may take place within a turn, away from the active site of
the polypeptide. Also changes such as deletions (e.g. removal of a
segment of the polypeptide, or in the corresponding nucleic acid
sequence encoding such polypeptide, which does not affect the
active site) and additions (e.g. addition of more peptides to the
polypeptide sequence without affecting the function of the active
site, such as the formation of GST-fusion proteins, or additions in
the corresponding nucleic acid sequence encoding such polypeptide
without affecting the function of the active site) are also within
the scope of the present invention. Such changes to the
polypeptides can be performed by those with ordinary skill in the
art using routine procedures and without undue experimentation.
Thus, all possible nucleic and/or amino acid sequences that can
readily be determined not to affect a significant activity of the
peptide or protein of the invention are also fully described
herein.
[0237] Other embodiments are within the following claims.
Sequence CWU 1
1
18 1 7382 DNA HUMAN 1 cagcggagtg cggcggcggc gacactgagt ggaaggcaaa
atggcggcgg cggcggcggt 60 ggcctggtgt taaggggaga gccaggtcct
cacgacccct gggacgggcc gcgctggccc 120 gcggcagccc ccccgttcgt
ctccccgctc tgccccacca gggatacttg gggttgctgg 180 gacggactct
ggccgcctca gcgtccgccc tcaggcccgt ggccgctgtc caggagctct 240
gctctcccct ccagagttaa ttatttatat tgtaaagaat tttaacagtc ctggggactt
300 ccttgaagga tcattttcac ttttgctcag aagaaagctc tggatctatc
aaataaagaa 360 gtccttcgtg tgggctacat atatagatgt tttcatgaag
aggagtgaaa agccagaagg 420 atatagacaa atgaggccta agacctttcc
tgccagtaac tatactgtca gtagccggca 480 aatgttacaa gaaattcggg
aatcccttag gaatttatct aaaccatctg atgctgctaa 540 ggctgagcat
aacatgagta aaatgtcaac cgaagatcct cgacaagtca gaaatccacc 600
caaatttggg acgcatcata aagccttgca ggaaattcga aactctctgc ttccatttgc
660 aaatgaaaca aattcttctc ggagtacttc agaagttaat ccacaaatgc
ttcaagactt 720 gcaagctgct ggatttgatg aggatatggt tatacaagct
cttcagaaaa ctaacaacag 780 aagtatagaa gcagcaattg aattcattag
taaaatgagt taccaagatc ctcgacgaga 840 gcagatggct gcagcagctg
ccagacctat taatgccagc atgaaaccag ggaatgtgca 900 gcaatcagtt
aaccgcaaac agagctggaa aggttctaaa gaatccttag ttcctcagag 960
gcatggcccg ccactagcag aaagtgtggc ctatcattct gagagtccca actcacagac
1020 agatgtagga agacctttgt ctggatctgg tatatcagca tttgttcaag
ctcaccctag 1080 caacggacag agagtgaacc ccccaccacc acctcaagta
aggagtgtta ctcctccacc 1140 acctccaaga ggccagactc cccctccaag
aggtacaact ccacctcccc cttcatggga 1200 accaaactct caaacaaagc
gctattctgg aaacatggaa tacgtaatct cccgaatctc 1260 tcctgtccca
cctggggcat ggcaagaggg ctatcctcca ccacctctca acacttcccc 1320
catgaatcct cctaatcaag gacagagagg cattagttct gttcctgttg gcagacaacc
1380 aatcatcatg cagagttcta gcaaatttaa ctttccatca gggagacctg
gaatgcagaa 1440 tggtactgga caaactgatt tcatgataca ccaaaatgtt
gtccctgctg gcactgtgaa 1500 tcggcagcca ccacctccat atcctctgac
agcagctaat ggacaaagcc cttctgcttt 1560 acaaacaggg ggatctgctg
ctccttcgtc atatacaaat ggaagtattc ctcagtctat 1620 gatggtgcca
aacagaaata gtcataacat ggaactatat aacattagtg tacctggact 1680
gcaaacaaat tggcctcagt catcttctgc tccagcccag tcatccccga gcagtgggca
1740 tgaaatccct acatggcaac ctaacatacc agtgaggtca aattctttta
ataacccatt 1800 aggaaataga gcaagtcact ctgctaattc tcagccttct
gctacaacag tcactgcaat 1860 tacaccagct cctattcaac agcctgtgaa
aagtatgcgt gtattaaaac cagagctaca 1920 gactgcttta gcacctacac
acccttcttg gataccacag ccaattcaaa ctgttcaacc 1980 cagtcctttt
cctgagggaa ccgcttcaaa tgtgactgtg atgccacctg ttgctgaagc 2040
tccaaactat caaggaccac caccacccta cccaaaacat ctgctgcacc aaaacccatc
2100 tgttcctcca tacgagtcaa tcagtaagcc tagcaaagag gatcagccaa
gcttgcccaa 2160 ggaagatgag agtgaaaaga gttatgaaaa tgttgatagt
ggggataaag aaaagaaaca 2220 gattacaact tcacctatta ctgttaggaa
aaacaagaaa gatgaagagc gaagggaatc 2280 tcgtattcaa agttattctc
ctcaagcatt taaattcttt atggagcaac atgtagaaaa 2340 tgtactcaaa
tctcatcagc agcgtctaca tcgtaaaaaa caattagaga atgaaatgat 2400
gcgggttgga ttatctcaag atgcccagga tcaaatgaga aagatgcttt gccaaaaaga
2460 atctaattac atccgtctta aaagggctaa aatggacaag tctatgtttg
tgaagataaa 2520 gacactagga ataggagcat ttggtgaagt ctgtctagca
agaaaagtag atactaaggc 2580 tttgtatgca acaaaaactc ttcgaaagaa
agatgttctt cttcgaaatc aagtcgctca 2640 tgttaaggct gagagagata
tcctggctga agctgacaat gaatgggtag ttcgtctata 2700 ttattcattc
caagataagg acaatttata ctttgtaatg gactacattc ctgggggtga 2760
tatgatgagc ctattaatta gaatgggcat ctttccagaa agtctggcac gattctacat
2820 agcagaactt acctgtgcag ttgaaagtgt tcataaaatg ggttttattc
atagagatat 2880 taaacctgat aatattttga ttgatcgtga tggtcatatt
aaattgactg actttggcct 2940 ctgcactggc ttcagatgga cacacgattc
taagtactat cagagtggtg accatccacg 3000 gcaagatagc atggatttca
gtaatgaatg gggggatccc tcaagctgtc gatgtggaga 3060 cagactgaag
ccattagagc ggagagctgc acgccagcac cagcgatgtc tagcacattc 3120
tttggttggg actcccaatt atattgcacc tgaagtgttg ctacgaacag gatacacaca
3180 gttgtgtgat tggtggagtg ttggtgttat tctttttgaa atgttggtgg
gacaacctcc 3240 tttcttggca caaacaccat tagaaacaca aatgaaggtt
atcaactggc aaacatctct 3300 tcacattcca ccacaagcta aactcagtcc
tgaagcttct gatcttatta ttaaactttg 3360 ccgaggaccc gaagatcgct
taggcaagaa tggtgctgat gaaataaaag ctcatccatt 3420 ttttaaaaca
attgacttct ccagtgacct gagacagcag tctgcttcat acattcctaa 3480
aatcacacac ccaacagata catcaaattt tgatcctgtt gatcctgata aattatggag
3540 tgatgataac gaggaagaaa atgtaaatga cactctcaat ggatggtata
aaaatggaaa 3600 gcatcctgaa catgcattct atgaatttac cttccgaagg
ttttttgatg acaatggcta 3660 cccatataat tatccgaagc ctattgaata
tgaatacatt aattcacaag gctcagagca 3720 gcagtcggat gaagatgatc
aaaacacagg ctcagagatt aaaaatcgcg atctagtata 3780 tgtttaacac
actagtaaat aaatgtaatg aggatttgta aaagggcctg aaatgcgagg 3840
tgttttgagg ttctgagagt aaaattatgc aaatatgaca gagctatata tgtgtgctct
3900 gtgtacaata ttttattttc ctaaattatg ggaaatcctt ttaaaatgtt
aatttattcc 3960 agccgtttaa atcagtattt agaaaaaaat tgttataagg
aaagtaaatt atgaactgaa 4020 tattatagtc agttcttggt acttaaagta
cttaaaataa gtagtgcttt gtttaaaagg 4080 agaaacctgg tatctatttg
tatatatgct aaataatttt aaaatacaag agtttttgaa 4140 atttttttga
aagacagttt tagttttatc ttgctttaac caaatatgaa acataccccc 4200
tattttacag agctcttttt tcccctcata accttgtttt tggtagaaaa taagctagag
4260 aaattaagcc atcgtgttgg tgagtgttcc taggctaatg ataatctgta
taattcacat 4320 cctgaaacta aggaatacag ggttgaaaaa atattaatat
gtttgtcaga aggaaaaata 4380 atgcatttat cttccccccc accccccgcc
ccatggaata tttaatctat ttaatcttct 4440 tgcatttatt tctcaagaat
tactggcttt aaaagaagcc aaagcactac tagctttttt 4500 tccatattgg
tatttttgat gctgcttcca attttaaaag ggaacaaagc tgccataaat 4560
cgaaatgttc aatactaaaa gctaaaatat ttctcaccat cctaagcaga taattatttt
4620 aattttcata tacttttcct gtatagtaac tattttgatt atatcatcaa
tgttacctgt 4680 ttcctctttc agaacagtgc tgcatataca gattgttatt
ggcaaaggaa aatctggcta 4740 tctggcaata ttttacctaa gcgcagatta
attggtgaaa aaattaactc ttaagatggc 4800 cattaataat taggaaagtt
tacagagtgg tcttagtaga aaattcaagt cctcctaatt 4860 tatttaaggt
tcaataatgc gttcaacatg cctgttatgt ataacgctta ggttctaagg 4920
aagattaagg tttcatacca aaatacatgt agcttatctt ttaggaaggg gaaaaaggct
4980 ccattttgac catagtaaaa tttgtgttgt gttttatttc cttttcttaa
gctccactga 5040 taagggattg tttttatcaa aagttactat ttgtagattg
gaggcataat tttagtgatt 5100 ttcatacttt tagctttctt cgcataaaag
ctaattgaaa ccgtatatgt agtaaaatta 5160 aaggcagagc tgttgcagtt
gaattggaga gttagggcaa agaacactta ttagcccaca 5220 cttcccacct
ttctacaggt ggtcctttca gagctcagcc tgaaaaccca ctactgtgtt 5280
atcgtgcgtc ttttggggtt agtggttctt ttgagaatct gaaggaagct gtggactctt
5340 cctagaaaaa aaaaccacac atacacatac aatgttgcat gcagtttcaa
gggattttgg 5400 acatattgaa acctatcaca ggctgtaggt tatggacctc
tgtgccatga gaaaattgat 5460 acattaaact aagaactttg tttttaactt
accaatcact actcagcaca tcttatataa 5520 gctgataatt tgtgatggaa
aaggtctgta gcatgtgata taaggtgacc ttatgaatgc 5580 ctctcttgct
ggtacattaa gttgttttaa tatatcattt ggaggggact gaaatgttag 5640
gctcattaca agcttgatac agaaatattt ctgaaggatt tctaatcaga attgtaaaac
5700 aatgtgctat catgaaatcg cagtcttcac ctcatggttc atggaacatt
tggttagtcc 5760 cataaaatcc tatgcaaaac aaagtagttc aagaattttt
aggtgggtag tcacatttat 5820 aaggtattcc tcttactctt tgggcttttt
cagtctgatt tatttaaatt ttcatttagt 5880 tgttttactt ttggactaag
gtgcaataca gtagaagata actttgttac atttatgttg 5940 taggaaaact
aaggtgctgt ctcctccccc ttcccttccc acaaaatctg tattccccct 6000
attgctgaaa tgtaacagac actacaaatt ttgtattctt tttttgtttt ttgttttgag
6060 acagggtctc actctgtcac ccaggctgga gggcagtggc gcttcacagc
tcactgcatc 6120 ctcaaccttg ggggctcacg cagtcctccc gcctcagcct
cccaagtagc tgggcatgcg 6180 ccaccaagcc cagctaattt ttgtatcttt
agtagagatg gggtttcgcc atgttgccca 6240 ggttggtgtg gaattcctgg
gctccagtta tatgcccacc tcagcctccc aaagtgctgg 6300 gattacagac
gtgacccacc gcgcctggcg caaatatgta ttcttttaaa atttcctctg 6360
atactataag ctttttgcat ttatctgaag cagtatacat gcctttggta tcagcaattt
6420 taacagtttg gatatactta tcagctatct tattccaaaa ctacatctac
ttcttccagt 6480 atagaatctg gtgcttcctg accaaaaaga tgagaaaaac
aatgttaaaa atatagatgc 6540 tttccattga aatggagtga aaacattggt
tctatatgtt ttcttttaaa ataattttct 6600 tattaaaaac ttgctgtctt
tattatactt acccttttta tgcatatcaa tagtatttat 6660 aagatgtgtt
ctataattat gtaattgtag atactgttat gcattgtcca gtgacatcat 6720
aaggcaggcc ctactgctgt atcttttcta ccttcttatt tgtaatagaa actatagaat
6780 gtatgactaa aaagtcactt tgagattgac ttttttaaaa agttattacc
ttctgctgtt 6840 gcaaagtgca aaactgtgag tggaattgtt ttattctgac
ttaatgtgtt agaaattaga 6900 gaatacagtg ggaggatttt tagacattgc
tgctgctgtt acccaaggta ttttagataa 6960 aaaattttta ataaacatcc
ctttggtatt taaagtggaa catttagcct gttcatttta 7020 atctaaagca
aaaagtaatt tgggtcaaaa tattggtata tttgtaaagc gccttaatat 7080
atccctttgt ggaaggcact acacagttta cttttatatt gtattgtgta tataagtatt
7140 ttgtattaaa attgaatcag tggcaacatt aaagttttat aaaatcatgc
tttgttagaa 7200 aaagaattac agctttgcaa tataactaat tgtttcgcat
aattctgaat gtaatagata 7260 tgaataatca gcctgtgttt ttaatgaact
tatttgtatt ttcccaatca ttttctctag 7320 tgtaatgttt gctgggataa
taaaaaaaat tcaaatcttt cgaaaaaaaa aaaaaaaaaa 7380 aa 7382 2 5276 DNA
HUMAN 2 gccccgggaa gatggagcag tcgccgtcca cgccaccgcc gccgcccggg
gctcccccgt 60 ccctgcgggg ccagcagcag ctccagccac cagtgcccgg
tctcccggcg cgagaggccc 120 gggagccgcc ggccaggacg cccccgaggg
tgtagaccgc gccccctgga gagagtgata 180 atcttcaaaa tgaagacttt
ggaaaatttt aggttctcta taggaactac aaaaatggaa 240 ggaaagaaca
ttttcaaaag gaaattattt tgaaagtatg tttacaacaa actgatacta 300
ttgacagttt tttttttaaa taataaaaca ctttaagaag attgtattta tggtaaaagg
360 aaactggact aacaatgagg ccaaagactt ttcctgccac gacttattct
ggaaatagcc 420 ggcagcgact gcaagagatt cgtgaggggt taaagcagcc
atccaagtct tcggttcagg 480 ggctacccgc aggaccaaac agtgacactt
ccctggatgc caaagtcctg gggagcaaag 540 atgccaccag gcagcagcag
cagatgagag ccaccccaaa gttcggacct tatcagaaag 600 ccttgaggga
aatcagatat tccttgttgc cttttgctaa tgaatcgggc acctctgcag 660
ctgcagaagt gaaccggcaa atgctgcagg aactggtgaa cgcaggatgc gaccaggaga
720 tggctggccg agctctcaag cagactggca gcaggagcat cgaggccgcc
ctggagtaca 780 tcagcaagat gggctacctg gacccgagga atgagcagat
tgtgcgggtc attaagcaga 840 cctccccagg aaaggggctc atgccaaccc
cagtgacgcg gaggcccagc ttcgaaggaa 900 ccggcgattc gtttgcgtcc
taccaccagc tgagcggtac cccctacgag ggcccaagct 960 tcggcgctga
cggccccacg gcgctggagg agatgccgcg gccgtacgtg gactaccttt 1020
tccccggagt cggcccccac gggcccggcc accagcacca gcacccaccc aagggctacg
1080 gtgccagcgt agaggcagca ggggcacact tcccgctgca gggcgcgcac
tacgggcggc 1140 cgcacctgct ggtgcctggg gaacccctgg gctacggagt
gcagcgcagc ccctccttcc 1200 agagcaagac gccgccggag accgggggtt
acgccagcct gcccacgaag ggccagggag 1260 gaccgccagg cgccggcctc
gctttcccac cccctgccgc cgggctctac gtgccgcacc 1320 cacaccacaa
gcaggccggt cccgcggccc accagctgca tgtgctgggc tcccgcagcc 1380
aggtgttcgc cagcgacagc cccccgcaga gcctgctcac tccctcgcgg aacagcctca
1440 acgtggacct gtatgaattg ggcagcacct ccgtccagca gtggccggct
gccaccctgg 1500 cccgccggga ctccctgcag aagccgggcc tggaggcgcc
gccgcgcgcg cacgtggcct 1560 tccggcctga ctgcccagtg cccagcagga
ccaactcctt caacagccac cagccgcggc 1620 ccggtccgcc tggcaaggcc
gagccctccc tgcccgcccc caacaccgtg acggctgtca 1680 cggccgcgca
catcttgcac ccggtgaaga gcgtgcgtgt gctgaggccg gagccgcaga 1740
cggctgtggg gccctcgcac cccgcctggg tgcccgcgcc tgccccggcc cccgcccccg
1800 cccccgcccc ggctgcggag ggcttggacg ccaaggagga gcatgccctg
gcgctgggcg 1860 gcgcaggcgc cttcccgctg gacgtggagt acggaggccc
agaccggagg tgcccgcctc 1920 cgccctaccc gaagcacctg ctgctgcgca
gcaagtcgga gcagtacgac ctggacagcc 1980 tgtgcgcagg catggagcag
agcctccgtg cgggccccaa cgagcccgag ggcggcgaca 2040 agagccgcaa
aagcgccaag ggggacaaag gcggaaagga taaaaagcag attcagacct 2100
ctcccgttcc cgtccgcaaa aacagcagag acgaagagaa gagagagtca cgcatcaaga
2160 gctactcgcc atacgccttt aagttcttca tggagcagca cgtggagaat
gtcatcaaaa 2220 cctaccagca gaaggttaac cggaggctgc agctggagca
agaaatggcc aaagctggac 2280 tctgtgaagc tgagcaggag cagatgcgga
agatcctcta ccagaaagag tctaattaca 2340 acaggttaaa gagggccaag
atggacaagt ctatgtttgt caagatcaaa accctgggga 2400 tcggtgcctt
tggagaagtg tgccttgctt gtaaggtgga cactcacgcc ctgtacgcca 2460
tgaagaccct aaggaaaaag gatgtcctga accggaatca ggtggcccac gtcaaggccg
2520 agagggacat cctggccgag gcagacaatg agtgggtggt caaactctac
tactccttcc 2580 aagacaaaga cagcctgtac tttgtgatgg actacatccc
tggtggggac atgatgagcc 2640 tgctgatccg gatggaggtc ttccctgagc
acctggcccg gttctacatc gcagagctga 2700 ctttggccat tgagagtgtc
cacaagatgg gcttcatcca ccgagacatc aagcctgata 2760 acattttgat
agatctggat ggtcacatta aactcacaga tttcggcctc tgcactgggt 2820
tcaggtggac tcacaattcc aaatattacc agaaagggag ccatgtcaga caggacagca
2880 tggagcccag cgacctctgg gatgatgtgt ctaactgtcg gtgtggggac
aggctgaaga 2940 ccctagagca gagggcgcgg aagcagcacc agaggtgcct
ggcacattca ctggtgggga 3000 ctccaaacta catcgcaccc gaggtgctcc
tccgcaaagg gtacactcaa ctctgtgact 3060 ggtggagtgt tggagtgatt
ctcttcgaga tgctggtggg gcagccgccc tttttggcac 3120 ctactcccac
agaaacccag ctgaaggtga tcaactggga gaacacgctc cacattccag 3180
cccaggtgaa gctgagccct gaggccaggg acctcatcac caagctgtgc tgctccgcag
3240 accaccgcct ggggcggaat ggggccgatg acctgaaggc ccaccccttc
ttcagcgcca 3300 ttgacttctc cagtgacatc cggaagcagc cagcccccta
cgttcccacc atcagccacc 3360 ccatggacac ctcgaatttc gaccccgtag
atgaagaaag cccttggaac gatgccagcg 3420 aaggtagcac caaggcctgg
gacacactca cctcgcccaa taacaagcat cctgagcacg 3480 cattttacga
attcaccttc cgaaggttct ttgatgacaa tggctacccc tttcgatgcc 3540
caaagccttc aggagcagaa gcttcacagg ctgagagctc agatttagaa agctctgatc
3600 tggtggatca gactgaaggc tgccagcctg tgtacgtgta gatgggggcc
aggcaccccc 3660 accactcgct gcctcccagg tcagggtccc ggagccggtg
ccctcacagg ccaataggga 3720 agccgagggc tgttttgttt taaattagtc
cgtcgattac ttcacttgaa attctgctct 3780 tcaccaagaa aacccaaaca
ggacactttt gaaaacagga ctcagcatcg ctttcaatag 3840 gcttttcagg
accttcactg cattaaaaca atatttttga aaatttagta cagtttagaa 3900
agagcactta ttttgtttat atccattttt tcttactaaa ttatagggat taactttgac
3960 aaatcatgct gctgttattt tctacatttg tattttatcc atagcactta
ttcacattta 4020 ggaaaagaca taaaaactga agaacattga tgagaaatct
ctgtgcaata atgtaaaaaa 4080 aaaaaaagat aacactctgc tcaatgtcac
ggagaccatt ttatccacac aatggttttt 4140 gttttttatt ttttcccatg
tttcaaaatt gtgatataat gatataatgt taaaagctgc 4200 tttttttggc
tttttgcata tctagtataa taggaagtgt gagcaaggtg atgatgtggc 4260
tgtgatttcc gacgtctggt gtgtggagag tactgcatga gcagagttct tctattataa
4320 aattaccata tcttgccatt cacagcaggt cctgtgaata cgtttttact
gagtgtcttt 4380 aaatgaggtg ttctagacag tgtgctgata atgtattgtg
cgggtgacct cttcgctatg 4440 attgtatctc ttactgtttt gttaaagaaa
tgcagatgtg taactgagaa gtgatttgtg 4500 tgtgtgtctt ggttgtgatt
ggattctttg gggggggggg aactgaaaca tttgtcatat 4560 actgaactta
tatacatcaa aagggattaa tacagcgatg ccaaaaagtt taatcacgga 4620
cacacgtccg tttctgtagt ccgtatgctc tttcattctt ggtagagctg gtatgtggaa
4680 tgccatacct ctgaccctac tacttacctt tttactgaca gactgcccac
actgaaagct 4740 tcagtgaatg ttcttagtcc tgttttcttc tgttactgtc
aggaaactga gtgatctaat 4800 ggttctctca cttttttttt gttcttttag
tgtactttgg aagtatcaaa tcttaacttg 4860 gtttaaacaa tacatattcc
taacctttgt aaaaaagcaa agattcttca aaatgacatt 4920 gaaataaaaa
gtaagccata cgtattttct tagaagtata gatgtatgtg cgtgtataca 4980
cacacacaca cacacacaga gataaacaca atattcctta tttcaaatta gtatgattcc
5040 tatttaaagt gatttatatt tgagtaaaaa gttcaattct tttttgcttt
ttaaaaaatc 5100 tgatgcttca taattttcat tatattattc cacatatttt
tccttgaagt tcttagcata 5160 atgtatccat tacttagtat atatctaggc
aacaacactt agaagtttat cagtgtttaa 5220 actaaaaaaa taaagattcc
tgtgtactgg ttcaaaaaaa aaaaaaaaaa aaaaaa 5276 3 1130 PRT HUMAN 3 Met
Lys Arg Ser Glu Lys Pro Glu Gly Tyr Arg Gln Met Arg Pro Lys 1 5 10
15 Thr Phe Pro Ala Ser Asn Tyr Thr Val Ser Ser Arg Gln Met Leu Gln
20 25 30 Glu Ile Arg Glu Ser Leu Arg Asn Leu Ser Lys Pro Ser Asp
Ala Ala 35 40 45 Lys Ala Glu His Asn Met Ser Lys Met Ser Thr Glu
Asp Pro Arg Gln 50 55 60 Val Arg Asn Pro Pro Lys Phe Gly Thr His
His Lys Ala Leu Gln Glu 65 70 75 80 Ile Arg Asn Ser Leu Leu Pro Phe
Ala Asn Glu Thr Asn Ser Ser Arg 85 90 95 Ser Thr Ser Glu Val Asn
Pro Gln Met Leu Gln Asp Leu Gln Ala Ala 100 105 110 Gly Phe Asp Glu
Asp Met Val Ile Gln Ala Leu Gln Lys Thr Asn Asn 115 120 125 Arg Ser
Ile Glu Ala Ala Ile Glu Phe Ile Ser Lys Met Ser Tyr Gln 130 135 140
Asp Pro Arg Arg Glu Gln Met Ala Ala Ala Ala Ala Arg Pro Ile Asn 145
150 155 160 Ala Ser Met Lys Pro Gly Asn Val Gln Gln Ser Val Asn Arg
Lys Gln 165 170 175 Ser Trp Lys Gly Ser Lys Glu Ser Leu Val Pro Gln
Arg His Gly Pro 180 185 190 Pro Leu Ala Glu Ser Val Ala Tyr His Ser
Glu Ser Pro Asn Ser Gln 195 200 205 Thr Asp Val Gly Arg Pro Leu Ser
Gly Ser Gly Ile Ser Ala Phe Val 210 215 220 Gln Ala His Pro Ser Asn
Gly Gln Arg Val Asn Pro Pro Pro Pro Pro 225 230 235 240 Gln Val Arg
Ser Val Thr Pro Pro Pro Pro Pro Arg Gly Gln Thr Pro 245 250 255 Pro
Pro Arg Gly Thr Thr Pro Pro Pro Pro Ser Trp Glu Pro Asn Ser 260 265
270 Gln Thr Lys Arg Tyr Ser Gly Asn Met Glu Tyr Val Ile Ser Arg Ile
275 280 285 Ser Pro Val Pro Pro Gly Ala Trp Gln Glu Gly Tyr Pro Pro
Pro Pro 290 295 300 Leu Asn Thr Ser Pro Met Asn Pro Pro Asn Gln Gly
Gln Arg Gly Ile 305 310 315 320 Ser Ser Val Pro Val Gly Arg Gln Pro
Ile Ile Met Gln Ser Ser Ser 325 330 335 Lys Phe Asn Phe Pro Ser Gly
Arg Pro Gly Met Gln Asn Gly Thr Gly 340 345 350 Gln Thr Asp Phe Met
Ile His Gln Asn Val Val Pro Ala Gly Thr Val 355 360 365 Asn Arg Gln
Pro Pro Pro Pro
Tyr Pro Leu Thr Ala Ala Asn Gly Gln 370 375 380 Ser Pro Ser Ala Leu
Gln Thr Gly Gly Ser Ala Ala Pro Ser Ser Tyr 385 390 395 400 Thr Asn
Gly Ser Ile Pro Gln Ser Met Met Val Pro Asn Arg Asn Ser 405 410 415
His Asn Met Glu Leu Tyr Asn Ile Ser Val Pro Gly Leu Gln Thr Asn 420
425 430 Trp Pro Gln Ser Ser Ser Ala Pro Ala Gln Ser Ser Pro Ser Ser
Gly 435 440 445 His Glu Ile Pro Thr Trp Gln Pro Asn Ile Pro Val Arg
Ser Asn Ser 450 455 460 Phe Asn Asn Pro Leu Gly Asn Arg Ala Ser His
Ser Ala Asn Ser Gln 465 470 475 480 Pro Ser Ala Thr Thr Val Thr Ala
Ile Thr Pro Ala Pro Ile Gln Gln 485 490 495 Pro Val Lys Ser Met Arg
Val Leu Lys Pro Glu Leu Gln Thr Ala Leu 500 505 510 Ala Pro Thr His
Pro Ser Trp Ile Pro Gln Pro Ile Gln Thr Val Gln 515 520 525 Pro Ser
Pro Phe Pro Glu Gly Thr Ala Ser Asn Val Thr Val Met Pro 530 535 540
Pro Val Ala Glu Ala Pro Asn Tyr Gln Gly Pro Pro Pro Pro Tyr Pro 545
550 555 560 Lys His Leu Leu His Gln Asn Pro Ser Val Pro Pro Tyr Glu
Ser Ile 565 570 575 Ser Lys Pro Ser Lys Glu Asp Gln Pro Ser Leu Pro
Lys Glu Asp Glu 580 585 590 Ser Glu Lys Ser Tyr Glu Asn Val Asp Ser
Gly Asp Lys Glu Lys Lys 595 600 605 Gln Ile Thr Thr Ser Pro Ile Thr
Val Arg Lys Asn Lys Lys Asp Glu 610 615 620 Glu Arg Arg Glu Ser Arg
Ile Gln Ser Tyr Ser Pro Gln Ala Phe Lys 625 630 635 640 Phe Phe Met
Glu Gln His Val Glu Asn Val Leu Lys Ser His Gln Gln 645 650 655 Arg
Leu His Arg Lys Lys Gln Leu Glu Asn Glu Met Met Arg Val Gly 660 665
670 Leu Ser Gln Asp Ala Gln Asp Gln Met Arg Lys Met Leu Cys Gln Lys
675 680 685 Glu Ser Asn Tyr Ile Arg Leu Lys Arg Ala Lys Met Asp Lys
Ser Met 690 695 700 Phe Val Lys Ile Lys Thr Leu Gly Ile Gly Ala Phe
Gly Glu Val Cys 705 710 715 720 Leu Ala Arg Lys Val Asp Thr Lys Ala
Leu Tyr Ala Thr Lys Thr Leu 725 730 735 Arg Lys Lys Asp Val Leu Leu
Arg Asn Gln Val Ala His Val Lys Ala 740 745 750 Glu Arg Asp Ile Leu
Ala Glu Ala Asp Asn Glu Trp Val Val Arg Leu 755 760 765 Tyr Tyr Ser
Phe Gln Asp Lys Asp Asn Leu Tyr Phe Val Met Asp Tyr 770 775 780 Ile
Pro Gly Gly Asp Met Met Ser Leu Leu Ile Arg Met Gly Ile Phe 785 790
795 800 Pro Glu Ser Leu Ala Arg Phe Tyr Ile Ala Glu Leu Thr Cys Ala
Val 805 810 815 Glu Ser Val His Lys Met Gly Phe Ile His Arg Asp Ile
Lys Pro Asp 820 825 830 Asn Ile Leu Ile Asp Arg Asp Gly His Ile Lys
Leu Thr Asp Phe Gly 835 840 845 Leu Cys Thr Gly Phe Arg Trp Thr His
Asp Ser Lys Tyr Tyr Gln Ser 850 855 860 Gly Asp His Pro Arg Gln Asp
Ser Met Asp Phe Ser Asn Glu Trp Gly 865 870 875 880 Asp Pro Ser Ser
Cys Arg Cys Gly Asp Arg Leu Lys Pro Leu Glu Arg 885 890 895 Arg Ala
Ala Arg Gln His Gln Arg Cys Leu Ala His Ser Leu Val Gly 900 905 910
Thr Pro Asn Tyr Ile Ala Pro Glu Val Leu Leu Arg Thr Gly Tyr Thr 915
920 925 Gln Leu Cys Asp Trp Trp Ser Val Gly Val Ile Leu Phe Glu Met
Leu 930 935 940 Val Gly Gln Pro Pro Phe Leu Ala Gln Thr Pro Leu Glu
Thr Gln Met 945 950 955 960 Lys Val Ile Asn Trp Gln Thr Ser Leu His
Ile Pro Pro Gln Ala Lys 965 970 975 Leu Ser Pro Glu Ala Ser Asp Leu
Ile Ile Lys Leu Cys Arg Gly Pro 980 985 990 Glu Asp Arg Leu Gly Lys
Asn Gly Ala Asp Glu Ile Lys Ala His Pro 995 1000 1005 Phe Phe Lys
Thr Ile Asp Phe Ser Ser Asp Leu Arg Gln Gln Ser Ala 1010 1015 1020
Ser Tyr Ile Pro Lys Ile Thr His Pro Thr Asp Thr Ser Asn Phe Asp
1025 1030 1035 1040 Pro Val Asp Pro Asp Lys Leu Trp Ser Asp Asp Asn
Glu Glu Glu Asn 1045 1050 1055 Val Asn Asp Thr Leu Asn Gly Trp Tyr
Lys Asn Gly Lys His Pro Glu 1060 1065 1070 His Ala Phe Tyr Glu Phe
Thr Phe Arg Arg Phe Phe Asp Asp Asn Gly 1075 1080 1085 Tyr Pro Tyr
Asn Tyr Pro Lys Pro Ile Glu Tyr Glu Tyr Ile Asn Ser 1090 1095 1100
Gln Gly Ser Glu Gln Gln Ser Asp Glu Asp Asp Gln Asn Thr Gly Ser
1105 1110 1115 1120 Glu Ile Lys Asn Arg Asp Leu Val Tyr Val 1125
1130 4 1088 PRT HUMAN 4 Met Arg Pro Lys Thr Phe Pro Ala Thr Thr Tyr
Ser Gly Asn Ser Arg 1 5 10 15 Gln Arg Leu Gln Glu Ile Arg Glu Gly
Leu Lys Gln Pro Ser Lys Ser 20 25 30 Ser Val Gln Gly Leu Pro Ala
Gly Pro Asn Ser Asp Thr Ser Leu Asp 35 40 45 Ala Lys Val Leu Gly
Ser Lys Asp Ala Thr Arg Gln Gln Gln Gln Met 50 55 60 Arg Ala Thr
Pro Lys Phe Gly Pro Tyr Gln Lys Ala Leu Arg Glu Ile 65 70 75 80 Arg
Tyr Ser Leu Leu Pro Phe Ala Asn Glu Ser Gly Thr Ser Ala Ala 85 90
95 Ala Glu Val Asn Arg Gln Met Leu Gln Glu Leu Val Asn Ala Gly Cys
100 105 110 Asp Gln Glu Met Ala Gly Arg Ala Leu Lys Gln Thr Gly Ser
Arg Ser 115 120 125 Ile Glu Ala Ala Leu Glu Tyr Ile Ser Lys Met Gly
Tyr Leu Asp Pro 130 135 140 Arg Asn Glu Gln Ile Val Arg Val Ile Lys
Gln Thr Ser Pro Gly Lys 145 150 155 160 Gly Leu Met Pro Thr Pro Val
Thr Arg Arg Pro Ser Phe Glu Gly Thr 165 170 175 Gly Asp Ser Phe Ala
Ser Tyr His Gln Leu Ser Gly Thr Pro Tyr Glu 180 185 190 Gly Pro Ser
Phe Gly Ala Asp Gly Pro Thr Ala Leu Glu Glu Met Pro 195 200 205 Arg
Pro Tyr Val Asp Tyr Leu Phe Pro Gly Val Gly Pro His Gly Pro 210 215
220 Gly His Gln His Gln His Pro Pro Lys Gly Tyr Gly Ala Ser Val Glu
225 230 235 240 Ala Ala Gly Ala His Phe Pro Leu Gln Gly Ala His Tyr
Gly Arg Pro 245 250 255 His Leu Leu Val Pro Gly Glu Pro Leu Gly Tyr
Gly Val Gln Arg Ser 260 265 270 Pro Ser Phe Gln Ser Lys Thr Pro Pro
Glu Thr Gly Gly Tyr Ala Ser 275 280 285 Leu Pro Thr Lys Gly Gln Gly
Gly Pro Pro Gly Ala Gly Leu Ala Phe 290 295 300 Pro Pro Pro Ala Ala
Gly Leu Tyr Val Pro His Pro His His Lys Gln 305 310 315 320 Ala Gly
Pro Ala Ala His Gln Leu His Val Leu Gly Ser Arg Ser Gln 325 330 335
Val Phe Ala Ser Asp Ser Pro Pro Gln Ser Leu Leu Thr Pro Ser Arg 340
345 350 Asn Ser Leu Asn Val Asp Leu Tyr Glu Leu Gly Ser Thr Ser Val
Gln 355 360 365 Gln Trp Pro Ala Ala Thr Leu Ala Arg Arg Asp Ser Leu
Gln Lys Pro 370 375 380 Gly Leu Glu Ala Pro Pro Arg Ala His Val Ala
Phe Arg Pro Asp Cys 385 390 395 400 Pro Val Pro Ser Arg Thr Asn Ser
Phe Asn Ser His Gln Pro Arg Pro 405 410 415 Gly Pro Pro Gly Lys Ala
Glu Pro Ser Leu Pro Ala Pro Asn Thr Val 420 425 430 Thr Ala Val Thr
Ala Ala His Ile Leu His Pro Val Lys Ser Val Arg 435 440 445 Val Leu
Arg Pro Glu Pro Gln Thr Ala Val Gly Pro Ser His Pro Ala 450 455 460
Trp Val Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 465
470 475 480 Ala Glu Gly Leu Asp Ala Lys Glu Glu His Ala Leu Ala Leu
Gly Gly 485 490 495 Ala Gly Ala Phe Pro Leu Asp Val Glu Tyr Gly Gly
Pro Asp Arg Arg 500 505 510 Cys Pro Pro Pro Pro Tyr Pro Lys His Leu
Leu Leu Arg Ser Lys Ser 515 520 525 Glu Gln Tyr Asp Leu Asp Ser Leu
Cys Ala Gly Met Glu Gln Ser Leu 530 535 540 Arg Ala Gly Pro Asn Glu
Pro Glu Gly Gly Asp Lys Ser Arg Lys Ser 545 550 555 560 Ala Lys Gly
Asp Lys Gly Gly Lys Asp Lys Lys Gln Ile Gln Thr Ser 565 570 575 Pro
Val Pro Val Arg Lys Asn Ser Arg Asp Glu Glu Lys Arg Glu Ser 580 585
590 Arg Ile Lys Ser Tyr Ser Pro Tyr Ala Phe Lys Phe Phe Met Glu Gln
595 600 605 His Val Glu Asn Val Ile Lys Thr Tyr Gln Gln Lys Val Asn
Arg Arg 610 615 620 Leu Gln Leu Glu Gln Glu Met Ala Lys Ala Gly Leu
Cys Glu Ala Glu 625 630 635 640 Gln Glu Gln Met Arg Lys Ile Leu Tyr
Gln Lys Glu Ser Asn Tyr Asn 645 650 655 Arg Leu Lys Arg Ala Lys Met
Asp Lys Ser Met Phe Val Lys Ile Lys 660 665 670 Thr Leu Gly Ile Gly
Ala Phe Gly Glu Val Cys Leu Ala Cys Lys Val 675 680 685 Asp Thr His
Ala Leu Tyr Ala Met Lys Thr Leu Arg Lys Lys Asp Val 690 695 700 Leu
Asn Arg Asn Gln Val Ala His Val Lys Ala Glu Arg Asp Ile Leu 705 710
715 720 Ala Glu Ala Asp Asn Glu Trp Val Val Lys Leu Tyr Tyr Ser Phe
Gln 725 730 735 Asp Lys Asp Ser Leu Tyr Phe Val Met Asp Tyr Ile Pro
Gly Gly Asp 740 745 750 Met Met Ser Leu Leu Ile Arg Met Glu Val Phe
Pro Glu His Leu Ala 755 760 765 Arg Phe Tyr Ile Ala Glu Leu Thr Leu
Ala Ile Glu Ser Val His Lys 770 775 780 Met Gly Phe Ile His Arg Asp
Ile Lys Pro Asp Asn Ile Leu Ile Asp 785 790 795 800 Leu Asp Gly His
Ile Lys Leu Thr Asp Phe Gly Leu Cys Thr Gly Phe 805 810 815 Arg Trp
Thr His Asn Ser Lys Tyr Tyr Gln Lys Gly Ser His Val Arg 820 825 830
Gln Asp Ser Met Glu Pro Ser Asp Leu Trp Asp Asp Val Ser Asn Cys 835
840 845 Arg Cys Gly Asp Arg Leu Lys Thr Leu Glu Gln Arg Ala Arg Lys
Gln 850 855 860 His Gln Arg Cys Leu Ala His Ser Leu Val Gly Thr Pro
Asn Tyr Ile 865 870 875 880 Ala Pro Glu Val Leu Leu Arg Lys Gly Tyr
Thr Gln Leu Cys Asp Trp 885 890 895 Trp Ser Val Gly Val Ile Leu Phe
Glu Met Leu Val Gly Gln Pro Pro 900 905 910 Phe Leu Ala Pro Thr Pro
Thr Glu Thr Gln Leu Lys Val Ile Asn Trp 915 920 925 Glu Asn Thr Leu
His Ile Pro Ala Gln Val Lys Leu Ser Pro Glu Ala 930 935 940 Arg Asp
Leu Ile Thr Lys Leu Cys Cys Ser Ala Asp His Arg Leu Gly 945 950 955
960 Arg Asn Gly Ala Asp Asp Leu Lys Ala His Pro Phe Phe Ser Ala Ile
965 970 975 Asp Phe Ser Ser Asp Ile Arg Lys Gln Pro Ala Pro Tyr Val
Pro Thr 980 985 990 Ile Ser His Pro Met Asp Thr Ser Asn Phe Asp Pro
Val Asp Glu Glu 995 1000 1005 Ser Pro Trp Asn Asp Ala Ser Glu Gly
Ser Thr Lys Ala Trp Asp Thr 1010 1015 1020 Leu Thr Ser Pro Asn Asn
Lys His Pro Glu His Ala Phe Tyr Glu Phe 1025 1030 1035 1040 Thr Phe
Arg Arg Phe Phe Asp Asp Asn Gly Tyr Pro Phe Arg Cys Pro 1045 1050
1055 Lys Pro Ser Gly Ala Glu Ala Ser Gln Ala Glu Ser Ser Asp Leu
Glu 1060 1065 1070 Ser Ser Asp Leu Val Asp Gln Thr Glu Gly Cys Gln
Pro Val Tyr Val 1075 1080 1085 5 23 DNA HUMAN The letter "y" stands
for c or t. The letter "n" stands for a, c, g or t. The letter "r"
stands for a or g. The letter "h" stands for a, c or t. 5
caygtnaara thacngaytt ygg 23 6 20 DNA HUMAN The letter "r" stands
for a or g. The letter "d" stands for a, g or t. The letter "y"
stands for c or t. 6 ggrtcdatca tccagcaytt 20 7 6 PRT HUMAN 7 Lys
Ile Thr Asp Phe Gly 1 5 8 6 PRT HUMAN 8 Lys Cys Trp Met Ile Asp 1 5
9 27 DNA HUMAN The letter "r" stands for a or g. The letter "d"
stands for a, g or t. The letter "n" stands for a, c, g or t. The
letter "s" stands for c or g. The letter "w" stands for for a or t.
9 tccraacagd atnacnccna cnswcca 27 10 27 DNA HUMAN The letter "y"
stands for c or t. The letter "n" stands for a, c, g or t. The
letter "m" stands for a or c. 10 ttyggnytnt gyacnggntt ymgntgg 27
11 9 PRT HUMAN 11 Phe Gly Leu Cys Thr Gly Phe Arg Trp 1 5 12 9 PRT
HUMAN 12 Trp Ser Val Gly Val Ile Leu Phe Glu 1 5 13 1088 PRT HUMAN
13 Met His Pro Ala Gly Glu Lys Arg Gly Gly Arg Pro Asn Asp Lys Tyr
1 5 10 15 Thr Ala Glu Ala Leu Glu Ser Ile Lys Gln Asp Leu Thr Arg
Phe Glu 20 25 30 Val Gln Asn Asn His Arg Asn Asn Gln Asn Tyr Thr
Pro Leu Arg Tyr 35 40 45 Thr Ala Thr Asn Gly Arg Asn Asp Ala Leu
Thr Pro Asp Tyr His His 50 55 60 Ala Lys Gln Pro Met Glu Pro Pro
Pro Ser Ala Ser Pro Ala Pro Asp 65 70 75 80 Val Val Ile Pro Pro Pro
Pro Ala Ile Val Gly Gln Pro Gly Ala Gly 85 90 95 Ser Ile Ser Val
Ser Gly Val Gly Val Gly Val Val Gly Val Ala Asn 100 105 110 Gly Arg
Val Pro Lys Met Met Thr Ala Leu Met Pro Asn Lys Leu Ile 115 120 125
Arg Lys Pro Ser Ile Glu Arg Asp Thr Ala Ser Ser His Tyr Leu Arg 130
135 140 Cys Ser Pro Ala Leu Asp Ser Gly Ala Gly Ser Ser Arg Ser Asp
Ser 145 150 155 160 Pro His Ser His His Thr His Gln Pro Ser Ser Arg
Thr Val Gly Asn 165 170 175 Pro Gly Gly Asn Gly Gly Phe Ser Pro Ser
Pro Ser Gly Phe Ser Glu 180 185 190 Val Ala Pro Pro Ala Pro Pro Pro
Arg Asn Pro Thr Ala Ser Ser Ala 195 200 205 Ala Thr Pro Pro Pro Pro
Val Pro Pro Thr Ser Gln Ala Tyr Val Lys 210 215 220 Arg Arg Ser Pro
Ala Leu Asn Asn Arg Pro Pro Ala Ile Ala Pro Pro 225 230 235 240 Thr
Gln Arg Gly Asn Ser Pro Val Ile Thr Gln Asn Gly Leu Lys Asn 245 250
255 Pro Gln Gln Gln Leu Thr Gln Gln Leu Lys Ser Leu Asn Leu Tyr Pro
260 265 270 Gly Gly Gly Ser Gly Ala Val Val Glu Pro Pro Pro Pro Tyr
Leu Ile 275 280 285 Gln Gly Gly Ala Gly Gly Ala Ala Pro Pro Pro Pro
Pro Pro Ser Tyr 290 295 300 Thr Ala Ser Met Gln Ser Arg Gln Ser Pro
Thr Gln Ser Gln Gln Ser 305 310 315 320 Asp Tyr Arg Lys Ser Pro Ser
Ser Gly Ile Tyr Ser Ala Thr Ser Ala 325 330 335 Gly Ser Pro Ser Pro
Ile Thr Val Ser Leu Pro Pro Ala Pro Leu Ala 340 345 350 Lys Pro Gln
Pro Arg Val Tyr Gln Ala Arg Ser Gln Gln Pro Ile Ile 355 360 365 Met
Gln Ser Val Lys Ser Thr Gln Val Gln Lys Pro Val Leu Gln Thr 370 375
380 Ala Val Ala Pro Gln Ser Pro Ser Ser Ala Ser Ala Ser Asn Ser Pro
385 390 395 400 Val His Val Leu Ala Ala Pro Pro Ser Tyr Pro Gln Lys
Ser Ala Ala 405 410 415 Val Val Gln Gln Gln Gln Gln Ala Ala Ala Ala
Ala His Gln Gln Gln 420
425 430 His Gln His Gln Gln Ser Lys Pro Ala Thr Pro Thr Thr Pro Pro
Leu 435 440 445 Val Gly Leu Asn Ser Lys Pro Asn Cys Leu Glu Pro Pro
Ser Tyr Ala 450 455 460 Lys Ser Met Gln Ala Lys Ala Ala Thr Val Val
Gln Gln Gln Gln Gln 465 470 475 480 Gln Gln Gln Gln Gln Gln Val Gln
Gln Gln Gln Val Gln Gln Gln Gln 485 490 495 Gln Gln Gln Gln Gln Gln
Leu Gln Ala Leu Arg Val Leu Gln Ala Gln 500 505 510 Ala Gln Arg Glu
Arg Asp Gln Arg Glu Arg Glu Arg Asp Gln Gln Lys 515 520 525 Leu Ala
Asn Gly Asn Pro Gly Arg Gln Met Leu Pro Pro Pro Pro Tyr 530 535 540
Gln Ser Asn Asn Asn Asn Asn Ser Glu Ile Lys Pro Pro Ser Cys Asn 545
550 555 560 Asn Asn Asn Ile Gln Ile Ser Asn Ser Asn Leu Ala Thr Thr
Pro Pro 565 570 575 Ile Pro Pro Ala Lys Tyr Asn Asn Asn Ser Ser Asn
Thr Gly Ala Asn 580 585 590 Ser Ser Gly Gly Ser Asn Gly Ser Thr Gly
Thr Thr Ala Ser Ser Ser 595 600 605 Thr Ser Cys Lys Lys Ile Lys His
Ala Ser Pro Ile Pro Glu Arg Lys 610 615 620 Lys Ile Ser Lys Glu Lys
Glu Glu Glu Arg Lys Glu Phe Arg Ile Arg 625 630 635 640 Trp Ala Arg
Thr His Ser Pro Gln Ala Phe Lys Phe Phe Met Glu Gln 645 650 655 His
Ile Glu Asn Val Ile Lys Ser Tyr Arg Gln Arg Thr Tyr Arg Lys 660 665
670 Asn Gln Leu Glu Lys Glu Met His Lys Val Gly Leu Pro Asp Gln Thr
675 680 685 Gln Ile Glu Met Arg Lys Met Leu Asn Gln Lys Glu Ser Asn
Tyr Ile 690 695 700 Arg Leu Lys Arg Ala Lys Met Asp Lys Ser Met Phe
Val Lys Leu Lys 705 710 715 720 Pro Ile Gly Val Gly Ala Phe Gly Glu
Val Thr Leu Val Ser Lys Ile 725 730 735 Asp Thr Ser Asn His Leu Tyr
Ala Met Lys Thr Leu Arg Lys Ala Asp 740 745 750 Val Leu Lys Arg Asn
Gln Val Ala His Val Lys Ala Glu Arg Asp Ile 755 760 765 Leu Ala Glu
Ala Asp Asn Asn Trp Val Val Lys Leu Tyr Tyr Ser Phe 770 775 780 Gln
Asp Lys Asp Asn Leu Tyr Phe Val Met Asp Tyr Ile Pro Gly Gly 785 790
795 800 Asp Leu Met Ser Leu Leu Ile Lys Leu Gly Ile Phe Glu Glu Glu
Leu 805 810 815 Ala Arg Phe Tyr Ile Ala Glu Val Thr Cys Ala Val Asp
Ser Val His 820 825 830 Lys Met Gly Phe Ile His Arg Asp Ile Lys Pro
Asp Asn Ile Leu Ile 835 840 845 Asp Arg Asp Gly His Ile Lys Leu Thr
Asp Phe Gly Leu Cys Thr Gly 850 855 860 Phe Arg Trp Thr His Asn Ser
Lys Tyr Tyr Gln Glu Asn Gly Asn His 865 870 875 880 Ser Arg Gln Asp
Ser Met Glu Pro Trp Glu Glu Tyr Ser Glu Asn Gly 885 890 895 Pro Lys
Pro Thr Val Leu Glu Arg Arg Arg Met Arg Asp His Gln Arg 900 905 910
Val Leu Ala His Ser Leu Val Gly Thr Pro Asn Tyr Ile Ala Pro Glu 915
920 925 Val Leu Glu Arg Ser Gly Tyr Thr Gln Leu Cys Asp Tyr Trp Ser
Val 930 935 940 Gly Val Ile Leu Tyr Glu Met Leu Val Gly Gln Pro Pro
Phe Leu Ala 945 950 955 960 Asn Ser Pro Leu Glu Thr Gln Gln Lys Val
Ile Asn Trp Glu Lys Thr 965 970 975 Leu His Ile Pro Pro Gln Ala Glu
Leu Ser Arg Glu Ala Thr Asp Leu 980 985 990 Ile Arg Arg Leu Cys Ala
Ser Ala Asp Lys Arg Leu Gly Lys Ser Val 995 1000 1005 Asp Glu Val
Lys Ser His Asp Phe Phe Lys Gly Ile Asp Phe Ala Asp 1010 1015 1020
Met Arg Lys Gln Lys Ala Pro Tyr Ile Pro Glu Ile Lys His Pro Thr
1025 1030 1035 1040 Asp Thr Ser Asn Phe Asp Pro Val Asp Pro Glu Lys
Leu Arg Ser Asn 1045 1050 1055 Asp Ser Thr Met Ser Ser Gly Asp Asp
Val Asp Gln Asn Asp Arg Thr 1060 1065 1070 Phe His Gly Phe Phe Glu
Phe Thr Phe Arg Arg Phe Phe Asp Asp Lys 1075 1080 1085 14 10 PRT
HUMAN 14 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 1 5 10 15 14 PRT
HUMAN 15 Ile Ser Lys Pro Ser Lys Glu Asp Gln Pro Ser Leu Pro Lys 1
5 10 16 17 PRT HUMAN 16 Asp Asp Gln Asn Thr Gly Ser Glu Ile Lys Asn
Arg Asp Leu Val Tyr 1 5 10 15 Val 17 16 PRT HUMAN 17 Pro Ser Gly
Lys Asn Ser Arg Asp Glu Glu Lys Arg Glu Ser Arg Ile 1 5 10 15 18 15
PRT HUMAN 18 Ser Asp Leu Val Asp Gln Thr Glu Gly Cys Gln Pro Val
Tyr Val 1 5 10 15
* * * * *