U.S. patent application number 11/224297 was filed with the patent office on 2006-03-09 for human-derived bradeion proteins, dna coding for the proteins, and uses thereof.
This patent application is currently assigned to SECRETARY OF AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY. Invention is credited to Manami Tanaka, Tomoo Tanaka.
Application Number | 20060052584 11/224297 |
Document ID | / |
Family ID | 18176196 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060052584 |
Kind Code |
A1 |
Tanaka; Manami ; et
al. |
March 9, 2006 |
Human-derived bradeion proteins, DNA coding for the proteins, and
uses thereof
Abstract
A human-derived bradeion protein, which has the following
properties: (i) it is a transmembranous protein; (ii) it has a
structure characteristic of growth hormone and cytokine receptors
even in a structure of its transmembranous portion when its
structure is determined by a hydrophobicity analysis according to
Kyte-Doolittle method; (iii) it is expressed at a high level in a
human adult brain, and in less amount in the heart, while it is not
expressed in other adult organs or fetus; (iv) it induces
programmed cell death (apoptosis) when over-expressed in a cultured
human nerve cell lines; (v) it induces termination of cell division
and aging when over-expressed in a cultured human normal cell; (vi)
it is located in cytoplasm, and forms an intracellular aggregate
when overexpressed; and (vii) besides human adult neurons, it is
specifically expressed in a human colorectal cancer cell line or in
a skin cancer cell line, or an analogue thereof.
Inventors: |
Tanaka; Manami; (Ibaraki,
JP) ; Tanaka; Tomoo; (Kanagawa, JP) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
SECRETARY OF AGENCY OF INDUSTRIAL
SCIENCE AND TECHNOLOGY
|
Family ID: |
18176196 |
Appl. No.: |
11/224297 |
Filed: |
September 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10190555 |
Jul 9, 2002 |
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11224297 |
Sep 13, 2005 |
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09440936 |
Nov 16, 1999 |
6423504 |
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10190555 |
Jul 9, 2002 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/6.14; 435/69.1; 536/23.5 |
Current CPC
Class: |
G01N 33/57419 20130101;
G01N 33/5743 20130101; C07K 14/715 20130101; C12Q 2600/158
20130101; C07K 14/4747 20130101; C07K 14/71 20130101; C12Q 1/6886
20130101 |
Class at
Publication: |
530/350 ;
435/006; 435/069.1; 435/320.1; 435/325; 536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C07K 14/72 20060101
C07K014/72; C07K 14/82 20060101 C07K014/82 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 16, 1998 |
JP |
325380/1998 |
Claims
1. A human-derived bradeion protein, which has the following
properties: (i) it is a transmembranous protein; (ii) it has a
structure characteristic of growth hormone and cytokine receptors
even in a structure of its transmembranous portion when its
structure is determined by a hydrophobicity analysis according to
Kyte-Doolittle method; (iii) it is expressed at a high level in a
human adult brain, and in less amount in the heart, while it is not
expressed in other adult organs or fetus; (iv) it induces
programmed cell death (apoptosis) when over-expressed in a cultured
human nerve cell lines; (v) it induces termination of cell division
and aging when over-expressed in a cultured human normal cell; (vi)
it is located in cytoplasm, and forms an intracellular aggregate
when overexpressed; and (vii) besides human adult neurons, it is
specifically expressed in a human colorectal cancer cell line or in
a skin cancer cell line, or an analogue thereof.
2. The human-derived bradeion protein or an analogue thereof
according to claim 1, wherein the programmed cell death (apoptosis)
is induced when DNA coding for the bradeion protein or the analogue
thereof is gene-transferred into a cultured cancer cell.
3. The human-derived bradeion protein or an analogue thereof
according to claim 1, comprising an amino acid sequence shown in
SEQ ID NO: 2, or an amino acid sequence with deletions,
substitutions or additions of at least one amino acid residue in
the amino acid sequence shown in SEQ ID NO: 2.
4. The human-derived bradeion protein or an analogue thereof
according to claim 1, comprising an amino acid sequence shown in
SEQ ID NO: 4, or an amino acid sequence with deletions,
substitutions or additions of at least one amino acid residue in
the amino acid sequence shown in SEQ ID NO: 4.
5. DNA comprising a nucleotide sequence coding for the bradeion
protein or the analogue thereof according to claim 1, or a fragment
thereof.
6. The DNA or a fragment thereof according to claim 5, comprising
the nucleotide sequence 129-1943 of SEQ ID NO: 1, or a nucleotide
sequence of at least 15 consecutive nucleotides derived from the
nucleotide sequence 129-1943 of SEQ ID NO: 1.
7. The DNA or a fragment thereof according to claim 5, comprising
the nucleotide sequence 129-1562 of SEQ ID NO: 3, or a nucleotide
sequence of at least 15 consecutive nucleotides derived from the
nucleotide sequence 129-1562 of SEQ ID NO: 3.
8. The DNA according to claim 5, which hybridizes with DNA
comprising the nucleotide sequence 129-1943 of SEQ ID NO: 1 under
stringent conditions.
9. The DNA according to claim 5, which hybridizes with DNA
comprising the nucleotide sequence 129-1562 of SEQ ID NO: 3 under
stringent conditions.
10. A vector comprising the isolated DNA according to claim 17.
11. A host cell transformed or transfected with the vector of claim
10.
12. A host cell according to claim 11, which is a prokaryotic or
eukaryotic cell.
13. An antibody immunologically reactive with the bradeion protein
or the analogue thereof according to claim 1.
14. A method for detecting a cancer, comprising detecting the
cancer by using the DNA or the fragment thereof according to claim
5 or the antibody according to claim 13 as a tumor-specific
marker.
15. The method according to claim 14, wherein the cancer is human
colorectal cancer or human malignant melanoma.
16. The method according to claim 14 or 15, wherein the detection
is conducted by hybridization or immunoassay.
17. An isolated DNA that encodes a protein characterized by at
least 90% homology with a human-derived bradeion a protein that (A)
comprises an amino acid sequence of SEQ ID NO:2 and (B) has the
following properties: (i) it is a transmembranous protein; (ii) it
has a transmembrane portion, an extracellular portion, and a
cytoplasmic portion in its molecule as determined by a
hydrophobicity analysis according to Kyte-Doolittle method; (iii)
it is expressed in the human adult normal brain and heart; the
expression level thereof in the heart being about 10% or lower of
that in the brain, while it is not expressed in other adult organs
or fetus; (iv) it induces programmed cell death when over-expressed
in a cultured human brain-derived undifferentiated nerve cell line;
(v) it induces termination of cell division and aging when
overexpressed in a cultured human brain-derived differentiated
nerve cell; (vi) it is located in cytoplasm in the course of the
induced cell death, and forms an intracellular aggregate when
overexpressed; and (vii) it is expressed in a human colorectal
cancer cell line or in a human malignant melanoma cell line, but
not in leukemia, lymphoma and lung carcinoma.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a protein involved in
long-term survival of cranial nerve-cell, to DNA encoding the
protein, and to uses thereof. More particularly, the present
invention relates to human-derived bradeion protein or derivatives
thereof, to DNA encoding the protein or the derivatives thereof, to
a vector containing the DNA, to a host cell transformed or
transfected with the vector, to an antibody immunologically
reactive with the protein or the derivatives thereof, and to uses
of the DNA or the antibody for detecting a cancer.
BACKGROUND OF THE INVENTION
[0002] Cranial nerve cells (neurons) are main elements for
controlling survival of higher order animals. Once the neurons are
produced, they do not divide at all and only gradually exfoliate or
go through necrosis. Exfoliation of the neurons occurs in the
normal state but is particularly accelerated by genetic diseases,
brain ischemia, or status epilepticus, or under conditions of poor
nutrition and low oxygen. Some disorders of cranial nerves
associated with aging (e.g., dementia) result from deficiency of an
absolute amount of functional neurons caused by accumulation of
exfoliated neurons. Thus, the monitoring and control of the
exfoliation, as well as regeneration of the functions of neurons,
are the most demanding subject to be solved among the aging
problems.
[0003] Cranial nerve cells do not divide at all after the induction
phase of differentiation in the process of development, and
maintain their functions or is accompanied by gradual deterioration
of their functions until the end of the life-time of the
individual. They are presumed to have specific
division-interrupting and function-maintaining mechanisms although
these mechanisms have not yet been clarified. In the central
nervous system, there exist numbers of unknown proteins and
signaling substances, particularly stimulating substances and
receptors thereof involved in brain-specific signal transduction,
but their details are yet unclear.
[0004] Numbers of researches have been conducted worldwide on such
an important element that controls the survival of the cranial
nerve cells. However, none of the elements was clarified in the
substance or molecule level, and, prior to everything, it was
necessary to develop techniques for analysis. Recently, the group
of Dr. Masashi Yanagisawa and his colleagues of the University of
Texas, Medical Research Center (authorized by the Howard Hughes
Foundation) has succeeded in developing a technique for randomly
screening neuropeptides and receptors thereof by using cultured
cells, and they have found a substance (named orexin) that directly
binds to and stimulates the aperitive center in the hypothalamus,
and identified functions of the substance's receptor (Cell, 92,
573-585, 1998). However, such a systematic screening of substances
is rare, and currently, stimulating factors involved in
brain-specific signal transduction and receptors thereof are not
yet fully clarified.
[0005] Under such circumstances, the present inventors have now
constructed an improved expression gene (cDNA) library, developed a
systematic screening technique, and succeeded in extraction and
selection of genes specific for cranial nerve cells, thereby
accomplishing the present invention.
[0006] Thus, one object of the present invention is to provide a
bradeion protein involved in long-term survival of cranial nerve
cells, and DNA coding for the bradeion protein.
[0007] Another object of the present invention is to provide a
vector containing the above-mentioned DNA, and a host cell
transformed or transfected with the vector.
[0008] Still another object of the present invention is to use the
DNA or an antibody to the protein for detecting cancers.
SUMMARY OF THE INVENTION
[0009] The present invention provides a human-derived bradeion
protein, which has the following properties: [0010] (i) it is a
transmembranous protein; [0011] (ii) it has a structure
characteristic of growth hormone and cytokine receptors (even in a
structure of its transmembranous portion) when its structure is
determined by a hydrophobicity analysis according to Kyte-Doolittle
method; [0012] (iii) it is expressed at a high level in the human
adult brain, in less amount in the heart, while it is not expressed
in other adult organs or fetus; [0013] (iv) it induces programmed
cell death (apoptosis) when over-expressed in cultured human cell
lines; [0014] (v) it induces termination of cell division and aging
when over-expressed in cultured human normal cells; [0015] (vi) it
is located in cytoplasm, and forms an intracellular aggregate when
overexpressed; and [0016] (vii) besides human adult neurons, it is
specifically expressed in a human colorectal cancer cell line or in
a skin cancer cell line, or an analogue thereof.
[0017] The proteins of the invention include Bradeion .alpha. and
Bradeion .beta. proteins having the amino acid sequences shown in
SEQ ID NOS:2 and 4, respectively. These proteins are not the
consequence of alternative splicing, but coded in the adjacent area
(chromosome 17q23) of human genome. In addition to the
above-described properties, Bradeion .alpha. induces programmed
cell death when DNA coding for Bradeion .alpha. or an analogue
thereof is introduced into a cultured cancer cell.
[0018] The term "analogue" as used herein refers to a protein that
has properties substantially equivalent to those of the
human-derived bradeion proteins (for example, at least the
properties of (i), (ii), (iii), (vi) and (vii)), or a protein
having an amino acid sequence with deletions, substitutions or
additions of at least one amino acid residue in the amino acid
sequence shown in SEQ ID NO:2 or 4, or a protein having. an amino
acid sequence that is substantially the same as that shown in SEQ.
ID NO:2 or 4. Preferably, the analogue of the invention has at
least 90%, preferably at least 95%, more preferably at least 97%
homology with the amino acid sequence of SEQ ID NO:2 or 4. The
analogue of the invention also includes human bradeion proteins
modified or mutated in the amino acid level, and bradeion proteins
from non-human mammals having properties substantially equivalent
to those from humans. Only Bradeion .beta. (similar to the
human-derived Bradeion .beta.) was found, for example, in a mouse
brain and its homology with the human Bradeion .beta. was 94%. The
analogues of the invention may be obtained through DNA recombinant
techniques by artificial modifications (e.g., site-directed
mutagenesis), as long as the original bioactivity of the human
bradeion is not impaired. The analogues of the invention may or may
not contain a sugar chain, or they may be chemically modified with
an aqueous polymer such as polyethylene glycol.
[0019] Moreover, the present invention provides DNAs comprising
nucleotide sequences coding for the above-defined bradeion proteins
or analogues thereof, and fragments of the DNAs.
[0020] Specific examples of such DNAs or fragments thereof include:
DNA comprising the sequence of the nucleotides 129-1943 of SEQ ID
NO:1 (i.e., DNA encoding human Bradeion .alpha.); DNA fragments
having at least 15, preferably at least 20, more preferably at
least 30 consecutive nucleotides derived from the nucleotides
129-1943 of SEQ ID NO:1; DNA comprising the full-length nucleotide
sequence shown in SEQ ID NO:1; DNA comprising the sequence of the
nucleotides 129-1562 of SEQ ID NO:3 (i.e., DNA encoding human
Bradeion .beta.); DNA fragments having at least 15, preferably at
least 20, more preferably at least 30 consecutive nucleotides
derived from the nucleotides 129-1562 of SEQ ID NO:3; and DNA
comprising the full-length nucleotide sequence shown in SEQ ID
NO:3. In addition, DNA that can hybridize with one of the
above-mentioned DNAs under stringent conditions is also encompassed
in the present invention. The stringent conditions as mentioned
herein refer to such conditions that allow hybridization only when
there is at least 90% homology, preferably at least 95% homology,
more preferably at least 97% homology with the nucleotide sequence
shown in SEQ ID NO:1 (positions 129-1943) or SEQ ID NO:3 (positions
129-1562). Generally, such conditions allow hybridization at a
temperature that is lower by about 5.degree. C.-30.degree. C.,
preferably by about 10.degree. C.-25.degree. C. than the melting
temperature (Tm) of the complete hybrid. Stringent conditions that
may be used are described in J. Sambrook et al., Molecular Cloning,
A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press (1989), esp. "Conditions for Hybridization of Oligonucleotide
Probes". The DNA and fragments thereof according to the present
invention can be used not only for the expression of the bradeion
proteins but also as a probe for hybridization or as a primer for
PCR.
[0021] The present invention further provides vectors containing
DNAs coding for bradeion proteins or analogues thereof, or
fragments of the DNAs. The vector usually contains a promoter that
is capable of operably expressing the DNAs. In addition to the
promoter, the vector may contain at least one other element such as
an origin of replication, an enhancer, a ribosome-binding site, a
transcription termination factor (terminator), a selective marker,
an RNA splicing site, or a polyadenylation signal.
[0022] The present invention further provides a host cell that has
been transformed or transfected with such a vector. The host cell
may be a prokaryotic or eukaryotic cell, preferably an eukaryotic
cell, more preferably a mammalian cell such as a human cell
line.
[0023] The present invention also provides an antibody that is
immunologically reactive with the above-defined bradeions or
analogues thereof. The antibody is preferably one that can
specifically immuno-react with the bradeion proteins or analogues
thereof, and is a polyclonal or monoclonal antibody.
[0024] The invention further provides a method for detecting a
cancer, comprising detecting the cancer by using the above-defined
DNAs or fragments thereof or the above-defined antibodies as tumor
markers. Herein, the cancer includes, but is not limited to, a
human colorectal cancer and a human skin cancer. The detection can
be conducted by hybridization or immunoassay.
[0025] This specification includes all or-part of the contents as
disclosed in the specification and/or drawings of Japanese Patent
Application No. 10-325380, which is a priority document of the
present application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A and 1B are photographs showing the results of a
hydropathy analysis by the Kyte-Doolittle method, giving
distributions of hydrophobic and hydrophilic portions in Bradeion
.alpha. together with distributions in IL2, IL3, IL4 and growth
hormone receptors for comparison;
[0027] FIGS. 2A-C are photographs taken with a confocal laser
microscope showing images of labeled cells observed by
gene-transfer and over-expression of Bradeion .alpha. and Bradeion
.beta. genes in cultured NT2 neuron (human undifferentiated nerve
cell) and HeLa cell. FIG. 2A shows photographs for NT2 neuron; FIG.
2B shows photographs for NT2 neuron and HeLa; and FIG. 2C shows
electron microscopic images of the cells shown in FIG. 2B (18 and
24 hours after the introduction). FIG. 2A shows locations of
bradeion genes identified with EGFP (Enhanced Green Fluorescent
Protein; CLONTECH Lab., Inc.) (left), locations of bradeion genes
in mitochondria (center), and overlaid images of the left and
center images (right).
[0028] FIG. 3A is a photograph showing expression of equal amounts
of Bradeion .alpha. and Bradeion .beta. genes in human cancer cell
lines, giving the results of Northern blot analysis using
radiolabeled bradeion genes. Lane 1 shows expression in
polymyelocytic leukemia, HL60; Lane 2 shows expression in HeLaS3;
Lane 3 shows expression in chronicmyologenous leukemia, K-562; Lane
4 shows expression in lymphoblastic leukemia, MOLT-4; Lane 5 shows
expression in Burkitt's lymphoma, Raji; Lane 6 shows expression in
colorectal adenocarcinoma, SW480 21, 22; Lane 7 shows expression in
lung carcinoma, A549; and Lane 8 shows expression in melanoma,
G361. The result of Northern blot analysis using a .beta.-actin
gene is shown underneath as a positive control.
[0029] FIG. 3B shows cancer-specific expression of bradeion genes
in colorectal adenocarcinoma cell (T1 to T10); skin cancer cells
(T11 to T13); and normal cells (N1 to N2). All specimens are from
humans. In the figure, "*" refers to the case where both Bradeion
.alpha. and Bradeion .beta. genes are detected without gene
mutation; "ND" refers to the case where detection was impossible
due to denaturation of RNA; "Ad (well)" refers to a well
differentiated adenocarcinoma; "Ad (mod)" refers to a moderately
differentiated adenocarcinoma; "Muc" refers to a mucinouscarcinoma;
and "MM" refers to malignant melanoma. Dukes' stage is based on the
Dukes' classification. Codon 12 of human K-ras gene (whose wild
type sequence is GGT) is indicated if it has been mutated. This
mutation is heterozygous.
[0030] FIG. 3C is photographs of the results of in situ
hybridization of specimens from human cancer tissue, showing
stained tissues for T13 and T8 (FIG. 3B) (Antisense: positive
control, Sense: negative control).
DETAILED DESCRIPTION OF THE INVENTION
[0031] cDNA coding for bradeion proteins of the invention may be
obtained as follows.
[0032] First, brain tissue is homogenized in a phenol or
phenol-chloroform solution containing guanidine isothiocyanate, and
subjected to high-speed centrifugation to be separated into an
aqueous layer and an organic layer. Total RNA contained in the
aqueous layer is precipitated and collected by adding isopropanol,
or is collected through sucrose or cesium chloride density-gradient
centrifugation. The obtained total RNA is subjected to
oligo(dT)-cellulose chromatography to purify mRNA (i.e., poly(A)
RNA) therefrom.
[0033] Then, cDNA is synthesized from the mRNA in the presence of a
reverse transcriptase. The cDNA is provided with suitable
restriction sites and inserted into a phage or plasmid vector
having the identical restriction sites. The thus-obtained vector is
used to transform or transfect E.coli to produce a cDNA
library.
[0034] Since the cloned cDNA library includes various DNA fragments
with information other than that of the DNA of interest, it is
necessary to select the DNA of interest. For this purpose, plaque
hybridization or colony hybridization may usually be employed.
According to such methods, plaques (in the case of a phage vector)
or colonies (in the case of a plasmid vector) formed on agar are
transferred to a nitrocellulose membrane or a nylon membrane. After
being treated with an alkaline solution, they are bound to a
radioactive (.sup.32P) or fluorescence labeling DNA probe that is
capable of hybridizing with the DNA of interest, and exposed onto
an X-ray film, thereby detecting and collecting a plaque or colony
containing the DNA of interest. Alternatively, the obtained set of
clones may be exposed to an inducer such as isopropyl
1-thio-.beta.-D-galactoside (hereinafter, referred to as "IPTG") to
forcibly express proteins. The proteins are then transferred to a
nylon membrane or a cellulose membrane, and a specific antibody for
the protein of interest is used to immunologically select the
corresponding clones.
[0035] The cDNA of interest collected from the plaques or colonies
that positively reacts with the probe is sequenced by
Maxiam-Gilbert method or Sanger-Coulson method.
[0036] For cloning and sequencing, for example, methods described
in Sambrook et al., Molecular Cloning (supra), Ausubel et al.,
Current Protocols in Molecular Biology, Green Publishing Company
Assoc. and John Wiley Interscience, NY, 1992, etc. may be used.
[0037] Specifically, as will be described later in Examples, cDNA
library from human adult brain is constructed and thereafter cDNA
coding for the bradeion proteins of the invention are collected
from the positive clones. As the result of the sequencing analysis,
two types of bradeion genes, i.e., Bradeion .alpha. and Bradeion
.beta. genes, were found which were presumably produced due to
alternative splicing. The nucleotide sequences of these genes are
shown in SEQ ID NOS: 1 and 3, respectively, where the coding
regions were at the positions 129-1943 and 129-1562, respecitively.
The Bradeion .alpha. and Bradeion .beta. proteins have amino acid
sequences shown in SEQ ID NOS:2 and 4, respectively, as identified
from their nucleotide sequences. The Bradeion .alpha. DNA has 1815
nucleotides and codes for a protein having 605 amino acids. The
Bradeion .beta. DNA has 1434 nucleotides and codes for a protein
having 478 amino acids. Met at each position 1 of the amino acid
sequences of SEQ ID NOS:2 and 4 may be present or absent. When
these nucleotide and amino acid sequences were compared to all
sequences deposited with the GenBank, it was found that the
Bradeion .alpha. and Bradeion .beta. genes and proteins were
novel.
[0038] Bradeion .alpha. and Bradeion .beta. proteins were found to
have a structure characteristic of an interleukin receptor even in
a structure of its transmembranous portion, when subjected to the
hydropathy analysis according to Kyte-Doolittle method (J. Mol.
Biol., 157 (1): 105-132, 1982). Thus, it has the structure of a
transmembrane-type receptor that is presumably involved in the
intracellular signaling mechanism. Bradeion .alpha. and Bradeion
.beta. proteins are also similar to the relationship of the tumor
suppression genes p53/p73 in that there are two types of expression
modes, i.e., .alpha.- and .beta.-types, and in that either of them
is prevalent in various organisms. Formation of an intracellular
aggregate is very similar to that seen in human nerve retroplasia
caused by a triplet repeat gene expressed substance (Igarashi et
al., Nature Genetics, 111-117, 1998; Martindale et al., Nature
Genetics, 150-154, 1998). Accordingly, it is assumed that the
Bradeion .alpha. and Bradeion .beta. proteins are greatly
associated with specific termination of the division of human nerve
cells and/or with maintenance of the function of the nerve cells
after development/differentiation in the normal gene expression
state.
[0039] The human-derived bradeion proteins or analogues thereof of
the invention may be obtained, for example, by using gene
recombinant techniques as follows.
[0040] Taking account of degeneracy of the genetic codes, a
hybridization probe having at least 15, preferably about 20 to
about 50 consecutive nucleotides is constructed based on the
nucleotide sequence shown in SEQ ID NO:1 or 3 or the amino acid
sequence shown in SEQ ID NO:2 or 4. By using this hybridization
probe, DNAs coding for the bradeion proteins are screened from a
genomic DNA library or cDNA library derived from human or non-human
mammal brain tissue. The library may be produced by using
commercially available vector such as .lamda. ZAPII or
pBluescript.RTM. cloning vector (Stratagene Cloning Systems). The
plaque or colony containing the DNA of interest is selected through
plaque hybridization or colony hybridization.
[0041] Alternatively, a DNA sequence generally having 15 to 100
consecutive nucleotides complementary to the nucleotides 129-1943
of SEQ ID NO:1 or the nucleotides 129-1562 of SEQ ID NO: 3, is
produced as a primer. This primer can be used to conduct polymerase
chain reaction (PCR) in the genomic DNA library or cDNA library
derived from human or non-human mammalian brain tissue, thereby
specifically amplifying the DNA of interest. PCR can be conducted
through at least 20 cycles, preferably at least 30 cycles of:
denaturation at 94.degree. C. for 1 min.; annealing at 57.degree.
C. for 2 min.; and elongation at 70.degree. C. for 3 min. For the
PCR, see the techniques described in Protein Nucleic acid and
Enzyme, "Frontier of PCR method--Basic to Applied Techniques" vol.
4(5), April, 1996 Supplement, Kyoritsu Shuppan, Tokyo, Japan.
[0042] The cloned or amplified DNA of interest is collected and
introduced into an available suitable expression vector. The
obtained vector is used to transform a suitable host cell, which is
thereafter cultured in a proper medium for expression of the DNA,
to isolate and purify the protein of interest.
[0043] Examples of the analogue of the present invention include: a
protein having a substantially equivalent properties to the
human-derived bradeion (especially, at least the above-mentioned
properties of (i), (ii), (iii), (vi) and (vii)); or a protein
having an amino acid sequence with deletions, substitutions or
additions of at least one amino acid residue in the amino acid
sequence shown in SEQ ID NO:2 or 4; or a protein having an amino
acid sequence that is substantially the same as that shown in SEQ
ID NO:2 or 4. Preferably, the analogue has at least 90%, preferably
at least 95%, more preferably at least 97% homology with the amino
acid sequence shown in SEQ ID NO:2 or 4. This means that the amino
acid sequence of the analogue may include a modification chosen
from deletion, substitution or addition of at least one amino acid
as long as the analogue has properties substantially equivalent to
those of the human Bradeion .alpha. or Bradeion .beta.. The
mutation may be introduced into the amino acid sequence shown in
SEQ ID NO:2 or 4 through genetic engineering, for example, by
well-known techniques such as site-directed mutagenesis/PCR method
(S. N. Ho et al., Gene, 77, 51, 1989); and methods described in
Kaoru Saigo and Yumiko Sano (trans.), Current Protocols compact
version, Experimental Protocols in Molecular Biology I, June 1997,
Maruzen, Tokyo, Japan). Examples of the substitution include
substitutions between hydrophobic amino acids (Ala, Val, Leu, Ile,
etc.), Ser and Thr, Asp and Glu, Asn and Gln, Lys and Arg, and Phe
and Tyr. Examples of the addition include an addition of Met to the
N-terminus of a mature protein as seen in products expressed in a
bacterial host, and an addition of His-tag, or Met-Lys or Met-Arg
sequence to the N-terminus for facilitating induction of a mature
protein. It is also possible to truncate some amino acid residues
at the carboxyl or amino terminus of the bradeion protein to an
extent where bioactivity is not impaired.
[0044] Proteins without sugar chains are produced when a
prokaryotic cell such as bacterium is used as the host cell for
expressing DNAs coding for the bradeion proteins or analogues
thereof through genetic engineering. On the other hand, products
with sugar chains may be produced when the DNAs are expressed in an
eurokaryotic cell such as fungus, yeast, insect, plant, or
mammalian cell. For example, sugar chains may be formed by
introducing an N-binding sugar chain site (generally,
Asn-Xaa-Thr/Ser where Xaa is any amino acid other than Pro) into
the sequence. In any event, the obtained analogue should have
bioactivity substantially equivalent to the human bradeions.
[0045] Amino groups at the N-terminus or .epsilon.-amino groups of
lysine of the human bradeion proteins or analogues thereof of the
invention may be chemically bound to an aqueous polymer such as
polyethylene glycol (PEG). Pegylation is generally known to reduce
or suppress antigenicity or immunogenisity upon in vivo
administration of a pegylated product.
[0046] The present invention further provides an expression vector
containing the above-described DNA, and a host cell transformed or
transfected with the vector.
[0047] The vector of the invention may be in the form of, for
example, plasmid, phage, or virus. Other types of vectors may also
be used as long as they are replicable in a host cell. For example,
bacterium plasmids (e.g., pBR322, pKC30, pCFM536, etc.), phage DNAs
(e.g., .lamda. phage, etc.), yeast plasmids (e.g., pG-1, etc.), or
viral DNAs for mammal host cells (e.g., baculovirus, vaccinia
virus, adenovirus, SV40 and its derivatives, etc.) may be used.
[0048] The vector usually contains a replication origin, a
selective marker, a promoter, and, if necessary, may contain an
enhancer, a transcription termination sequence (terminator), a
ribosome-binding site, a polyadenylation signal, etc.
[0049] Where the vector is used for E.coli, the replication origin
is derived from ColE1, R factor, or F factor. Where the vector is
used for yeast, the replication origin is derived from, for
example, 2 .mu.m DNA or ARS1. Where the vector is used for a
mammalian cell, the replication origin is derived from, for
example, SV40, adenovirus, or bovine papilloma virus.
[0050] The promoter is a regulator sequence for directing a
synthesis of mRNA coding for the DNA of the invention.
Representative examples of the promoter include adenovirus or SV40
promoter, E.coli lac or trp promoter, phage .lamda.P.sub.L
promoter, ADH, PHO5, GPD, PGK or AOX1 promoter (for yeast), and a
promoter derived from nuclear polyhedrosis virus (for Bombyx mori
cell).
[0051] The selective marker is a gene for providing a phenotype to
the host in order to select transformed host cells. Exemplary
selective markers include kanamycin-resistant gene,
ampicillin-resistant gene, tetracycline-resistant gene, and the
like (when the vector is used for E.coli); Leu2, Trp1, Ura3 genes,
and the like (when the vector is used for yeasts); and
neomycin-resistant gene, thymidine kynase gene, dihydrofolate
reductase gene, and the like (when the vector is used for mammalian
cells).
[0052] Commercially available vectors may be used such as pQE70,
pQE60, pQE-9 (Qiagen), pBluescript II KS, ptrc99a, pKK223-3,
pDR540, pRIT2T (Pharmacia), and pET-11a (Novagen) as bacterium
vectors; and pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG and pSVL
SV40 (Pharmacia) as eukaryote vectors.
[0053] The DNA of the invention may be introduced into the vector
by any means. The vector preferably contains a polylinker with
various restriction sites, or a unique restriction site. The DNA of
the invention is inserted into a particular restriction site(s) of
the vector where it has been cleaved with a particular restriction
endonuclease(s).
[0054] The expression vector containing the DNA of the invention
with a regulatory sequence is used to transform or transfect a
suitable host cell, thereby expressing and producing the human
bradeion protein of the invention or an analogue thereof in the
host cell.
[0055] The host cell is, for example, a bacterial cell (e.g.,
E.coli, streptomyces, or Bacillus subtilis), an eukaryotic cell
(e.g., Aspergillus strain), an yeast cell (e.g., Saccharomyces
cerevisiae, or methanol-assimilating yeast), an insect cell (e.g.,
Drosophila S2 or Spodoptera Sf9), and a mammalian cell including
cultured human cell (e.g., CHO, COS, BHK, 3T3, or C127).
[0056] Transformation or tranfection may be conducted by a known
method such as calcium chloride/rubidium chloride method, calcium
phosphate method, DEAE-dextran-mediated transfection, or
electroporation.
[0057] The human-derived bradeion or an analogue thereof of the
invention can be obtained by culturing the host cells which have
been transformed or transfected as described above under the
control of the promoter, and by collecting the produced protein of
interest. The host cell is amplified or grown to a proper cell
density. Then, the promoter is induced by shifting the temperature
or by chemical induction (with IPTG, etc.). The cell is further
cultured for a predetermined period. Where the protein of interest
is secreted extracellularly, it can directly be obtained from the
medium. Where the protein of interest is present intracellularly,
the cell can be disrupted by physical means (e.g., sonication or
mechanical disruption) or by chemical means (e.g., lyzozyme or
cytolytic agent). Then the protein of interest is purified. The
protein may partially or completely be purified from the culture
medium containing the recombinant cells or an extracted solution
thereof, by using routine techniques such as ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, hydrophobic interaction chromatography, affinity
chromatography, gel filtration chromatography, HPLC,
electrophoresis, and chromatofocusing, alone or in combination.
[0058] The DNA of the invention was gene-transferred into several
human cell lines or cancer cell lines to examine the functions of
the bradeion proteins. As a result, in addition to the
above-described findings, the following facts were found out:
[0059] (1) when the DNA of the invention is gene-transferred into
NT2 neuron (Stratagene), which is a cultured human undifferentiated
nerve cell, using Superfect reagent (Qiagen) and over-expressed,
the cell death is induced within 18-24 hours. [0060] (2) when the
DNA is similarly over-expressed in cultured human normal cells,
termination of the aging and cell division is induced. [0061] (3)
when the DNA is gene-transferred into a cultured cancer cell,
Bradeion .alpha. and Bradeion .beta. genes induce the programmed
cell death. [0062] (4) in the course of the induced cell death, the
bradeion protein is present in cytoplasm, and forms an
intracellular aggregate.
[0063] As a result of studies using cultured human cancer cell
lines, the expression of bradeion was detected to be specific for
colorectal cancer and malignant melanoma (skin cancer), suggesting
that it has strong preferability for tissues and cell types in
which bradeion genes are expressed.
[0064] Based on the above-described findings, the bradeion proteins
of the invention seem to allow survival of cranial nerve cells of
the central nervous system in non-dividing state via
neuro-stimulating transmission. It seems to be important that
over-expression of the bradeion genes controls cell fate: apoptosis
or carcinogenesis, and that normally the expression ratio of the
.alpha.-type to the .beta.-type is maintained (at a ratio of 10:1
in a normal cranial nerve cell). It was also suggested that
depending on changes in the expression ratio (e.g., 1:1), it may
induce development of cancer. Accordingly, the bradeion proteins
are presumed to play an important role in controlling cell fate
(termination, apoptosis, and/or carcinogenesis), and also to
determine long-term survival of cranial nerve cells in non-dividing
state in human adult central nervous system. Thus, the bradeion
proteins are useful for monitoring exfoliated nerve cells
associated with aging, studying necrosis of nerve cells that occurs
during brain ischemia and status epilepticus, and understanding the
survival mechanism of neurons and pathology of brain. The proteins
are also useful for producing novel medicines for treating genetic
diseases, and may be applied to genetic diagnosis and gene therapy
of cancers.
[0065] Thus, the proteins of the invention or fragments thereof,
antibodies to them, or the DNAs of the invention or fragments
thereof may be used for detecting or diagnosing cancers (e.g.,
human colorectal carcinoma and human skin cancer), for determining
the cell fate, and as a targeting molecule in colorectal cancer and
malignant melanoma.
[0066] For the above purposes, a polyclonal or monoclonal antibody
that is specific to the proteins of the invention is useful. The
antibody, preferably the monoclonal antibody, may be used for
diagnosis, vaccination and drug delivery systems.
[0067] Such antibodies may be produced, for example, by methods
described in Suguru Matsubasi et al., Biochemical Experimental
Methods 15, Introduction to Immunological Experiments, 1982, Japan
Scientific Societies Press, Tokyo, Japan; Tatsuo Iwasaki et al.,
Monoclonal antibody -Hybridoma and ELISA, 1987, Kodansha
Scientific, Tokyo, Japan.
[0068] The polyclonal antibody can be obtained as follows. First,
an antigen solution containing the protein of the invention or a
fragment thereof as an antigen is mixed with complete Freund's
adjuvant to form an emulsion. The emulsion is then subcutaneously
injected into a mammal such as rabbit, mouse, goat, bovine or
equine. After about 2 weeks, an antigen solution emulsified in
incomplete Freund's adjuvant is similarly injected into the animal,
which is then boosted if necessary, and blood is drawn from the
animal to collect anti-serum as the polyclonal antibody. The
anti-serum is further subjected to ammonium sulfate precipitation
or ion exchange chromatography using DEAE cellulose to obtain an
IgG fraction. The IgG is subjected to an affinity chromatography on
CNBr-activated Sephadex or Sepharose bound to the protein of the
invention or a fragment thereof to mono-specifically purify the
antibody of interest that is bound through immunologic
reaction.
[0069] The monoclonal antibody can be obtained as follows. First,
an antigen-adjuvant emulsion is prepared as described above, which
is then intraperitoneally injected into a mouse (e.g., BALB/c) for
immunization. The spleen was removed from the mouse to collect
spleen cells, which are then fused with a myeloma cell (e.g., X63
or NS-1) in the presence of polyethylene glycol (e.g., PEG 400).
Then, an antibody-producing hybridoma is selected in an HAT medium
to obtain the monoclonal antibody of interest by a cloning method
or alternatively from ascites obtained from the mouse after the
intraperitoneal injection. Humanized monoclonal antibody may also
be prepared by using known techniques as described in Teng et al.,
Proc. Nal. Acad. Sci. USA, 1983, 80:7308-7312, and Kozbor et al.,
Immunology Today, 1983, 4 (3): 72-79.
[0070] The above-described antibody may be used for diagnosing, for
example, cancers in a standard immunoassay such as an enzymatic
immunoassay, a radioimmunoassay or a fluorescent antibody
method.
[0071] Where the DNA of the invention or a fragment thereof is used
as a probe or a primer, at least 15, preferably at least 20, more
preferably at least 30 consecutive nucleotides derived from the
nucleotide sequence of SEQ ID NO:1 (the nucleotides 129-1943) or
SEQ ID NO:3 (the nucleotides 129-1562) can be prepared using an
automatic DNA synthesizer. The obtained nucleotide fragment is
labeled with an isotope, a fluorescent substance or the like,
thereby preparing a probe or a PCR primer for diagnosis. The
conditions employed for hybridization are, for example, described
in Sambrook et al., Molecular Cloning (supra), and F. M. Ausubel et
al., Short Protocols In Molecular Biology, Third Edition, John
Wiley & Sons.
EXAMPLES
[0072] Hereinafter, the present invention will be described by way
of examples. The present invention, however, is not intended to be
limited to these examples.
Example 1
Cloning and Sequencing of cDNA Coding for Human Bradeion
[0073] First, a cDNA library from a human adult brain was
constructed using the plasmid vector pCMV SPORT1 (Life
Technologies, Inc., USA) which is capable of linking with a CMV
promoter for expressing in an eukaryotic cell. The adult brain was
obtained from a 36-year-old white Caucasian American female, and
mRNA (poly(A) RNA) was extracted therefrom with TRIzol.RTM. reagent
(Life Technologies, Inc.) and purified with MESSAGEMAKER.RTM.
reagent (Life Technologies, Inc.). Then double stranded cDNA
synthesis and library construction were initiated by SuperScript
plasmid system.
[0074] The prepared mRNA (poly(A) RNA) was linked with NotI primer
adapter at its 3'-terminus. Then, a double stranded cDNA was
synthesized according to a standard method using Superscript II
reverse transcriptase and T4 DNA polymerase. The 5'-terminus of the
cDNA was linked with SalI adapter and 3'-terminus was treated with
NotI restriction enzyme so that the cDNA fragment had restriction
sites SalI and NotI at each end. The cDNA was separated in sizes by
gel filtration chromatography to select and fractionate cDNA having
a size of 1 kb or more. The obtained set of cDNA fragments was
inserted, by a standard method, into the plasmid vector pCMV SPORT1
that also had been cleaved with SalI and NotI, thereby producing
circular plasmids. These plasmids were introduced into E.coli DH12S
cells (Life Technologies, Inc.) by an electroporation method and
amplified to construct a library.
[0075] The resulting E.coli strains were grown on
ampicillin-containing LB agar medium to form colonies. Biodyne A
nylon membrane (Pall Corp., US) treated with 10 mM IPTG was placed
in close contact with the colonies and left at 37.degree. C. for 2
hours. The nylon membrane was reacted with the antibody CE5 that
specifically recognizes cranial nerve cells [Nature, 296, 34-38,
1982]. Positive clones were selected using picoBlue.RTM.
Immunoscreening Kit (Stratagene, US).
[0076] Plasmid DNA was collected from the obtained positive clones
to be used as a .sup.32P-labeled probe, which was hybridized with
nylon membranes with mRNAs specific for different human organs (MTN
blot, CLONTECH Lab., Inc.) to test whether the probe was specific
for the brain. The nucleotide sequences of the cDNAs were
determined by sequencing analysis and compared with homologous
sequences deposited with the GenBank. Only the one that was
completely novel was deposited with the GenBank as a sequence of
interest. The determined nucleotide sequence of the Bradeion
.alpha. cDNA is shown in SEQ ID NO: 1 (GenBank Accession No.
AB002110). The coding region was at the positions 129-1943. The
amino acid sequence of the Bradeion .alpha. determined based on
this nucleotide sequence is shown in SEQ ID NO:2. DNA containing
Bradeion .alpha. cDNA was deposited with the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology (Higashi 1-1-3, Tsukuba-shi, Ibaraki-ken 305-8566,
Japan) on Jul. 14, 1998 as FERM P-16897.
[0077] Based on the above-described sequence of Bradeion .alpha.
cDNA, 5'-terminal primers (described below) were synthesized to
systematically screen relevant genes. For this purpose, Gene
Trapper Positive Selection system (Life Technologies, Inc.) was
used to screen the above-described gene library with the
synthesized oligonucleotides and magnet beads. The sequences of the
oligonucleotides used were:
[0078] 5'-ctgagcaagttcgtgaaggatttc-3' (SEQ ID NO:5); and
[0079] 5'-cagtcctctgacaaccagcagta-3' (SEQ ID NO:6)
[0080] As a result, a gene was detected which was named Bradeion
.beta. and whose nucleotide sequence is shown in SEQ ID NO:3
(GenBank Accession No. AB008753). The coding region was at the
positions 129-1562. The amino acid sequence of Bradeion .beta.
determined based on this nucleotide sequence is shown in SEQ ID
NO:4.
Example 2
Characterization of Bradeion .alpha. and Bradeion .beta.
Proteins
(1) Hydropathy Analysis
[0081] The amino acid sequences of Bradeion .alpha. and .beta.
proteins determined in Example 1 were subjected to hydropathy
analysis by Kyte-Doolittle method (Kyte, J. and Doolittle, R. F.
J., J. Mol. Biol., 1982, 157 (1): 105-132). This analysis is one
method for predicting a high-order structure of a protein based on
its amino acid sequence, whereby the distributions of hydrophobic
and hydrophilic portions of the protein can be determined. This
analysis therefore allows to study the presence of a
three-dimentional structure or a transmembranous domain. FIGS. 1A
and 1B show the results of the analysis for the bradeion proteins
as well as the results for human-derived IL (interleukin) 2, IL3
and IL4 receptors and growth hormone receptor for comparison.
Referring to the figures, the proteins having the sequences of
growth hormone and cytokine receptors may be divided roughly into
three sections, i.e., an assembly of hydrophobic groups (calculated
as positive ("+") values) as a transmembranous portion (third
column from the left, shown in red), an extracellular portion
(second column from the left, shown in blue) preceding the
transmembranous portion, and a cytoplasmic portion (fourth column
from the left, shown in green) following the transmembranous
portion. This structure is common among all of the receptors
including the bradeion proteins of the invention. However, bradeion
proteins did not have a hydrophobic signal peptide (first column
from the left, shown in yellow).
[0082] As a result, the bradeion proteins of the invention were
found to be membrane proteins with a structure characteristic of an
interleukin receptor even in the structure of a transmembranous
portion in the amino acid sequences.
(2) Localization of Bradeion Proteins
[0083] Hybridization with the nylon membranes with mRNAs specific
for different human organs (MTN blot, CLONTECH Lab., Inc.)
indicated a high level expression only in the human adult brain,
and a low level of expression in the heart (.ltoreq.10% of the
expression level in the brain). No expression was seen in other
organs or in human fetus. Both of the .alpha.- and .beta.-types
were expressed in the adult brain. The difference in types was due
to the gene duplication in the adjacent area (17q23) of human
chromosome. A homologous gene sequence, but only one of the two
types of human bradeions (i.e., Bradeion .beta.) existed in the
mouse brain (94% homology).
(3) Experiment of Overexpression of Bradeion .alpha. and Bradeion
.beta. Genes in Cultured Human Cell Lines
[0084] Bradeion .alpha. and Bradeion .beta. DNAs were individually
gene-transferred into NT2 neuron (Stratagene, US), which is a
cultured human undifferentiated nerve cell line, with Superfect
reagent (QIAGEN, US) and over-expressed. The results are shown in
FIGS. 2A, 2B and 2C.
[0085] FIG. 2A shows images of the labeled cell observed 24 hours
after overexpression of Bradeion .alpha. gene (upper panels) and
Bradeion .beta. gene (lower panels). The left images show the
locations of Bradeion .alpha. and Bradeion .beta.. The center
images show their locations in mitochondria, and the right images
show the overlaid images of the left and center images. All of the
images were observed with a confocal laser microscope. As a result,
it was found that the location of Bradeion .alpha. was consistent
with the location in mitochondria (Note: yellow color indicates an
overlap of red and green.) while the location of Bradeion .beta.
was not consistent with the location in mitochondria.
[0086] Programmed cell death (apoptosis) was induced within 18-24
hours after the transfection of the Bradeion .alpha. gene. In the
course of the apoptosis, Bradeion .alpha. formed an intracellular
aggregate. FIG. 2B shows cell images observed at predetermined
points of time after the gene transfer. The left images show the
cultured human cell NT2 neuron (Stratagene) and the right images
show the human cancer cell line, HeLa. Both cell lines formed
intracellular aggregates, resulting in cell death. To confirm this
fact, the cells of FIG. 2B were observed with an electron
microscope. As shown in FIG. 2C, the presence of apoptosis
corpuscles specific for programmed cell death (apoptosis) was
confirmed.
(4) Correlation of Bradeion .alpha. and Bradeion .beta. Genes to
Cancers
[0087] Although the Bradeion .alpha. and Bradeion .beta. genes are
only expressed in the normal adult brain and the heart (about 10%
of the expression level of the brain), their expression was also
found in cultured human cancer cell lines. The results are shown in
FIGS. 3A, 3B and 3C.
[0088] FIG. 3A shows the results of Northern blot regarding
expression of Bradeion .alpha. and Bradeion .beta. genes in
different cultured human cancer cells. Specific expressions
(signals) were found only in Lane 8 (skin cancer cell line G361)
and Lane 6 (colorectal adenocarcinoma SW480).
[0089] Specimens from human patients (i.e., specimens from
pathologic tissues) were used for detection of the cancer-specific
expression. As shown in FIG. 3B, the specific expression was
observed in 10 specimens having colorectal adenocarcinoma (T1 to
T10; indicated as Ad), and in 3 specimens having skin cancer (T11
to T13; indicated as Muc and MM). FIG. 3C shows images of stained
cancer cells for confirming the cancer-specific expression.
[0090] The above results show that the Bradeion .alpha. and
Bradeion .beta. proteins and the genes encoding them can be used as
tumor-specific markers of colorectal cancer.
ADVANTAGEOUS EFFECT OF THE INVENTION
[0091] The bradeion proteins of the invention and the DNA encoding
them seem to allow survival of cranial nerve cells of the central
nervous system in non-dividing state via neuro-stimulating
transmission. Over-expression of the bradeion genes induces
apoptosis. Normally, the in vivo expression ratio of Bradeion
.alpha. and Bradeion .beta. proteins is maintained (at a ratio of
10:1 in a normal cranial nerve cell). It is also suggested that
depending on changes in the expression ratio (e.g., 1:1), the
development of a cancer may be induced. Accordingly, the bradeion
proteins and the DNA encoding them were presumed to act as a
cell-lifetime-prolonging, cancer-development-suppressing factor
that determines long-term survival of cranial nerve cells in
non-dividing state after the development/differentiation of the
cells. Thus, they are useful for monitoring exfoliated nerve cells
associated with the aging, studying the necrosis of nerve cells
that occurs during brain ischemia and status epilepticus, and
understanding the survival mechanism of the central nerve cells and
pathology of brain. They are also useful for producing novel
medicines for treating genetic diseases, and may be applied to
genetic diagnosis and gene therapy of cancers.
[0092] All publications (including patent application) cited herein
are incorporated herein by reference in their entirety.
[0093] The following are information on SEQ ID NOS:1 to 4 described
herein: TABLE-US-00001 gaaaggagca agccaggaag ccagacaaca acagcatcaa
aacaaggctg tttctgtgtg 60 SEQ ID NO:1 tgaggaactt tgcctgggag
ataaaattag acctagagct ttctgacagg gagtctgaag 120 cgtgggacat
ggaccgttca ctgggatggc aagggaattc tgtccctgag gacaggactg 180
aacctgggat caaccgtttc ctggaggaca ccacggatga tggagaactg agcaagttcg
240 tgaaggattt ctcaggaaat gcgagctgcc acccaccaga ggctaagacc
tgggcatcca 300 ggccccaagt cccggagcca aggccccagg ccccggacct
ctatgatgat gacctggagt 360 tcagaccccc ctcgcggccc cagtcctctg
acaaccagca gtacttctgt gccccagccc 420 ctctcagccc atctgccagg
ccccgcagcc catgggggga gcttgatccc tatgattcct 480 ctgaggtaga
gcctccagcc ctgcctttgc ctttcagtgg gctgctgcag gaagaccggg 540
ggcagggagc aggaatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtttgtgt
600 gtgtgtgtat ctgggaccca tttcagtcct gtgtcagccc tagctccaaa
atatctgccc 660 ccaagggcac tggaaatttg cagtttcagc aagggcagga
ggcccagctg gtggcctcag 720 atgggaactc acagaagtct ggcactgctt
ttttaaggct ggggcaaagg cctgaaaggg 780 agagaagatt ggcgctgggt
gccggggccc ctttggctcc tcaccgtgat gcattctgcc 840 ttcctgtcta
ctacgatgac aaggagtatg tgggctttgc aaccctcccc aaccaagtcc 900
accgaaagtc cgtgaagaaa ggctttgact ttaccctcat ggtggcagga gagtctggcc
960 tgggcaaatc cacacttgtc aatagcctct tcctcactga tctgtaccgg
gaccggaaac 1020 ttcttggtgc tgaagaaagg atcatgcaaa ctgtggagat
cactaagcat gcagtggaca 1080 tagaaaaaaa aggtgtgagg ctgcggctca
ccattgtgga cacaccaagt tttggggatg 1140 cagtcaacaa cacagagtgt
atgtctgact ggaagcctgt ggcagaatac attgatcagc 1200 agtttgagca
gtatttccga gacgagagtg gcctgaaccg aaagaacatc caagacaaca 1260
gggtgcactg ctgcctgtac ttcatctcac ccttcggcca tgggctccgg ccattggatg
1320 ttgaattcat gaaggccctg catcagcggg tcaacatcgt gcctatcctg
gctaaggcag 1380 acacactgac acctcccgaa gtggaccaca agaaacgcaa
aatccgggag gagattgagc 1440 attttggaat caagatctat caattcccag
actgtgactc tgatgaggat gaggacttca 1500 aattgcagga ccaagcccta
aaggaaagca tcccatttgc agtaattggc agcaacactg 1560 tagtagaggc
cagagggcgg cgagttcggg gtcgactcta cccctggggc atcgtggaag 1620
tggaaaaccc agggcactgc gactttgtga agctgaggac aatgctggta cgtacccaca
1680 tgcaggacct gaaggatgtg acacgggaga cacattatga gaactaccgg
gcacagtgca 1740 tccagagcat gacccgcctg gtggtgaatg aacggaatcg
caagtatgac cagaagccag 1800 gacaaagctg gcagggggag atcccaagcc
tagccttggg tgagaccaag ccctactttt 1860 gttcttctat aggccctggg
ctcaatctaa gcgggtgctg gggtcctcct cgccttatca 1920 acccttttct
ccctttagca aactgactcg ggaaagtggt accgacttcc ccatccctgc 1980
tgtcccacca gggacagatc cagaaactga gaagcttatc ccagagaaag attaggagct
2040 gcggcggata cacgagatac tacaccaaat accaaaacag ataaaggaga
actatttact 2100 ggctttcagc cctggatatt taaatctcct cctcttcttc
ctgtccatgc cggcccctcc 2160 cagcaccagc tctgctcagg ccccttcagc
tactgccact tcgccttaca tccctgctga 2220 ctgcccagag actcagagga
aataaagttt aataaatctg taggtggctt ctgg 2274 Met Asp Arg Ser Leu Gly
Trp Gln Gly Asn Ser Val Pro Glu Asp Arg SEQ ID NO:2 1 5 10 15 Thr
Glu Pro Gly Ile Asn Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly 20 25
30 Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His
35 40 45 Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro
Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu
Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln
Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro Leu Ser Pro Ser Ala Arg
Pro Arg Ser Pro Trp Gly Glu Leu 100 105 110 Asp Pro Tyr Asp Ser Ser
Glu Val Glu Pro Pro Ala Leu Pro Leu Pro 115 120 125 Phe Ser Gly Leu
Leu Gln Glu Asp Arg Gly Gln Gly Ala Gly Met Cys 130 135 140 Val Cys
Val Cys Val Cys Val Cys Val Cys Val Phe Val Cys Val Cys 145 150 155
160 Ile Trp Asp Pro Phe Gln Ser Cys Val Ser Pro Ser Ser Lys Ile Ser
165 170 175 Ala Pro Lys Gly Thr Gly Asn Leu Gln Phe Gln Gln Gly Gln
Glu Ala 180 185 190 Gln Leu Val Ala Ser Asp Gly Asn Ser Gln Lys Ser
Gly Thr Ala Phe 195 200 205 Leu Arg Leu Gly Gln Arg Pro Glu Arg Glu
Arg Arg Leu Ala Leu Gly 210 215 220 Ala Gly Ala Pro Leu Ala Pro His
Arg Asp Ala Phe Cys Leu Pro Val 225 230 235 240 Tyr Tyr Asp Asp Lys
Glu Tyr Val Gly Phe Ala Thr Leu Pro Asn Gln 245 250 255 Val His Arg
Lys Ser Val Lys Lys Gly Phe Asp Phe Thr Leu Met Val 260 265 270 Ala
Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu Val Asn Ser Leu Phe 275 280
285 Leu Thr Asn Leu Tyr Arg Asp Arg Lys Leu Leu Gly Ala Glu Glu Arg
290 295 300 Ile Met Gln Thr Val Glu Ile Thr Lys His Ala Val Asp Ile
Glu Lys 305 310 315 320 Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp
Thr Pro Ser Phe Gly 325 330 335 Asp Ala Val Asn Asn Thr Glu Cys Met
Ser Asp Trp Lys Pro Val Ala 340 345 350 Glu Tyr Ile Asp Gln Gln Phe
Glu Gln Tyr Phe Arg Asp Glu Ser Gly 355 360 365 Leu Asn Arg Lys Asn
Ile Gln Asp Asn Arg Val His Cys Cys Leu Tyr 370 375 380 Phe Ile Ser
Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val Glu Phe 385 390 395 400
Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro Ile Leu Ala Lys 405
410 415 Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg Lys
Ile 420 425 430 Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln
Phe Pro Asp 435 440 445 Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu
Gln Asp Gln Ala Leu 450 455 460 Lys Glu Ser Ile Pro Phe Ala Val Ile
Gly Ser Asn Thr Val Val Glu 465 470 475 480 Ala Arg Gly Arg Arg Val
Arg Gly Arg Leu Tyr Pro Trp Gly Ile Val 485 490 495 Glu Val Glu Asn
Pro Gly His Cys Asp Phe Val Lys Leu Arg Thr Met 500 505 510 Leu Val
Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg Glu Thr 515 520 525
His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr Arg Leu 530
535 540 Val Val Asn Glu Arg Asn Arg Lys Tyr Asp Gln Lys Pro Gly Gln
Ser 545 550 555 560 Trp Gln Gly Glu Ile Pro Ser Leu Ala Leu Gly Glu
Thr Lys Pro Tyr 565 570 575 Phe Cys Ser Ser Ile Gly Pro Gly Leu Asn
Leu Ser Gly Cys Trp Gly 580 585 590 Pro Pro Arg Leu Ile Asn Pro Phe
Leu Pro Leu Ala Asn 595 600 605 gaaaggagca agccaggaag ccagacaaca
acagcatcaa aacaaggctg tttctgtgtg 60 SEQ ID NO:3 tgaggaactt
tgcctgggag ataaaattag acctagagct ttctgacagg gagtctgaag 120
cgtgggacat ggaccgttca ctgggatggc aagggaattc tgtccctgag gacaggactg
180 aagctgggat caagcgtttc ctggaggaca ccacggatga tggagaactg
agcaagttcg 240 tgaaggattt ctcaggaaat gcgagctgcc acccaccaga
ggctaagacc tgggcatcca 300 ggccccaagt cccggagcca aggccccagg
ccccggacct ctatgatgat gacctggagt 360 tcagaccccc ctcgcggccc
cagtcctctg acaaccagca gtacttctgt gccccagccc 420 ctctcagccc
atctgccagg ccccgcagcc catggggcaa gcttgatccc tatgattcct 480
ctgaggatga caaggagtat gtgggctttg caaccctccc caaccaagtc caccgaaagt
540 ccgtgaagaa aggctttgac tttaccctca tggtggcagg agagtctggc
ctgggcaaat 600 ccacacttgt caatagcctc ttcctcactg atctgtaccg
ggaccggaaa cttcttggtg 660 ctgaagagag gatcatgcaa actgtggaga
tcactaagca tgcagtggac atagaagaga 720 agggtgtgag gctgcggctc
accattgtgg acacaccagg ttttggggat gcagtcaaca 780 acacagagtg
ctggaagcct gtggcagaat acattgatca gcagtttgag cagtatttcc 840
gagacgagag tggcctgaac cgaaagaaca tccaagacaa cagggtgcac tgctgcctgt
900 acttcatctc acccttcggc catgggctcc ggccattgga tgttgaattc
atgaaggccc 960 tgcatcagcg ggtcaacatc gtgcctatcc tggctaaggc
agacacactg acacctcccg 1020 aagtggacca caagaaacgc aaaatccggg
aggagattga gcattttgga atcaagatct 1080 atcaattccc agactgtgac
tctgatgagg atgaggactt caaattgcag gaccaagccc 1140 taaaggaaag
catcccattt gcagtaattg gcagcaacac tgtagtagag gccagagggc 1200
ggcgagttcg gggtcgactc tacccctggg gcatcgtgga agtggaaaac ccagggcact
1260 gcgactttgt gaagctgagg acaatgctgg tacgtaccca catgcaggac
ctgaaggatg 1320 tgacacggga gacacattat gagaactacc gggcacagtg
catccagagc atgacccgcc 1380 tggtggtgaa ggaacggaat cgcaacaaac
tgactcggga aagtggtacc gacttcccca 1440 tccctgctgt cccaccaggg
acagatccag aaactgagaa gcttatccga gagaaagatg 1500 aggagctgcg
gcggatgcag gagatgctac acaaaataca aaaacagatg aaggagaact 1560
attaactggc tttcagccct ggatatttaa atctcctcct cttcttcctg tccatgccgg
1620 cccctcccag caccagctct gctcaggccc cttcagctac tgccacttcg
cctaacatcc 1680
ctgctgactg cccagagact cagaggaaat aaagtttaat aaatctgtag gtggc 1735
Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg SEQ
ID NO:4 1 5 10 15 Thr Glu Ala Gly Ile Lys Arg Phe Leu Glu Asp Thr
Thr Asp Asp Gly 20 25 30 Glu Leu Ser Lys Phe Val Lys Asp Phe Ser
Gly Asn Ala Ser Cys His 35 40 45 Pro Pro Glu Ala Lys Thr Trp Ala
Ser Arg Pro Gln Val Pro Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp
Leu Tyr Asp Asp Asp Leu Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro
Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro
Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Lys Leu 100 105 110
Asp Pro Tyr Asp Ser Ser Glu Asp Asp Lys Glu Tyr Val Gly Phe Ala 115
120 125 Thr Leu Pro Asn Gln Val His Arg Lys Ser Val Lys Lys Gly Phe
Asp 130 135 140 Phe Thr Leu Met Val Ala Gly Glu Ser Gly Leu Gly Lys
Ser Thr Leu 145 150 155 160 Val Asn Ser Leu Phe Leu Thr Asp Leu Tyr
Arg Asp Arg Lys Leu Leu 165 170 175 Gly Ala Glu Glu Arg Ile Met Gln
Thr Val Glu Ile Thr Lys His Ala 180 185 190 Val Asp Ile Glu Glu Lys
Gly Val Arg Leu Arg Leu Thr Ile Val Asp 195 200 205 Thr Pro Gly Phe
Gly Asp Ala Val Asn Asn Thr Glu Cys Val Lys Pro 210 215 220 Val Ala
Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu 225 230 235
240 Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys Cys
245 250 255 Leu Tyr Phe Ile Ser Pro Phe Gly His Gly Leu Arg Pro Leu
Asp Val 260 265 270 Glu Phe Met Lys Ala Leu His Gln Arg Val Asn Ile
Val Pro Ile Leu 275 280 285 Ala Lys Ala Asp Thr Leu Thr Pro Pro Glu
Val Asp His Lys Lys Arg 290 295 300 Lys Ile Arg Glu Glu Ile Glu His
Phe Gly Ile Lys Ile Tyr Gln Phe 305 310 315 320 Pro Asp Cys Asp Ser
Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln 325 330 335 Ala Leu Lys
Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn Thr Val 340 345 350 Val
Glu Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly 355 360
365 Ile Val Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys Leu Arg
370 375 380 Thr Met Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val
Thr Arg 385 390 395 400 Glu Thr His Tyr Glu Asn Tyr Arg Ala Gln Cys
Ile Gln Ser Met Thr 405 410 415 Arg Leu Val Val Lys Glu Arg Asn Arg
Asn Lys Leu Thr Arg Glu Ser 420 425 430 Gly Thr Asp Phe Pro Ile Pro
Ala Val Pro Pro Gly Thr Asp Pro Glu 435 440 445 Thr Glu Lys Leu Ile
Arg Glu Lys Asp Glu Glu Leu Arg Arg Met Asp 450 455 460 Glu Met Leu
His Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr 465 470 475
[0094]
Sequence CWU 1
1
6 1 2274 DNA Homo sapiens CDS (129)..(1943) 1 gaaaggagca agccaggaag
ccagacaaca acagcatcaa aacaaggctg tttctgtgtg 60 tgaggaactt
tgcctgggag ataaaattag acctagagct ttctgacagg gagtctgaag 120 cgtgggac
atg gac cgt tca ctg gga tgg caa ggg aat tct gtc cct gag 170 Met Asp
Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu 1 5 10 gac agg act
gaa cct ggg atc aac cgt ttc ctg gag gac acc acg gat 218 Asp Arg Thr
Glu Pro Gly Ile Asn Arg Phe Leu Glu Asp Thr Thr Asp 15 20 25 30 gat
gga gaa ctg agc aag ttc gtg aag gat ttc tca gga aat gcg agc 266 Asp
Gly Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser 35 40
45 tgc cac cca cca gag gct aag acc tgg gca tcc agg ccc caa gtc ccg
314 Cys His Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro
50 55 60 gag cca agg ccc cag gcc ccg gac ctc tat gat gat gac ctg
gag ttc 362 Glu Pro Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu
Glu Phe 65 70 75 aga ccc ccc tcg cgg ccc cag tcc tct gac aac cag
cag tac ttc tgt 410 Arg Pro Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln
Gln Tyr Phe Cys 80 85 90 gcc cca gcc cct ctc agc cca tct gcc agg
ccc cgc agc cca tgg ggg 458 Ala Pro Ala Pro Leu Ser Pro Ser Ala Arg
Pro Arg Ser Pro Trp Gly 95 100 105 110 gag ctt gat ccc tat gat tcc
tct gag gta gag cct cca gcc ctg cct 506 Glu Leu Asp Pro Tyr Asp Ser
Ser Glu Val Glu Pro Pro Ala Leu Pro 115 120 125 ttg cct ttc agt ggg
ctg ctg cag gaa gac cgg ggg cag gga gca gga 554 Leu Pro Phe Ser Gly
Leu Leu Gln Glu Asp Arg Gly Gln Gly Ala Gly 130 135 140 atg tgt gtg
tgt gtg tgt gtg tgt gtg tgt gtg tgt gtg ttt gtg tgt 602 Met Cys Val
Cys Val Cys Val Cys Val Cys Val Cys Val Phe Val Cys 145 150 155 gtg
tgt atc tgg gac cca ttt cag tcc tgt gtc agc cct agc tcc aaa 650 Val
Cys Ile Trp Asp Pro Phe Gln Ser Cys Val Ser Pro Ser Ser Lys 160 165
170 ata tct gcc ccc aag ggc act gga aat ttg cag ttt cag caa ggg cag
698 Ile Ser Ala Pro Lys Gly Thr Gly Asn Leu Gln Phe Gln Gln Gly Gln
175 180 185 190 gag gcc cag ctg gtg gcc tca gat ggg aac tca cag aag
tct ggc act 746 Glu Ala Gln Leu Val Ala Ser Asp Gly Asn Ser Gln Lys
Ser Gly Thr 195 200 205 gct ttt tta agg ctg ggg caa agg cct gaa agg
gag aga aga ttg gcg 794 Ala Phe Leu Arg Leu Gly Gln Arg Pro Glu Arg
Glu Arg Arg Leu Ala 210 215 220 ctg ggt gcc ggg gcc cct ttg gct cct
cac cgt gat gca ttc tgc ctt 842 Leu Gly Ala Gly Ala Pro Leu Ala Pro
His Arg Asp Ala Phe Cys Leu 225 230 235 cct gtc tac tac gat gac aag
gag tat gtg ggc ttt gca acc ctc ccc 890 Pro Val Tyr Tyr Asp Asp Lys
Glu Tyr Val Gly Phe Ala Thr Leu Pro 240 245 250 aac caa gtc cac cga
aag tcc gtg aag aaa ggc ttt gac ttt acc ctc 938 Asn Gln Val His Arg
Lys Ser Val Lys Lys Gly Phe Asp Phe Thr Leu 255 260 265 270 atg gtg
gca gga gag tct ggc ctg ggc aaa tcc aca ctt gtc aat agc 986 Met Val
Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu Val Asn Ser 275 280 285
ctc ttc ctc act gat ctg tac cgg gac cgg aaa ctt ctt ggt gct gaa
1034 Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu Gly Ala
Glu 290 295 300 gaa agg atc atg caa act gtg gag atc act aag cat gca
gtg gac ata 1082 Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys His
Ala Val Asp Ile 305 310 315 gaa aaa aaa ggt gtg agg ctg cgg ctc acc
att gtg gac aca cca agt 1130 Glu Lys Lys Gly Val Arg Leu Arg Leu
Thr Ile Val Asp Thr Pro Ser 320 325 330 ttt ggg gat gca gtc aac aac
aca gag tgt atg tct gac tgg aag cct 1178 Phe Gly Asp Ala Val Asn
Asn Thr Glu Cys Met Ser Asp Trp Lys Pro 335 340 345 350 gtg gca gaa
tac att gat cag cag ttt gag cag tat ttc cga gac gag 1226 Val Ala
Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu 355 360 365
agt ggc ctg aac cga aag aac atc caa gac aac agg gtg cac tgc tgc
1274 Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys
Cys 370 375 380 ctg tac ttc atc tca ccc ttc ggc cat ggg ctc cgg cca
ttg gat gtt 1322 Leu Tyr Phe Ile Ser Pro Phe Gly His Gly Leu Arg
Pro Leu Asp Val 385 390 395 gaa ttc atg aag gcc ctg cat cag cgg gtc
aac atc gtg cct atc ctg 1370 Glu Phe Met Lys Ala Leu His Gln Arg
Val Asn Ile Val Pro Ile Leu 400 405 410 gct aag gca gac aca ctg aca
cct ccc gaa gtg gac cac aag aaa cgc 1418 Ala Lys Ala Asp Thr Leu
Thr Pro Pro Glu Val Asp His Lys Lys Arg 415 420 425 430 aaa atc cgg
gag gag att gag cat ttt gga atc aag atc tat caa ttc 1466 Lys Ile
Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe 435 440 445
cca gac tgt gac tct gat gag gat gag gac ttc aaa ttg cag gac caa
1514 Pro Asp Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp
Gln 450 455 460 gcc cta aag gaa agc atc cca ttt gca gta att ggc agc
aac act gta 1562 Ala Leu Lys Glu Ser Ile Pro Phe Ala Val Ile Gly
Ser Asn Thr Val 465 470 475 gta gag gcc aga ggg cgg cga gtt cgg ggt
cga ctc tac ccc tgg ggc 1610 Val Glu Ala Arg Gly Arg Arg Val Arg
Gly Arg Leu Tyr Pro Trp Gly 480 485 490 atc gtg gaa gtg gaa aac cca
ggg cac tgc gac ttt gtg aag ctg agg 1658 Ile Val Glu Val Glu Asn
Pro Gly His Cys Asp Phe Val Lys Leu Arg 495 500 505 510 aca atg ctg
gta cgt acc cac atg cag gac ctg aag gat gtg aca cgg 1706 Thr Met
Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg 515 520 525
gag aca cat tat gag aac tac cgg gca cag tgc atc cag agc atg acc
1754 Glu Thr His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met
Thr 530 535 540 cgc ctg gtg gtg aat gaa cgg aat cgc aag tat gac cag
aag cca gga 1802 Arg Leu Val Val Asn Glu Arg Asn Arg Lys Tyr Asp
Gln Lys Pro Gly 545 550 555 caa agc tgg cag ggg gag atc cca agc cta
gcc ttg ggt gag acc aag 1850 Gln Ser Trp Gln Gly Glu Ile Pro Ser
Leu Ala Leu Gly Glu Thr Lys 560 565 570 ccc tac ttt tgt tct tct ata
ggc cct ggg ctc aat cta agc ggg tgc 1898 Pro Tyr Phe Cys Ser Ser
Ile Gly Pro Gly Leu Asn Leu Ser Gly Cys 575 580 585 590 tgg ggt cct
cct cgc ctt atc aac cct ttt ctc cct tta gca aac 1943 Trp Gly Pro
Pro Arg Leu Ile Asn Pro Phe Leu Pro Leu Ala Asn 595 600 605
tgactcggga aagtggtacc gacttcccca tccctgctgt cccaccaggg acagatccag
2003 aaactgagaa gcttatccca gagaaagatt aggagctgcg gcggatacac
gagatactac 2063 accaaatacc aaaacagata aaggagaact atttactggc
tttcagccct ggatatttaa 2123 atctcctcct cttcttcctg tccatgccgg
cccctcccag caccagctct gctcaggccc 2183 cttcagctac tgccacttcg
ccttacatcc ctgctgactg cccagagact cagaggaaat 2243 aaagtttaat
aaatctgtag gtggcttctg g 2274 2 605 PRT Homo sapiens 2 Met Asp Arg
Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg 1 5 10 15 Thr
Glu Pro Gly Ile Asn Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly 20 25
30 Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His
35 40 45 Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro
Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu
Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln
Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro Leu Ser Pro Ser Ala Arg
Pro Arg Ser Pro Trp Gly Glu Leu 100 105 110 Asp Pro Tyr Asp Ser Ser
Glu Val Glu Pro Pro Ala Leu Pro Leu Pro 115 120 125 Phe Ser Gly Leu
Leu Gln Glu Asp Arg Gly Gln Gly Ala Gly Met Cys 130 135 140 Val Cys
Val Cys Val Cys Val Cys Val Cys Val Phe Val Cys Val Cys 145 150 155
160 Ile Trp Asp Pro Phe Gln Ser Cys Val Ser Pro Ser Ser Lys Ile Ser
165 170 175 Ala Pro Lys Gly Thr Gly Asn Leu Gln Phe Gln Gln Gly Gln
Glu Ala 180 185 190 Gln Leu Val Ala Ser Asp Gly Asn Ser Gln Lys Ser
Gly Thr Ala Phe 195 200 205 Leu Arg Leu Gly Gln Arg Pro Glu Arg Glu
Arg Arg Leu Ala Leu Gly 210 215 220 Ala Gly Ala Pro Leu Ala Pro His
Arg Asp Ala Phe Cys Leu Pro Val 225 230 235 240 Tyr Tyr Asp Asp Lys
Glu Tyr Val Gly Phe Ala Thr Leu Pro Asn Gln 245 250 255 Val His Arg
Lys Ser Val Lys Lys Gly Phe Asp Phe Thr Leu Met Val 260 265 270 Ala
Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu Val Asn Ser Leu Phe 275 280
285 Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu Gly Ala Glu Glu Arg
290 295 300 Ile Met Gln Thr Val Glu Ile Thr Lys His Ala Val Asp Ile
Glu Lys 305 310 315 320 Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp
Thr Pro Ser Phe Gly 325 330 335 Asp Ala Val Asn Asn Thr Glu Cys Met
Ser Asp Trp Lys Pro Val Ala 340 345 350 Glu Tyr Ile Asp Gln Gln Phe
Glu Gln Tyr Phe Arg Asp Glu Ser Gly 355 360 365 Leu Asn Arg Lys Asn
Ile Gln Asp Asn Arg Val His Cys Cys Leu Tyr 370 375 380 Phe Ile Ser
Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val Glu Phe 385 390 395 400
Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro Ile Leu Ala Lys 405
410 415 Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg Lys
Ile 420 425 430 Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln
Phe Pro Asp 435 440 445 Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu
Gln Asp Gln Ala Leu 450 455 460 Lys Glu Ser Ile Pro Phe Ala Val Ile
Gly Ser Asn Thr Val Val Glu 465 470 475 480 Ala Arg Gly Arg Arg Val
Arg Gly Arg Leu Tyr Pro Trp Gly Ile Val 485 490 495 Glu Val Glu Asn
Pro Gly His Cys Asp Phe Val Lys Leu Arg Thr Met 500 505 510 Leu Val
Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg Glu Thr 515 520 525
His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr Arg Leu 530
535 540 Val Val Asn Glu Arg Asn Arg Lys Tyr Asp Gln Lys Pro Gly Gln
Ser 545 550 555 560 Trp Gln Gly Glu Ile Pro Ser Leu Ala Leu Gly Glu
Thr Lys Pro Tyr 565 570 575 Phe Cys Ser Ser Ile Gly Pro Gly Leu Asn
Leu Ser Gly Cys Trp Gly 580 585 590 Pro Pro Arg Leu Ile Asn Pro Phe
Leu Pro Leu Ala Asn 595 600 605 3 1735 DNA Homo sapiens CDS
(129)..(1562) 3 gaaaggagca agccaggaag ccagacaaca acagcatcaa
aacaaggctg tttctgtgtg 60 tgaggaactt tgcctgggag ataaaattag
acctagagct ttctgacagg gagtctgaag 120 cgtgggac atg gac cgt tca ctg
gga tgg caa ggg aat tct gtc cct gag 170 Met Asp Arg Ser Leu Gly Trp
Gln Gly Asn Ser Val Pro Glu 1 5 10 gac agg act gaa gct ggg atc aag
cgt ttc ctg gag gac acc acg gat 218 Asp Arg Thr Glu Ala Gly Ile Lys
Arg Phe Leu Glu Asp Thr Thr Asp 15 20 25 30 gat gga gaa ctg agc aag
ttc gtg aag gat ttc tca gga aat gcg agc 266 Asp Gly Glu Leu Ser Lys
Phe Val Lys Asp Phe Ser Gly Asn Ala Ser 35 40 45 tgc cac cca cca
gag gct aag acc tgg gca tcc agg ccc caa gtc ccg 314 Cys His Pro Pro
Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro 50 55 60 gag cca
agg ccc cag gcc ccg gac ctc tat gat gat gac ctg gag ttc 362 Glu Pro
Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe 65 70 75
aga ccc ccc tcg cgg ccc cag tcc tct gac aac cag cag tac ttc tgt 410
Arg Pro Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys 80
85 90 gcc cca gcc cct ctc agc cca tct gcc agg ccc cgc agc cca tgg
ggc 458 Ala Pro Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp
Gly 95 100 105 110 aag ctt gat ccc tat gat tcc tct gag gat gac aag
gag tat gtg ggc 506 Lys Leu Asp Pro Tyr Asp Ser Ser Glu Asp Asp Lys
Glu Tyr Val Gly 115 120 125 ttt gca acc ctc ccc aac caa gtc cac cga
aag tcc gtg aag aaa ggc 554 Phe Ala Thr Leu Pro Asn Gln Val His Arg
Lys Ser Val Lys Lys Gly 130 135 140 ttt gac ttt acc ctc atg gtg gca
gga gag tct ggc ctg ggc aaa tcc 602 Phe Asp Phe Thr Leu Met Val Ala
Gly Glu Ser Gly Leu Gly Lys Ser 145 150 155 aca ctt gtc aat agc ctc
ttc ctc act gat ctg tac cgg gac cgg aaa 650 Thr Leu Val Asn Ser Leu
Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys 160 165 170 ctt ctt ggt gct
gaa gag agg atc atg caa act gtg gag atc act aag 698 Leu Leu Gly Ala
Glu Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys 175 180 185 190 cat
gca gtg gac ata gaa gag aag ggt gtg agg ctg cgg ctc acc att 746 His
Ala Val Asp Ile Glu Glu Lys Gly Val Arg Leu Arg Leu Thr Ile 195 200
205 gtg gac aca cca ggt ttt ggg gat gca gtc aac aac aca gag tgc tgg
794 Val Asp Thr Pro Gly Phe Gly Asp Ala Val Asn Asn Thr Glu Cys Trp
210 215 220 aag cct gtg gca gaa tac att gat cag cag ttt gag cag tat
ttc cga 842 Lys Pro Val Ala Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr
Phe Arg 225 230 235 gac gag agt ggc ctg aac cga aag aac atc caa gac
aac agg gtg cac 890 Asp Glu Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp
Asn Arg Val His 240 245 250 tgc tgc ctg tac ttc atc tca ccc ttc ggc
cat ggg ctc cgg cca ttg 938 Cys Cys Leu Tyr Phe Ile Ser Pro Phe Gly
His Gly Leu Arg Pro Leu 255 260 265 270 gat gtt gaa ttc atg aag gcc
ctg cat cag cgg gtc aac atc gtg cct 986 Asp Val Glu Phe Met Lys Ala
Leu His Gln Arg Val Asn Ile Val Pro 275 280 285 atc ctg gct aag gca
gac aca ctg aca cct ccc gaa gtg gac cac aag 1034 Ile Leu Ala Lys
Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys 290 295 300 aaa cgc
aaa atc cgg gag gag att gag cat ttt gga atc aag atc tat 1082 Lys
Arg Lys Ile Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr 305 310
315 caa ttc cca gac tgt gac tct gat gag gat gag gac ttc aaa ttg cag
1130 Gln Phe Pro Asp Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu
Gln 320 325 330 gac caa gcc cta aag gaa agc atc cca ttt gca gta att
ggc agc aac 1178 Asp Gln Ala Leu Lys Glu Ser Ile Pro Phe Ala Val
Ile Gly Ser Asn 335 340 345 350 act gta gta gag gcc aga ggg cgg cga
gtt cgg ggt cga ctc tac ccc 1226 Thr Val Val Glu Ala Arg Gly Arg
Arg Val Arg Gly Arg Leu Tyr Pro 355 360 365 tgg ggc atc gtg gaa gtg
gaa aac cca ggg cac tgc gac ttt gtg aag 1274 Trp Gly Ile Val Glu
Val Glu Asn Pro Gly His Cys Asp Phe Val Lys 370 375 380 ctg agg aca
atg ctg gta cgt acc cac atg cag gac ctg aag gat gtg 1322 Leu Arg
Thr Met Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val 385 390 395
aca cgg gag aca cat tat gag aac tac cgg gca cag tgc atc cag agc
1370 Thr Arg Glu Thr His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln
Ser 400 405 410 atg acc cgc ctg gtg gtg aag gaa cgg aat cgc aac aaa
ctg act cgg 1418 Met Thr Arg Leu Val Val Lys Glu Arg Asn Arg Asn
Lys Leu Thr Arg 415 420 425 430 gaa agt ggt acc gac ttc ccc atc cct
gct gtc cca cca ggg aca gat 1466 Glu Ser Gly Thr Asp Phe Pro Ile
Pro Ala Val Pro Pro Gly Thr Asp 435 440 445 cca gaa act gag aag ctt
atc cga gag aaa gat gag gag ctg cgg cgg 1514 Pro Glu Thr Glu Lys
Leu Ile Arg Glu Lys Asp Glu Glu Leu Arg Arg 450 455 460 atg cag gag
atg cta cac aaa
ata caa aaa cag atg aag gag aac tat 1562 Met Gln Glu Met Leu His
Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr 465 470 475 taactggctt
tcagccctgg atatttaaat ctcctcctct tcttcctgtc catgccggcc 1622
cctcccagca ccagctctgc tcaggcccct tcagctactg ccacttcgcc taacatccct
1682 gctgactgcc cagagactca gaggaaataa agtttaataa atctgtaggt ggc
1735 4 478 PRT Homo sapiens 4 Met Asp Arg Ser Leu Gly Trp Gln Gly
Asn Ser Val Pro Glu Asp Arg 1 5 10 15 Thr Glu Ala Gly Ile Lys Arg
Phe Leu Glu Asp Thr Thr Asp Asp Gly 20 25 30 Glu Leu Ser Lys Phe
Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His 35 40 45 Pro Pro Glu
Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro 50 55 60 Arg
Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe Arg Pro 65 70
75 80 Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala
Pro 85 90 95 Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp
Gly Lys Leu 100 105 110 Asp Pro Tyr Asp Ser Ser Glu Asp Asp Lys Glu
Tyr Val Gly Phe Ala 115 120 125 Thr Leu Pro Asn Gln Val His Arg Lys
Ser Val Lys Lys Gly Phe Asp 130 135 140 Phe Thr Leu Met Val Ala Gly
Glu Ser Gly Leu Gly Lys Ser Thr Leu 145 150 155 160 Val Asn Ser Leu
Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu 165 170 175 Gly Ala
Glu Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys His Ala 180 185 190
Val Asp Ile Glu Glu Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp 195
200 205 Thr Pro Gly Phe Gly Asp Ala Val Asn Asn Thr Glu Cys Trp Lys
Pro 210 215 220 Val Ala Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe
Arg Asp Glu 225 230 235 240 Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp
Asn Arg Val His Cys Cys 245 250 255 Leu Tyr Phe Ile Ser Pro Phe Gly
His Gly Leu Arg Pro Leu Asp Val 260 265 270 Glu Phe Met Lys Ala Leu
His Gln Arg Val Asn Ile Val Pro Ile Leu 275 280 285 Ala Lys Ala Asp
Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg 290 295 300 Lys Ile
Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe 305 310 315
320 Pro Asp Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln
325 330 335 Ala Leu Lys Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn
Thr Val 340 345 350 Val Glu Ala Arg Gly Arg Arg Val Arg Gly Arg Leu
Tyr Pro Trp Gly 355 360 365 Ile Val Glu Val Glu Asn Pro Gly His Cys
Asp Phe Val Lys Leu Arg 370 375 380 Thr Met Leu Val Arg Thr His Met
Gln Asp Leu Lys Asp Val Thr Arg 385 390 395 400 Glu Thr His Tyr Glu
Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr 405 410 415 Arg Leu Val
Val Lys Glu Arg Asn Arg Asn Lys Leu Thr Arg Glu Ser 420 425 430 Gly
Thr Asp Phe Pro Ile Pro Ala Val Pro Pro Gly Thr Asp Pro Glu 435 440
445 Thr Glu Lys Leu Ile Arg Glu Lys Asp Glu Glu Leu Arg Arg Met Gln
450 455 460 Glu Met Leu His Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr
465 470 475 5 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 5 ctgagcaagt tcgtgaagga tttc 24 6 23 DNA Artificial
Sequence Description of Artificial Sequence Primer 6 cagtcctctg
acaaccagca gta 23
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