U.S. patent application number 10/411207 was filed with the patent office on 2004-01-01 for method for examining central nervous system diseases and method for screening therapeutic agents.
This patent application is currently assigned to Tanabe Seiyaku Co., Ltd.. Invention is credited to Sato, Naoya, Takagi, Tsutomu, Tohyama, Masaya.
Application Number | 20040002100 10/411207 |
Document ID | / |
Family ID | 15244561 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040002100 |
Kind Code |
A1 |
Takagi, Tsutomu ; et
al. |
January 1, 2004 |
Method for examining central nervous system diseases and method for
screening therapeutic agents
Abstract
The present invention relates to a method for screening and
identifying therapeutic agents or preventive agents for central
nervous system diseases which comprises assaying a suppressing
effect of a test substance on an expression of a splicing variant
transcribed from presenilin-2 gene and to a method for examining
central nervous system diseases which comprises detecting an
expression of a splicing variant transcribed from presenilin-2 gene
in a test sample originated from an animal individual.
Inventors: |
Takagi, Tsutomu; (Osaka,
JP) ; Sato, Naoya; (Osaka, JP) ; Tohyama,
Masaya; (Osaka, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Tanabe Seiyaku Co., Ltd.
|
Family ID: |
15244561 |
Appl. No.: |
10/411207 |
Filed: |
April 11, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10411207 |
Apr 11, 2003 |
|
|
|
09700847 |
Nov 21, 2000 |
|
|
|
6579679 |
|
|
|
|
09700847 |
Nov 21, 2000 |
|
|
|
PCT/JP99/02627 |
May 20, 1999 |
|
|
|
Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/6883 20130101; C07K 14/4711 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 1998 |
JP |
139408/1998 |
Claims
1. A method for screening and identifying a therapeutic agent or
preventive agent for central nervous system disease which comprises
assaying a suppressing effect of a test substance on an expression
of a splicing variant transcribed from presenilin-2 gene.
2. The method according to claim 1, wherein expression of a
splicing variant is the expression of a splicing variant in nerve
cell.
3. The method according to claim 2, wherein expression of a
splicing variant is induced by exposure of nerve cell to oxidative
stress.
4. The method according to claim 1, wherein expression of a
splicing variant is the expression of a splicing variant in animal
brain tissue.
5. The method according to claim 1, wherein said splicing variant
is a splicing variant lacking exon 5 of presenilin-2 gene.
6. The method according to claim 5, wherein said splicing variant
is a splicing variant lacking exon 5 and exon 8 of presenilin-2
gene.
7. The method according to claim 1, wherein said splicing variant
is a splicing variant lacking exon 3 of presenilin-2 gene.
8. The method according to claim 1, wherein said splicing variant
is a splicing variant lacking exon 3 and exon 4 of presenilin-2
gene and having a portion of an intron sequence.
9. The method according to any one of claims 1 to 6, wherein said
central nervous system disease is Alzheimer's disease.
10. A therapeutic agent or preventive agent for Alzheimer's disease
having an effect of suppressing an expression of a splicing variant
transcribed from presenilin-2 gene and lacking exon 5.
11. A therapeutic agent or preventive agent for central nervous
system disease screened or identified by any one of the methods
according to claims 1 to 8.
12. A therapeutic agent or preventive agent for Alzheimer's disease
screened or identified by any one of the methods according to
claims 1 to 8.
13. A method for detecting a splicing variant lacking exon 5 of
presenilin-2 gene comprising detecting nucleic acid containing at
least the 705th and 706th bases in the base sequence described in
SEQ ID NO: 2.
14. A method for detecting a splicing variant lacking exon 3 of
presenilin-2 gene comprising detecting nucleic acid containing at
least the 329th and 330th bases in the base sequence described in
SEQ ID NO: 3.
15. A method for detecting a splicing variant lacking exon 3 and
exon 4 of presenilin-2 gene and having a portion of an intron
comprising detecting nucleic acid containing at least the 330th to
409th bases in the base sequence described in SEQ ID NO: 4.
16. A nucleic acid having a base sequence which is identical or
complementary to a splicing variant lacking exon 5 of presenilin-2
gene.
17. The nucleic acid according to claim 16 having a base sequence
selected from (1) the base sequence described in SEQ ID NO: 2, (2)
the base sequence lacking the 995th to 1093rd bases in SEQ ID NO:
2, and (3) a base sequence complementary to said (1) or (2).
18. A recombinant plasmid containing the nucleic acid according to
claim 15.
19. A polypeptide originated from a splicing variant lacking exon 5
of presenilin-2 gene, said polypeptide either (1) having an amino
acid sequence described in the bottom of SEQ ID NO: 5, or (2)
having an amino acid sequence in which one or a plurality of the
amino acid residues are added, deleted or substituted in an amino
acid sequence described in SEQ ID NO: 5.
20. A DNA coding for the polypeptide according to claim 19.
21. A DNA according to claim 20 having the base sequence described
in the top of SEQ ID NO: 5.
22. A recombinant plasmid containing the DNA according to claim 20.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel splicing variants
of presenilin-2 along with a method for screening and a method for
identifying therapeutic agents for central nervous system diseases.
The present invention also relates to a method for examining
central nervous system diseases.
BACKGROUND ART
[0002] In the genome DNA of eucaryotes, sequences corresponding to
mature mRNA are frequently present separated at several locations.
In such cases, gene transcription is performed continuously over
the entire region, including those sequences corresponding to
mature mRNA, resulting in the production of precursor mRNA
(pre-mRNA) (transcription products also containing portions not
required by mature mRNA). This precursor mRNA is then subjected to
processing to become mature mRNA. During the course of processing,
simultaneous to the addition of a cap structure and poly A, those
portions not required by the mRNA (introns or intervening
sequences) are cut out, while the portions corresponding to the
mature mRNA (exons) are joined to form mature mRNA. These cutting
and joining processes are referred to as "splicing". Splicing is a
complicated and delicate process in which a plurality of severing
and coupling processes are regulated by the involvement of a large
protein-RNA aggregate referred to as a spliceosome. For example,
although different types of mature mRNA, namely "splicing
variants", are frequently formed due to a mutation and so forth
that occurs in the boundary region between an intron and exon on
the genome, some of these variants are known to give rise to
functionally abnormal mutant proteins that are capable of causing
disease. Research has been conducted thus far on splicing variants
in causative genes in order to elucidate the mechanism of
pathogenesis for various diseases and disorders, and splicing
variants have also been used as diagnostic markers for certain
diseases.
[0003] On the other hand, the amyloid precursor protein (APP) gene
on chromosome 21, the presenilin-1 (PS-1) gene on chromosome 14 and
the presenilin-2 (PS-2) gene on chromosome 1 have been previously
determined to be the major causative genes of familial Alzheimer's
disease (FAD). Subsequently, research has been conducted while
focusing on mutations and splicing variants and so forth discovered
in these causative genes on the relationship between these and the
mechanism of pathogenesis of Alzheimer's disease. However, very
little has been determined for the mechanism of pathogenesis of
sporadic Alzheimer's disease, which constitutes the majority of
Alzheimer's disease.
[0004] For example, mRNA originating in the brain tissue of
familiar or sporadic Alzheimer's disease patients and normal
subjects was analyzed for presenilin-1 gene (Sherrington et al.,
Nature, Vol. 375, pp. 754-760, 1995), and the existence of various
splicing variants has been reported (Clark et al,. Nature Genetics,
Vol. 11, pp. 219-222, 1995; Anwar et al., Journal of
Neurochemistry, Vol. 66, pp. 1774-1777, 1996).
[0005] Similar to presenilin-1 gene, presenelin-2 gene (Levy-Lahad
et al., Genomics, Vol. 34, pp. 198-204, 1996; Levy-Lahad et al.,
Science, Vol. 269, pp. 973-977, 1995; Rogaev et al., Nature, Vol.
376, pp. 775-778, 1995) is composed of 12 exons, and 10 of these
exons (exons 3-12) are known to code proteins. In addition, the
existence of splicing variants lacking exon 8 as well as splicing
variants simultaneously lacking exon 3 and exon 4 has been reported
in normal human tissue (Prihar et al., NeuroReport, Vol. 7, pp.
1680-1684, 1996). However, there have been no splicing variants
specific to Alzheimer's disease (and particularly sporadic
Alzheimer's disease) reported for presenilin-2 gene.
[0006] An object of the present invention is to provide a novel
method for screening and a novel method for identifying therapeutic
agents for central nervous system diseases (such as Alzheimer's
disease). In addition, an object of the present invention is to
provide a novel method for examining central nervous system
diseases (such as Alzheimer's disease). In addition, another object
of the present invention is to provide a novel splicing variant
originating in presenilin-2 that is useful in research on central
nervous system diseases.
[0007] The present inventors found that expression of abnormal
presenilin-2 mRNA splicing variants not found in the normal state
((i) splicing variant lacking exon 5, (ii) splicing variant lacking
exon 3, and (iii) splicing variant lacking both exon 3 and exon 4
and retaining a portion of an intron sequence) is induced by
exposure to oxidative stress or .beta.-amyloid stimulation in the
culture system of nerve cells. In addition, when the expression in
human brain tissue was investigated for one of these splicing
variants that is lacking exon 5, the present inventors made the
unique discovery that this splicing variant lacking exon 5 is
present at high frequency in the brain tissue of sporadic
Alzheimer's disease patients, thereby leading to completion of the
present invention.
DISCLOSURE OF THE INVENTION
[0008] Namely, the present invention is a method for screening
therapeutic agents or preventive agents for central nervous system
diseases, and a method for identifying the same, which comprises
assaying the suppressing effect of a test substance on the
expression of a splicing variant transcribed from presenilin-2
gene.
[0009] In addition, the present invention is a method for examining
central nervous system diseases which comprises detecting the
expression of a splicing variant transcribed from presenilin-2 gene
in a test sample originated from an animal individual.
[0010] Moreover, the present invention is a nucleic acid having a
base sequence originating in a splicing variant transcribed from
presenilin-2 gene (in the following, referred to as a presenilin-2
splicing variant) and a polypeptide coded by said splicing
variant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic drawing showing a PCR primer of PS-2
gene and PCR products originating in various splicing variants.
[0012] FIG. 2 is an electrophoresis drawing (in which black and
white colors have been reversed) showing induction of expression of
PS-2 splicing variant in human nerve cells. In the drawing, N
represents a control, H represents cells subjected to hypoxia, R
represents cells subjected to reoxygenation following exposure to
hypoxia, A.beta..sub.25-35 represents cells cultured with the
addition at 0.5 .mu.M or 5 .mu.M of A.beta..sub.25-35 (partial
peptide consisting of the 25th to 35th amino acid residues of
.beta.-amyloid protein), while H.sub.2O.sub.2 represents cells to
which were added 44 .mu.M or 440 .mu.M hydrogen peroxide.
[0013] FIG. 3 is a drawing showing the cDNA sequence containing all
exons of PS-2 (SEQ ID NO: 1), PCR primer and the positions of each
exon.
[0014] FIG. 4 is a drawing showing the sequence in the vicinity of
the splicing site of a splicing variant lacking exon 3 and exon 4
of PS-2 (residues 297-806 of SEQ ID NO: 4) and having a portion of
an intron sequence.
[0015] FIG. 5 is an electrophoresis drawing (in which black and
white colors have been reversed) showing the effect of a drug on
induction of expression of PS-2 splicing variant in human nerve
cells. In the drawing, N represents a control, H represents cells
subjected to hypoxia, CHX represents cells subjected to hypoxia
following addition of cycloheximide at 0.2 .mu.g/ml or 1 .mu.g/ml,
DPI represents cells subjected to hypoxia following addition of
diphenylene iodonium chloride at 5 .mu.M or 20 .mu.M, and NAC
represents cells subjected to hypoxia following addition of
N-acetyl-L-cysteine at 2 .mu.M or 10 .mu.M.
BEST MODE FOR CARRYING OUT THE INVENTION
[0016] In the present invention, a splicing variant lacking exon 5
refers to a splicing variant having a region in which exon 4 and
exon 6 are joined. A splicing variant lacking exon 3 refers to a
splicing variant having a region in which exon 2 and exon 4 are
joined. A splicing variant lacking exon 3 and exon 4 while having a
portion of an intron sequence refers to a splicing variant in which
a sequence originating in an intron (intron located between exon 4
and exon 5) is added downstream from exon 2 and having an exon 5
region downstream from that.
[0017] SEQ.ID.NO: 1 of the Sequence Listing described later and
FIG. 3 indicate the cDNA sequence (including all exons) of the
previously reported human presenilin-2 gene. SEQ.ID.NOS: 2, 3 and 4
indicate cDNA sequences corresponding to human presenilin-2
splicing variants (mRNA) newly discovered by the present inventors.
SEQ.ID.NO: 5 indicates the sequence of a mutant polypeptide coded
by a splicing variant.
[0018] SEQ.ID.NO: 2 indicates the cDNA sequence of a splicing
variant lacking exon 5 of human prenesilin-2 gene. The junction
site of exon 4 and exon 6 in this splicing variant is located
between bases 705th and 706th of SEQ.ID.NO: 2. The present
inventors also discovered a splicing variant that is lacking exon 8
in addition to lacking exon 5, and the cDNA of such a splicing
variant has a base sequence lacking bases 995th to 1093rd (region
equivalent to exon 8) in SEQ.ID.NO: 2.
[0019] SEQ.ID.NO: 3 indicates a cDNA sequence of a splicing variant
lacking exon 3 of human presenilin-2 gene. The junction site of
exon 2 and exon 4 is located between bases 329th and 330th of
SEQ.ID.NO: 3.
[0020] SEQ.ID.NO: 4 indicates the cDNA sequence of a splicing
variant that is lacking exon 3 and exon 4 of human presenilin-2
gene while simultaneously having a portion of an intron sequence.
The sequence originating in an intron (intron 4) present at the
junction region of exon 2 and exon 5 exists from bases 330th to
409th of SEQ.ID.NO: 4.
[0021] SEQ.ID.NO: 5 indicates the base sequence (upper part of
SEQ.ID.NO: 5) of the translation region of cDNA (SEQ.ID. NO: 2) of
a splicing variant lacking exon 5 of human presenilin-2 gene, and
the amino acid sequence (SEQ.ID.NO: 6) of a mutant polypeptide
coded by the above base sequence.
[0022] As a result of performing homology searching on all
sequences contained in known DNA databases (GenBank and EMBL) and
protein databases (NBRF and SWISS-PROT) for the base sequences or
amino acid sequences indicated in the above SEQ.ID.NOS: 2 through
6, there were no sequences that were completely identical, and
these sequences are considered to be novel sequences.
[0023] The methods of the present invention (examination method,
screening method and identification method) are applied to
Alzheimer's disease (familial or sporadic) (Alzheimer's senile
dementia) as well as other central nervous system diseases such as
diffuse Levi's microsomia and boxer's brain, and are particularly
preferably applied for Alzheimer's disease, and especially sporadic
Alzheimer's disease. The methods of the present invention are
applied to human diseases as well as diseases and disease models of
other mammals such as monkeys, dogs, rats and mice.
[0024] The methods of screening and identification of the present
invention are carried out by assaying the effect of suppressing
expression of a splicing variant of presenilin-2. More
specifically, screening or identification of a therapeutic agent or
preventive agent of central nervous system diseases can be carried
out by, for example, culturing cells or tissue in the presence of a
test substance or administering a test substance into an animal
individual, followed by detecting the expressed amount of
presenilin-2 splicing variant in the cells or tissue.
[0025] In the case of performing screening or identification using
a culture system, cells are cultured under conditions of, for
example, exposure to oxidative stress (exposure to hypoxia,
reoxygenation following exposure to hypoxia, addition of hydrogen
peroxide, etc.) or .beta.-amyloid stimulation. Since expression of
abnormal presenilin-2 splicing variants like (i) through (iii)
below, not observed in the normal state, are induced by culturing
conditions like those described above, these culturing systems can
be used as an in vitro pathological model to investigate the
effects of the test substance.
[0026] (i) Expression of a splicing variant lacking exon 5 is
induced by culturing cells with exposure to hypoxia. A variant
further lacking exon 8 is also contained in the expressed splicing
variant lacking exon 5.
[0027] (ii) Expression of a splicing variant lacking exon 3,
and
[0028] (iii) expression of a splicing variant lacking exon 3 and
exon 4 and having a portion of an intron sequence are induced by
reoxygenation following exposure to hypoxia, by addition of
hydrogen peroxide, and by .beta.-amyloid stimulation.
[0029] In the case the amount of expression of abnormal splicing
variants is decreased by the presence of a test substance in the
culture system as compared with the absence of that test substance,
that test substance is determined to have an effect that normalizes
the pathological state.
[0030] The splicing variant of presenilin-2 that is lacking exon 5
has actually been specifically found in the brain tissue of
Alzheimer's disease patients. Thus, a system that induces the
splicing variant lacking exon 5, for example, a cell culture system
that subjects cells to oxidative stress such as exposure to
hypoxia, can be preferably used for screening or identifying
therapeutic agents for Alzheimer's disease (and particularly
sporadic Alzheimer's disease).
[0031] Central nervous system cells (nerve cells, glia cells,
astrocytes, etc.) originating in mammals (humans, monkeys, etc.) as
well as undifferentiated cells that can be induced into
differentiating into nervous system cells and so forth can be used
for the cells used in the culture system. The cells may be primary
culture cells separated from animal tissue, or cancerated or
immortalized cell lines. Examples of cell lines include human
neuroblastoma SK--N--SH cells (ATCC HTB-11), human neuroblastoma
IMR-32 cells (ATCC CCL-127), human neuroblastoma SKN-MC cells (ATCC
HTB-10), and human kidney transformed cells 293 (ATCC
CRL-1573).
[0032] Exposure to hypoxia (hypoxic treatment) can be performed by,
for example, culturing cells for at least about 48 hours under
normal oxygen conditions (about 20% O.sub.2 and about 5% CO.sub.2),
transferring to an incubator under low oxygen conditions (O.sub.2
concentration: 1% or less, about 95% N.sub.2 and about 5% CO.sub.2)
and culturing for about 12-48 hours. Reoxygenation can be performed
by returning to normal culture conditions and culturing the cells
after subjected to hypoxic treatment as the above (Japanese
Laid-Open Patent Publication No. 9-238685; Ogawa et al., Journal of
Clinical Investigation, Vol. 85, pp. 1090-1098, 1990). Hydrogen
peroxide addition can be performed by adding hydrogen peroxide to
the medium to a final concentration of 44-440 .mu.M and culturing
for about 2 hours. In addition, .beta.-amyloid stimulation can be
performed by adding .beta.-amyloid protein or its partial peptide
(for example, A.beta..sub.25-35 (partial peptide consisting of the
25th to 35th amino acid residues of .beta.-amyloid protein) to a
concentration of 0.5-5 .mu.M, and culturing for about 16 hours.
[0033] An abnormal splicing variant of presenilin-2 can also be
used for examination of central nervous system diseases. The
examination method of the present invention can be carried out by
detecting the expression of a splicing variant of presenilin-2 in a
test sample originated from an animal individual. More
specifically, for example, a splicing variant of presenilin-2 in
RNA extracted from a test sample may be detected. Alternatively, a
mutant polypeptide, which is originated from a splicing variant, in
a test sample may be detected.
[0034] In Alzheimer's disease patients (and particularly sporadic
Alzheimer's disease patients), a splicing variant lacking exon 5 of
presenilin-2 is found in brain tissue at a frequency that is
clearly higher than that in normal individuals. Thus, in the case a
splicing variant lacking exon 5 of presenilin-2 is detected, that
patient can be determined to have a high risk for Alzheimer's
disease.
[0035] Examples of test samples include tissue and humor originated
from an animal individual. Particularly preferable examples of body
tissue include brain tissue including the central nervous system
(such as nerve cells, glia cells and astrocytes) and fibroblasts.
Examples of humor include serum and plasma.
[0036] In the methods of the present invention, in order to detect
expression of a splicing variant of presenilin-2, RNA (mRNA) may be
extracted from cells or tissue, and a splicing variant of
presenilin-2 in that RNA may be detected. Alternatively, expression
of a splicing variant may be detected indirectly by detecting
mutant polypeptide coded by the splicing variant.
[0037] Presenilin-2 gene has previously been cloned from humans,
rats, mice and so forth, and its base sequence and amino acid
sequence have been determined ((Human) GeneBank/EMBL Accession No.
L43964 and No. U50871, SWISS-PROT Accession No. P49810; Genomics,
Vol. 34, pp. 198-204, 1996; Science, Vol. 269, pp. 973-977, 1995;
Nature, Vol. 376, pp. 775-778, 1995: (Rat) Gene, Vol. 197, pp.
383-387, 1997; GeneBank/EMBL Accession No. D83700: (Mouse)
GeneBank/EMBL Accession No. AF038935). Thus, this sequence
information can be used for detection of a splicing variant. In
addition, information on the base sequences of each of the splicing
variants of human origin shown in the Sequence Listing described
later can also be used.
[0038] In the case of detecting a splicing variant in RNA, RNA
(mRNA) is prepared from cells or tissue, and the junction site
along with the regions before and after them resulting from
abnormal splicing are detected using that mRNA or cDNA prepared
from that mRNA. An ordinary polymerase chain reaction (PCR) method,
RNAase protection assay method or Northern blot analysis can be
used for detection. By the use of these methods, fragments
resulting from abnormal splicing are detected based on fragment
size.
[0039] In the case of using the PCR method ("PCR Protocols", Innis
M A, Gelfad D H, Sninsky J J and White T J, eds., Academic Press,
San Diego, 1990), suitable primers (sense primer and anti-sense
primer) are designed and synthesized in order to amplify the
fragment including the junction site along with the regions before
and after them resulting from abnormal splicing. By using these
primers and using cDNA synthesized from mRNA as the template to
perform PCR and isolating the resulting PCR products by
electrophoresis and so forth as necessary, a splicing variant can
be detected by investigating the fragment size. In addition, the
base sequences of the PCR products can be determined to identify
the splicing variant in detail.
[0040] The RNAase protection assay method (Nucleic Acid Research,
Vol. 12, pp. 7035-7056, 1984) uses RNAase having the property of
specifically decomposing single-strand RNA but not decomposing
double-strand RNA, and the anti-sense strand RNA for the RNA to be
examined is normally used as a probe. After hybridizing the RNA to
be examined and the labeled RNA probe, the RNAase is allowed to act
to decompose the non-hybridized RNA. This is then separated by
electrophoresis and so forth to detect and quantify the fragments.
Preparation of the RNA probe is performed by, for example,
amplifying a suitable region desired to be used as a probe by PCR
and so forth using cDNA synthesized from mRNA as the template, and
connecting the resulting fragment in a suitable direction
downstream from a promoter in a plasmid vector to construct a
vector for RNA probe synthesis. This vector can be used with RNA
polymerase such as T3, T7, SP6 or others to produce the RNA probe
in vitro. In the case of detecting a splicing variant of
presenilin-2, the region used for the probe is a region that
contains the junction site at which abnormal splicing occurs. For
example, the region that includes the region from exon 4 to exon 6
can be selected.
[0041] In addition to the methods described above, junction sites
that are newly formed as a result of abnormal splicing can be
detected directly using a nucleic acid probe. In this case, DNA
(oligonucleotide) complementary to a region that contains the
junction site and the regions on both sides is designed and
synthesized based on sequence information of the junction site to
be detected and its surrounding sequences, and this labeled DNA is
then used as a probe. Using the above probe, northern blot analysis
or southern blot analysis and so forth is performed on mRNA or its
cDNA obtained from test cells or tissue, or on a DNA fragment
amplified by PCR by using these as templates, after which a
judgment is made as to whether or not nucleic acid that hybridizes
with the probe is present.
[0042] In the case of detecting a splicing variant indirectly by
detecting a mutant polypeptide, a method can be used in which, for
example, detection is made immunochemically using an antibody that
specifically recognizes the mutant polypeptide. An antibody that
reacts with the mutant polypeptide but does not cross-react with
normal presenilin-2 can be used as the specific antibody. An
antigen, for example, prepared from the mutant polypeptide using
gene recombination technology can be used for the antigen for
preparing the antibody. Alternatively, a synthetic peptide may be
used for the antigen. For example, in the case of desiring to
detect a mutant polypeptide coded by a splicing variant lacking
exon 5, a specific antibody can be prepared by using as an antigen
a synthetic peptide and so forth of a suitable length that contains
the sequence of 6 amino acids on the C-terminal side of SEQ.ID.NO:
5.
[0043] Mutant polypeptide or normal presenilin-2 can be prepared
using a recombinant expression vector in which cDNA to a splicing
variant or normal mRNA of presenilin-2 gene is linked to a suitable
vector plasmid.
[0044] cDNA of presenilin-2 gene can be isolated and obtained, for
example, by using as gene sources the cDNA of mRNA prepared from
animal tissue or cells (such as central nervous system cells). In
the case of obtaining cDNA of a splicing variant, cells can be used
that have been cultured under conditions such as the above
oxidative stress (exposure to hypoxia, subjecting to reoxygenation
following exposure to hypoxia, addition of hydrogen peroxide, etc.)
or .beta.-amyloid stimulation. cDNA fragments are obtained from
these gene sources by PCR using a primer designed on the basis of
known sequence information. Desired cDNA can also be obtained by
colony hybridization or plaque hybridization and so forth by using
as probes fragments amplified by PCR or synthetic oligonucleotides
and using a cDNA library prepared from the gene sources or a
commercially available library as necessary.
[0045] Examples of expression systems (host-vector systems) for
producing presenilin-2 or its mutant polypeptide include expression
systems such as mammalian cells, insect cells, yeast and bacteria.
In order to obtain functional protein, it is preferable to use
insect cells (Spodoptera frugiperda SF9, SF21, etc.) and mammalian
cells (monkey COS-7 cells, Chinese hamster CHO cells, human HeLa
cells, etc.) as hosts. An example of a vector in the case of using
insect cells for the host is Baculovirus vector. Examples of
vectors in the case of using mammalian cells for the host include
retrovirus vector, papilloma virus vector, vaccinia virus vector
and SV40 vector. Expression vectors can be constructed by inserting
cDNA into downstream of a suitable promoter in these vectors.
[0046] The desired polypeptide is produced by transforming host
cells with the resulting expression vector, and culturing the
transformant in a suitable medium. Polypeptide produced in the host
cells can be harvested by crushing the cells by a physical
technique using a frictional crushing device such as a Dyno Mill or
by a chemical technique such as lysozyme treatment, and isolating
from the resulting cell extract by known purification methods (such
as salting out method using inorganic salts, fractional
precipitation by organic solvent, adsorption/desorption methods
using ion exchange resin or various types of column chromatography,
gel filtration and methods using protein precipitant) or a suitable
combination thereof.
[0047] An example of cDNA used for the DNA that codes for
presenilin-2 or its mutant polypeptide is cDNA of human origin
having the base sequences shown in SEQ.ID.NOS: 1-4 of the Sequence
Listing. In addition, genes and their alleles originated from other
species that exist in nature may also be used. In addition, base
sequences are not limited to those existing in nature, but rather
DNA corresponding to the amino acid sequence of a polypeptide can
also be designed and used. In this case, there are one to six types
of the codon that codes each amino acid. The codon to be used may
be selected arbitrarily, and a sequence having the highest
expression efficiency can be designed in consideration of the codon
usage frequency and so forth of the host being used.
[0048] In addition, a portion of the amino acid sequence of the
mutant polypeptide originated from a splicing variant may be
modified or altered provided the modification or alteration is to a
degree to which the mutant polypeptide still substantially retains
the equivalent immunogenicity or function. For example, an example
of a polypeptide originated from a splicing variant lacking exon 5
of human presenilin is that having the amino acid sequence shown in
SEQ.ID.NO: 5, and other examples include those having sequences in
which one or a plurality of amino acids are added, deleted or
substituted in that amino acid sequence. The number of amino acids
that are added, deleted or substituted is normally from 1 to about
20, preferably from 1 to about 10, and more preferably from 1 to
about 5. In this polypeptide, the homology of the amino acid
sequence with the amino acid sequence shown in SEQ.ID.NO: 5 is
usually at least 80%, preferably at least 90%, and more preferably
at least 95%.
[0049] DNA that codes the designed amino acid sequence can be
obtained by DNA chemical synthesis, fragmentation and coupling of
the above cDNA or partial alteration of the base sequence and so
forth. Partial alteration and mutation introduction of an
artificial base sequence can be performed by site-specific
mutagenesis using a primer comprising synthetic oligonucleotide
that codes for the desired alteration (Mark, D. F. et al.,
Proceedings of National Academy of Sciences, Vol. 81, pp. 5662-5666
(1984)).
[0050] In addition, abnormal splicing variants of presenilin-2
(particularly a splicing variant lacking exon 5) along with its
detection method are useful in pathological research on central
nervous system diseases (particularly Alzheimer's disease).
In-depth analysis of function in cells after inserting a
recombinant expression plasmid containing cDNA of an abnormal
splicing variant into those cells, and analysis of the function and
action of a mutant polypeptide originated from a splicing variant
are considered to lead to elucidation of the mechanism of
pathogenesis.
[0051] Although the following provides a more detailed explanation
of the present invention through its Examples, these Examples do
not limit the present invention.
[0052] Furthermore, in the following Examples, unless clearly
indicated otherwise, each procedure was performed according to the
methods described in "Molecular Cloning" (Sambrook, J., Fritsch, E.
F. and Maniatis, T. eds., published in 1989 by Cold Spring Harbor
Laboratory Press), or in the case of using commercially available
reagents or kits, such reagents or kits were used according to the
instructions provided with the commercially available products.
EXAMPLES
Example 1
[0053] Detection of Splicing Variants in Human Nerve Cells
[0054] (1) Culturing of Human Nerve Cells and Preparation of
RNA
[0055] After culturing human neuroblastoma SK--N--SH cells (ATCC
HTB-11) in .alpha.-MEM medium (.alpha.-Minimum Essential Medium;
available from GIBCO CO.) containing 10% fetal bovine serum in a 10
cm diameter culture plates at 37.degree. C. for about 48 hours in a
CO.sub.2 incubator to a confluent state, the medium was replaced
with the same medium not containing fetal bovine serum followed by
additional culturing for about 2 hours.
[0056] The above cultured cells were additionally cultured under
the conditions (i) to (iv) indicated below (culturing was continued
for an additional 16 hours under the same conditions as above for
the control).
[0057] (i) Hypoxic treatment: The culture plate was transferred to
a low oxygen incubator (oxygen concentration: 1% or less, 95%
N.sub.2, 5% CO.sub.2) (available from Coy Laboratory Products Co.)
and incubated for 16 hours.
[0058] (ii) Reoxygenation treatment: After transferring the culture
plate to a low oxygen incubator (oxygen concentration: 1% or less,
95% N.sub.2, 5% CO.sub.2) and incubating for 16 hours, the cell
plate was returned to an ordinary CO.sub.2 incubator (oxygen
concentration: 20%, 75% N.sub.2, 5% CO.sub.2) and incubated for 4
hours.
[0059] (iii) .beta.-amyloid stimulation: A.beta..sub.25-35
(available from Sigma Co.) was added to the medium to a final
concentration of 0.5 .mu.M or 5 .mu.M followed by incubating for 16
hours.
[0060] (iv) Hydrogen peroxide (H.sub.2O.sub.2) addition: Hydrogen
peroxide (H.sub.2O.sub.2) was added to the medium to a final
concentration of 44 .mu.M or 440 .mu.M followed by incubating for 2
hours.
[0061] Each of the cells cultured in the manner described above
were collected and washed with phosphate buffered saline (PBS).
After suspending the cells in 700 .mu.l of cell lysing buffer (RLT
solution, available from QIAGEN Co.), the cells were crushed to
obtain a cell extract. Total RNA was then prepared from this cell
extract. Preparation of RNA was performed using an RNA preparation
kit (trade name: Rneasy Total RNA Kit, available from QIAGEN
Co.).
[0062] (2) Detection of Splicing Variants
[0063] Using the RNA (total RNA, 1 .mu.g) obtained in section (1)
above as template, this RNA was allowed to react for 1 hour at
42.degree. C. in 0.05 ml of a buffer (0.05 M Tris-HCl, pH 8.3,
0.075 M KCl, 0.003 M MgCl.sub.2, DTT and 0.0002 M
deoxy-nucleotides) containing oligo dT primer (50 pmole), random
oligonucleotide (5 pmole) and reverse transcriptase (available from
Promega Co., Moloney leukaemia virus reverse transcriptase) (200
units) to synthesize single-strand cDNA. Using the resulting
single-strand cDNA, splicing variants were detected by polymerase
chain reaction (PCR) in the manner described below.
[0064] Four types of PCR primers consisting of PS251 (SEQ.ID. NO:
7) (sense primer), PS253 (SEQ.ID.NO: 8) (sense primer), PS233
(SEQ.ID.NO: 9) (anti-sense primer) and PS231 (SEQ.ID. NO: 10)
(anti-sense primer) were used for the PCR primers for presenilin-2
(PS-2) gene.
[0065] The location and direction on PS-2 gene corresponding to
each primer are shown in FIG. 1 and FIG. 3.
[0066] The first round of PCR reaction was performed using the
single-strand cDNA obtained above as the template, and using PS251
(SEQ.ID.NO: 7) (sense primer) and PS231 (SEQ.ID.NO: 10) (anti-sense
primer) as the primers for amplifying the entire coding region
(about 1.6 kbp) of PS-2 gene. PCR was performed under conditions of
95.degree. C. for about 40 seconds for the denaturing reaction,
72.degree. C. for 1 minute for elongation reaction, and 60.degree.
C. for about 30 seconds for annealing (number of cycles: 30
cycles).
[0067] The second round of PCR was performed after diluting the
reaction mixture 1:5 and using 1 .mu.l of that diluted liquid as
template. PS251 (SEQ.ID.NO: 7) (sense primer) and PS233 (SEQ.ID.NO:
9) (anti-sense primer) were used as PCR primers for detecting
5'-terminal fragments of the coding region of PS-2, while PS253
(SEQ.ID.NO: 8) (sense primer) and PS231 (SEQ.ID.NO: 10) (anti-sense
primer) were used as PCR primers for detecting 3'-terminal
fragments. PCR was performed under the same conditions as described
above. The sizes of the DNA fragments were investigated by
performing polyacrylamide gel electrophoresis on the resulting PCR
products.
[0068] Those results are shown in FIG. 2. As shown in FIG. 2, as a
result of performing PCR in which the 5'-terminal fragment was
amplified, a single band not observed in the control was detected
in the sample originated from cells subjected to hypoxic treatment.
In addition, two bands each not observed in the control were
detected in the samples originated from cells subjected to
reoxygenation following exposure to hypoxia and in the cells
stimulated with .beta.-amyloid, while three bands not observed in
the control were observed in the sample originated from cells
cultured following addition of hydrogen peroxide. In contrast, as a
result of performing PCR in which the 3'-terminal fragment was
amplified, two major bands each were similarly confirmed in all of
the samples, including the control.
[0069] On the basis of these results, it was considered that a
5'-terminal splicing abnormality occurred due to hypoxic treatment,
and at least one type of splicing variant was induced. In addition,
it was considered that a 5'-terminal splicing abnormality also
occurred due to reoxygenation following exposure to hypoxia and due
to .beta.-amyloid stimulation, and two types and three types,
respectively, of splicing variants were induced.
[0070] (3) Analysis of PS-2 Splicing Variants
[0071] The base sequences of the PS-2 splicing variants found in
section (2) above were determined in the manner described below.
Agarose gel electrophoresis was performed on the PCR products in
the same manner as in section (2) above, and DNA fragments thought
to be originated from splicing variants (5 types for the
5'-terminal fragments and 3 types for the 3'-terminal fragments)
were collected from the gel followed by coupling each to vector
plasmid pGEM-T (available from Promega Co.). The base sequences of
the inserted fragment portions were determined by the dideoxy
method using the resulting recombinant plasmids. Base sequences
were determined using an automated sequencer (373A DNA Sequencing
System) (available from Applied Biosystems Co.).
[0072] Those splicing variants confirmed as a result of determining
the base sequences are schematically illustrated in FIG. 1.
[0073] Of the two types of fragments found on the 3'-terminal, one
was a normal splicing product, and the other was a splicing variant
lacking exon 8. The existence of a splicing variant lacking exon 8
in normal tissue has been previously reported, and this deletion is
considered not to have an effect on the function of PS-2
protein.
[0074] On the other hand, the splicing variant found in the
5'-terminal fragment following hypoxic treatment was determined to
be a splicing variant lacking exon 5. A splicing variant lacking
exon 5 has never been previously reported in normal tissue. The
cDNA base sequence corresponding to this splicing variant (lacking
exon 5) is shown in SEQ.ID. NO: 2 of the Sequence Listing. The
junction site of exon 4 and exon 6 in this splicing variant is
located between bases 705th and 706th of SEQ.ID.NO: 2. A frame
shift occurs downstream from this junction site due to deletion of
exon 5 resulting in the presence of an open reading frame at bases
350th-724th. The base sequence of this open reading frame along
with the amino acid sequence of the mutant polypeptide (124 amino
acid residues) coded here are shown in SEQ.ID.NO: 5-6. The mutant
polypeptide has a sequence in which additional five amino acid
residues resulted from the frame shift are added to the C-terminal
portion of the 119 amino acid residues N-terminal portion of normal
presenilin-2 protein (448 amino acid residues).
[0075] In addition, a splicing variant also lacking exon 8 was also
present among the splicing variants lacking exon 5, and the cDNA of
this splicing variant has a base sequence in which bases
995th-1093rd (region equivalent to exon 8) is lacking in SEQ.ID.NO:
2.
[0076] One of the splicing variants found in the 5'-terminal
fragments following reoxygenation after exposure to hypoxia,
.beta.-amyloid stimulation and addition of hydrogen peroxide was a
splicing variant lacking exon 3, while another was a splicing
variant which, in addition to lacking exon 3 and exon 4, was
spliced inside of intron 4 (intron between exon 4 and exon 5).
[0077] The cDNA sequence of the splicing variant lacking exon 3 is
shown in SEQ.ID.NO: 3. The junction site of exon 2 and exon 5 is
located between bases 329th and 330th of SEQ.ID.NO: 3.
[0078] In addition, the cDNA sequence of the splicing variant
lacking exons 3 and 4 and having a portion of an intron sequence is
shown in SEQ.ID.NO: 4 and FIG. 4. The sequence derived from the
intron (intron 4) present at the junction region of exon 2 and exon
5 is at bases 330th-409th of SEQ.ID.NO: 4.
[0079] Although a splicing variant simultaneously lacking exon 3
and exon 4 has been previously reported in normal tissue, a variant
lacking only exon 3 as well as a splicing variant lacking exons 3
and 4 while also having a portion of an intron sequence have not
been reported in the past.
Example 2
[0080] Effect of Drug on Induction of the Expression of PS-2
Splicing Variants in Human Nerve Cells
[0081] (1) Culturing of Human Nerve Cells Under Hypoxic Conditions
and Preparation of RNA
[0082] Human neuroblastoma SK--N--SH cells were cultured under
hypoxic conditions as indicated below in compliance with section
(1) of the above Example 1. Namely, after culturing in .alpha.-MEM
medium containing 10% fetal bovine serum in a CO.sub.2 incubator
(oxygen concentration: 20%) at 37.degree. C. to a confluent state
(about 48 hours), the medium was replaced with the same medium not
containing fetal bovine serum. Cycloheximide (final concentration:
0.2 .mu.g/ml or 1 .mu.g/ml), N-acetyl-L-cysteine (final
concentration: 2 .mu.M or 10 .mu.M) or diphenylene iodonium
chloride (final concentration: 5 .mu.M or 20 .mu.M) was added to
the medium. The culture was then transferred to a low oxygen
incubator (oxygen concentration: 1% or less) and cultured for an
additional 16 hours.
[0083] Each of the cells cultured in the manner described above
were collected and RNA (total RNA) was prepared from the cells
according to the same method as section (1) of the above Example
1.
[0084] (2) Detection of PS-2 Splicing Variants
[0085] Detection of PS-2 splicing variants was performed according
to the same method as in section (2) of the above Example 1 using
the RNA (total RNA) obtained in section (1) above. Namely,
single-strand cDNA was prepared using the above RNA as template,
and a first round of PCR was performed using the resulting cDNA as
template to amplify the DNA fragment containing the entire length
of the PS-2 coding region. PS251 (SEQ.ID.NO: 7) (sense primer) and
PS231 (SEQ.ID.NO: 10) (anti-sense primer) were used as PCR
primers.
[0086] After diluting the reaction liquid and using 1 .mu.l thereof
as template, a second round of PCR was performed to amplify the DNA
fragment containing the 5'-terminal of the PS-2 coding region.
PS251 (SEQ.ID.NO: 7) (sense primer) and PS233 (SEQ.ID.NO: 9)
(anti-sense primer) were used as PCR primers. Polyacrylamide gel
electrophoresis was performed on the resulting PCR product to
investigate the presence or absence of fragments derived from
splicing variants (such as a fragment having a length of about 640
bases originated from a splicing variant lacking exon 5).
[0087] Those results are shown in FIG. 5. As shown in FIG. 5,
expression of a splicing variant lacking exon 5 induced by hypoxic
treatment was inhibited by treatment with cycloheximide, a protein
synthesis inhibitor. On the basis of this finding, it was
considered that protein newly produced during hypoxic treatment is
involved in induction of expression of a splicing variant lacking
exon 5. In addition, expression of a splicing variant lacking exon
5 was also inhibited by the antioxidants, N-acetyl-L-cysteine or
diphenylene iodonium chloride. On the basis of these findings, it
was considered that some form of oxidative stress in a hypoxic
state, such as active oxygen, is involved in the expression of PS-2
splicing variant lacking exon 5 that is induced by hypoxic
treatment.
Example 3
[0088] Expression of PS-2 Splicing Variants in Human Brain
Tissue
[0089] Detection of PS-2 splicing variant lacking exon 5 was
performed for samples of human brain tissue. Human brain tissue
samples consisted of a total of 47 samples collected from brains of
sporadic AD patients (30 cases) and normal brain subjects (17
cases) of nearly the same age.
[0090] After homogenizing the brain tissue test samples (frozen
storage samples), RNA was prepared from the resulting extract in
the same manner as section (1) of the above Example 1. Detection of
PS-2 splicing variants was performed using PCR in the same manner
as section (2) of Example 1 and section (2) of Example 2 using the
resulting RNA.
[0091] Those results are shown in Table 1. A splicing variant
lacking exon 5 was expressed in 3 of 17 normal brains (17%). In
contrast, this splicing variant was expressed in 21 of 30 brains of
sporadic AD patients (70%). Thus, it was determined that a splicing
variant lacking exon 5 is expressed at a clearly higher frequency
in the brains of sporadic AD patients than in normal brains.
1 TABLE 1 Presence or absence of PS- 2 splicing No. Case Age
variant N1 N 81 - N2 N 80 - N3 N 80 - N4 N 75 - N5 N 91 + N6 N 79 -
N7 N 93 - N8 N 89 - N9 N 81 - N10 N 84 + N11 N 92 - N12 N 92 - N13
N 92 - N14 N 88 - N15 N 86 - N16 N 84 + N17 N 85 - A1 AD 68 + A2 AD
91 + A3 AD 80 + A4 AD 82 + A5 AD 78 + A6 AD 87 - A7 AD 79 + A8 AD
62 + A9 AD 71 - A10 AD 84 + A11 AD 88 + A12 AD 81 + A13 AD 74 - A14
AD 75 - A15 AD 63 + A16 AD 74 + A17 AD 64 + A18 AD 86 + A19 AD 69 -
A20 AD 82 + A21 AD 91 - A22 AD 91 + A23 AD 97 + A24 AD 89 + A25 AD
70 + A26 AD 75 + A27 AD 69 - A28 AD 77 - A29 AD 83 + A30 AD 81 -
AD: Sporadic Alzheimer's disease N: Normal
[0092] Industrial Applicability
[0093] According to the method of the present invention, screening
and identification of therapeutic agents or preventive agents for
central nervous system diseases such as sporadic Alzheimer's
disease, which had been difficult in the past, can be carried out
efficiently. Since the point of action of drugs discovered or
identified by the screening method of the present invention is
clearly determined, they are advantageous for development as
pharmaceuticals.
[0094] In addition, diagnosis of central nervous system diseases,
particularly sporadic Alzheimer's disease for which diagnosis was
difficult in the past, can be carried out preferably.
[0095] In addition, abnormal splicing variants of presenilin-2 and
method for detecting thereof are useful in pathological research on
central nervous system diseases (particularly Alzheimer's disease)
as well. In-depth analysis of the intracellular function of
abnormal splicing variants, and analysis of the function and action
of mutant polypeptides originated from splicing variants, lead to
elucidation of the mechanism of pathogenesis.
Sequence CWU 1
1
10 1 2144 DNA Homo sapiens CDS (350)..(1696) 1 gcatttccag
cagtgaggag acagccagaa gcaagctatt ggagctgaag gaacctgaga 60
cagaagctag tcccccctct gaattttact gatgaagaaa ctgaggccac agagctaaag
120 tgacttttcc caaggtcgcc cagcgaggac gtgggacttc tcagacgtca
ggagagtgat 180 gtgagggagc tgtgtgacca tagaaagtga cgtgttaaaa
accagcgctg ccctctttga 240 aagccaggga gcatcattca tttagcctgc
tgagaagaag aaaccaagtg tccgggattc 300 agacctctct gcggccccaa
gtgttcgtgg tgcttccaga ggcagggct atg ctc aca 358 Met Leu Thr 1 ttc
atg gcc tct gac agc gag gaa gaa gtg tgt gat gag cgg acg tcc 406 Phe
Met Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu Arg Thr Ser 5 10 15
cta atg tcg gcc gag agc ccc acg ccg cgc tcc tgc cag gag ggc agg 454
Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln Glu Gly Arg 20
25 30 35 cag ggc cca gag gat gga gag aac act gcc cag tgg aga agc
cag gag 502 Gln Gly Pro Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg Ser
Gln Glu 40 45 50 aac gag gag gac ggt gag gag gac cct gac cgc tat
gtc tgt agt ggg 550 Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr
Val Cys Ser Gly 55 60 65 gtt ccc ggg cgg ccg cca ggc ctg gag gaa
gag ctg acc ctc aaa tac 598 Val Pro Gly Arg Pro Pro Gly Leu Glu Glu
Glu Leu Thr Leu Lys Tyr 70 75 80 gga gcg aag cac gtg atc atg ctg
ttt gtg cct gtc act ctg tgc atg 646 Gly Ala Lys His Val Ile Met Leu
Phe Val Pro Val Thr Leu Cys Met 85 90 95 atc gtg gtg gta gcc acc
atc aag tct gtg cgc ttc tac aca gag aag 694 Ile Val Val Val Ala Thr
Ile Lys Ser Val Arg Phe Tyr Thr Glu Lys 100 105 110 115 aat gga cag
ctc atc tac acg aca ttc act gag gac aca ccc tcg gtg 742 Asn Gly Gln
Leu Ile Tyr Thr Thr Phe Thr Glu Asp Thr Pro Ser Val 120 125 130 ggc
cag cgc ctc ctc aac tcc gtg ctg aac acc ctc atc atg atc agc 790 Gly
Gln Arg Leu Leu Asn Ser Val Leu Asn Thr Leu Ile Met Ile Ser 135 140
145 gtc atc gtg gtt atg acc atc ttc ttg gtg gtg ctc tac aag tac cgc
838 Val Ile Val Val Met Thr Ile Phe Leu Val Val Leu Tyr Lys Tyr Arg
150 155 160 tgc tac aag ttc atc cat ggc tgg ttg atc atg tct tca ctg
atg ctg 886 Cys Tyr Lys Phe Ile His Gly Trp Leu Ile Met Ser Ser Leu
Met Leu 165 170 175 ctg ttc ctc ttc acc tat atc tac ctt ggg gaa gtg
ctc aag acc tac 934 Leu Phe Leu Phe Thr Tyr Ile Tyr Leu Gly Glu Val
Leu Lys Thr Tyr 180 185 190 195 aat gtg gcc atg gac tac ccc acc ctc
ttg ctg act gtc tgg aac ttc 982 Asn Val Ala Met Asp Tyr Pro Thr Leu
Leu Leu Thr Val Trp Asn Phe 200 205 210 ggg gca gtg ggc atg gtg tgc
atc cac tgg aag ggc cct ctg gtg ctg 1030 Gly Ala Val Gly Met Val
Cys Ile His Trp Lys Gly Pro Leu Val Leu 215 220 225 cag cag gcc tac
ctc atc atg atc agt gcg ctc atg gcc cta gtg ttc 1078 Gln Gln Ala
Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe 230 235 240 atc
aag tac ctc cca gag tgg tcc gcg tgg gtc atc ctg ggc gcc atc 1126
Ile Lys Tyr Leu Pro Glu Trp Ser Ala Trp Val Ile Leu Gly Ala Ile 245
250 255 tct gtg tat gat ctc gtg gct gtg ctg tgt ccc aaa ggg cct ctg
aga 1174 Ser Val Tyr Asp Leu Val Ala Val Leu Cys Pro Lys Gly Pro
Leu Arg 260 265 270 275 atg ctg gta gaa act gcc cag gag aga aat gag
ccc ata ttc cct gcc 1222 Met Leu Val Glu Thr Ala Gln Glu Arg Asn
Glu Pro Ile Phe Pro Ala 280 285 290 ctg ata tac tca tct gcc atg gtg
tgg acg gtt ggc atg gcg aag ctg 1270 Leu Ile Tyr Ser Ser Ala Met
Val Trp Thr Val Gly Met Ala Lys Leu 295 300 305 gac ccc tcc tct cag
ggt gcc ctc cag ctc ccc tac gac ccg gag atg 1318 Asp Pro Ser Ser
Gln Gly Ala Leu Gln Leu Pro Tyr Asp Pro Glu Met 310 315 320 gaa gaa
gac tcc tat gac agt ttt ggg gag cct tca tac ccc gaa gtc 1366 Glu
Glu Asp Ser Tyr Asp Ser Phe Gly Glu Pro Ser Tyr Pro Glu Val 325 330
335 ttt gag cct ccc ttg act ggc tac cca ggg gag gag ctg gag gaa gag
1414 Phe Glu Pro Pro Leu Thr Gly Tyr Pro Gly Glu Glu Leu Glu Glu
Glu 340 345 350 355 gag gaa agg ggc gtg aag ctt ggc ctc ggg gac ttc
atc ttc tac agt 1462 Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp
Phe Ile Phe Tyr Ser 360 365 370 gtg ctg gtg ggc aag gcg gct gcc acg
ggc agc ggg gac tgg aat acc 1510 Val Leu Val Gly Lys Ala Ala Ala
Thr Gly Ser Gly Asp Trp Asn Thr 375 380 385 acg ctg gcc tgc ttc gtg
gcc atc ctc att ggc ttg tgt ctg acc ctc 1558 Thr Leu Ala Cys Phe
Val Ala Ile Leu Ile Gly Leu Cys Leu Thr Leu 390 395 400 ctg ctg ctt
gct gtg ttc aag aag gcg ctg ccc gcc ctc ccc atc tcc 1606 Leu Leu
Leu Ala Val Phe Lys Lys Ala Leu Pro Ala Leu Pro Ile Ser 405 410 415
atc acg ttc ggg ctc atc ttt tac ttc tcc acg gac aac ctg gtg cgg
1654 Ile Thr Phe Gly Leu Ile Phe Tyr Phe Ser Thr Asp Asn Leu Val
Arg 420 425 430 435 ccg ttc atg gac acc ctg gcc tcc cat cag ctc tac
atc tga 1696 Pro Phe Met Asp Thr Leu Ala Ser His Gln Leu Tyr Ile
440 445 gggacatggt gtgccacagg ctgcaagctg cagggaattt tcattggatg
cagttgtata 1756 gttttacact ctagtgccat atatttttaa gacttttctt
tccttaaaaa ataaagtacg 1816 tgtttacttg gtgaggagga ggcagaacca
gctctttggt gccagctgtt tcatcaccag 1876 actttggctc ccgctttggg
gagcgcctcg cttcacggac aggaagcaca gcaggtttat 1936 ccagatgaac
tgagaaggtc agattagggc ggggagaaga gcatccggca tgagggctga 1996
gatgcgcaaa gagtgtgctc gggagtggcc cctggcacct gggtgctctg gctggagagg
2056 aaaagccagt tccctacgag gagtgttccc aatgctttgt ccatgatgtc
cttgttattt 2116 tattgccttt agaaactgag tcctgttc 2144 2 2002 DNA Homo
sapiens CDS (350)..(724) 2 gcatttccag cagtgaggag acagccagaa
gcaagctatt ggagctgaag gaacctgaga 60 cagaagctag tcccccctct
gaattttact gatgaagaaa ctgaggccac agagctaaag 120 tgacttttcc
caaggtcgcc cagcgaggac gtgggacttc tcagacgtca ggagagtgat 180
gtgagggagc tgtgtgacca tagaaagtga cgtgttaaaa accagcgctg ccctctttga
240 aagccaggga gcatcattca tttagcctgc tgagaagaag aaaccaagtg
tccgggattc 300 agacctctct gcggccccaa gtgttcgtgg tgcttccaga
ggcagggct atg ctc aca 358 Met Leu Thr 1 ttc atg gcc tct gac agc gag
gaa gaa gtg tgt gat gag cgg acg tcc 406 Phe Met Ala Ser Asp Ser Glu
Glu Glu Val Cys Asp Glu Arg Thr Ser 5 10 15 cta atg tcg gcc gag agc
ccc acg ccg cgc tcc tgc cag gag ggc agg 454 Leu Met Ser Ala Glu Ser
Pro Thr Pro Arg Ser Cys Gln Glu Gly Arg 20 25 30 35 cag ggc cca gag
gat gga gag aac act gcc cag tgg aga agc cag gag 502 Gln Gly Pro Glu
Asp Gly Glu Asn Thr Ala Gln Trp Arg Ser Gln Glu 40 45 50 aac gag
gag gac ggt gag gag gac cct gac cgc tat gtc tgt agt ggg 550 Asn Glu
Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val Cys Ser Gly 55 60 65
gtt ccc ggg cgg ccg cca ggc ctg gag gaa gag ctg acc ctc aaa tac 598
Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr Leu Lys Tyr 70
75 80 gga gcg aag cac gtg atc atg ctg ttt gtg cct gtc act ctg tgc
atg 646 Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys
Met 85 90 95 atc gtg gtg gta gcc acc atc aag tct gtg cgc ttc tac
aca gag aag 694 Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr
Thr Glu Lys 100 105 110 115 aat gga cag ctt tca tcc atg gct ggt tga
tcatgtcttc actgatgctg 744 Asn Gly Gln Leu Ser Ser Met Ala Gly 120
ctgttcctct tcacctatat ctaccttggg gaagtgctca agacctacaa tgtggccatg
804 gactacccca ccctcttgct gactgtctgg aacttcgggg cagtgggcat
ggtgtgcatc 864 cactggaagg gccctctggt gctgcagcag gcctacctca
tcatgatcag tgcgctcatg 924 gccctagtgt tcatcaagta cctcccagag
tggtccgcgt gggtcatcct gggcgccatc 984 tctgtgtatg atctcgtggc
tgtgctgtgt cccaaagggc ctctgagaat gctggtagaa 1044 actgcccagg
agagaaatga gcccatattc cctgccctga tatactcatc tgccatggtg 1104
tggacggttg gcatggcgaa gctggacccc tcctctcagg gtgccctcca gctcccctac
1164 gacccggaga tggaagaaga ctcctatgac agttttgggg agccttcata
ccccgaagtc 1224 tttgagcctc ccttgactgg ctacccaggg gaggagctgg
aggaagagga ggaaaggggc 1284 gtgaagcttg gcctcgggga cttcatcttc
tacagtgtgc tggtgggcaa ggcggctgcc 1344 acgggcagcg gggactggaa
taccacgctg gcctgcttcg tggccatcct cattggcttg 1404 tgtctgaccc
tcctgctgct tgctgtgttc aagaaggcgc tgcccgccct ccccatctcc 1464
atcacgttcg ggctcatctt ttacttctcc acggacaacc tggtgcggcc gttcatggac
1524 accctggcct cccatcagct ctacatctga gggacatggt gtgccacagg
ctgcaagctg 1584 cagggaattt tcattggatg cagttgtata gttttacact
ctagtgccat atatttttaa 1644 gacttttctt tccttaaaaa ataaagtacg
tgtttacttg gtgaggagga ggcagaacca 1704 gctctttggt gccagctgtt
tcatcaccag actttggctc ccgctttggg gagcgcctcg 1764 cttcacggac
aggaagcaca gcaggtttat ccagatgaac tgagaaggtc agattagggc 1824
ggggagaaga gcatccggca tgagggctga gatgcgcaaa gagtgtgctc gggagtggcc
1884 cctggcacct gggtgctctg gctggagagg aaaagccagt tccctacgag
gagtgttccc 1944 aatgctttgt ccatgatgtc cttgttattt tattgccttt
agaaactgag tcctgttc 2002 3 1983 DNA Homo sapiens 3 gcatttccag
cagtgaggag acagccagaa gcaagctatt ggagctgaag gaacctgaga 60
cagaagctag tcccccctct gaattttact gatgaagaaa ctgaggccac agagctaaag
120 tgacttttcc caaggtcgcc cagcgaggac gtgggacttc tcagacgtca
ggagagtgat 180 gtgagggagc tgtgtgacca tagaaagtga cgtgttaaaa
accagcgctg ccctctttga 240 aagccaggga gcatcattca tttagcctgc
tgagaagaag aaaccaagtg tccgggattc 300 agacctctct gcggccccaa
gtgttcgtga gaagccagga gaacgaggag gacggtgagg 360 aggaccctga
ccgctatgtc tgtagtgggg ttcccgggcg gccgccaggc ctggaggaag 420
agctgaccct caaatacgga gcgaagcacg tgatcatgct gtttgtgcct gtcactctgt
480 gcatgatcgt ggtggtagcc accatcaagt ctgtgcgctt ctacacagag
aagaatggac 540 agctcatcta cacgacattc actgaggaca caccctcggt
gggccagcgc ctcctcaact 600 ccgtgctgaa caccctcatc atgatcagcg
tcatcgtggt tatgaccatc ttcttggtgg 660 tgctctacaa gtaccgctgc
tacaagttca tccatggctg gttgatcatg tcttcactga 720 tgctgctgtt
cctcttcacc tatatctacc ttggggaagt gctcaagacc tacaatgtgg 780
ccatggacta ccccaccctc ttgctgactg tctggaactt cggggcagtg ggcatggtgt
840 gcatccactg gaagggccct ctggtgctgc agcaggccta cctcatcatg
atcagtgcgc 900 tcatggccct agtgttcatc aagtacctcc cagagtggtc
cgcgtgggtc atcctgggcg 960 ccatctctgt gtatgatctc gtggctgtgc
tgtgtcccaa agggcctctg agaatgctgg 1020 tagaaactgc ccaggagaga
aatgagccca tattccctgc cctgatatac tcatctgcca 1080 tggtgtggac
ggttggcatg gcgaagctgg acccctcctc tcagggtgcc ctccagctcc 1140
cctacgaccc ggagatggaa gaagactcct atgacagttt tggggagcct tcataccccg
1200 aagtctttga gcctcccttg actggctacc caggggagga gctggaggaa
gaggaggaaa 1260 ggggcgtgaa gcttggcctc ggggacttca tcttctacag
tgtgctggtg ggcaaggcgg 1320 ctgccacggg cagcggggac tggaatacca
cgctggcctg cttcgtggcc atcctcattg 1380 gcttgtgtct gaccctcctg
ctgcttgctg tgttcaagaa ggcgctgccc gccctcccca 1440 tctccatcac
gttcgggctc atcttttact tctccacgga caacctggtg cggccgttca 1500
tggacaccct ggcctcccat cagctctaca tctgagggac atggtgtgcc acaggctgca
1560 agctgcaggg aattttcatt ggatgcagtt gtatagtttt acactctagt
gccatatatt 1620 tttaagactt ttctttcctt aaaaaataaa gtacgtgttt
acttggtgag gaggaggcag 1680 aaccagctct ttggtgccag ctgtttcatc
accagacttt ggctcccgct ttggggagcg 1740 cctcgcttca cggacaggaa
gcacagcagg tttatccaga tgaactgaga aggtcagatt 1800 agggcgggga
gaagagcatc cggcatgagg gctgagatgc gcaaagagtg tgctcgggag 1860
tggcccctgg cacctgggtg ctctggctgg agaggaaaag ccagttccct acgaggagtg
1920 ttcccaatgc tttgtccatg atgtccttgt tattttattg cctttagaaa
ctgagtcctg 1980 ttc 1983 4 1848 DNA Homo sapiens 4 gcatttccag
cagtgaggag acagccagaa gcaagctatt ggagctgaag gaacctgaga 60
cagaagctag tcccccctct gaattttact gatgaagaaa ctgaggccac agagctaaag
120 tgacttttcc caaggtcgcc cagcgaggac gtgggacttc tcagacgtca
ggagagtgat 180 gtgagggagc tgtgtgacca tagaaagtga cgtgttaaaa
accagcgctg ccctctttga 240 aagccaggga gcatcattca tttagcctgc
tgagaagaag aaaccaagtg tccgggattc 300 agacctctct gcggccccaa
gtgttcgtgc aggtccaaaa tcactcaagg tggggagcct 360 cgaggagcag
tcagggccgg gagcatcagc cccttgcctt ctccctcagc atctacacga 420
cattcactga ggacacaccc tcggtgggcc agcgcctcct caactccgtg ctgaacaccc
480 tcatcatgat cagcgtcatc gtggttatga ccatcttctt ggtggtgctc
tacaagtacc 540 gctgctacaa gttcatccat ggctggttga tcatgtcttc
actgatgctg ctgttcctct 600 tcacctatat ctaccttggg gaagtgctca
agacctacaa tgtggccatg gactacccca 660 ccctcttgct gactgtctgg
aacttcgggg cagtgggcat ggtgtgcatc cactggaagg 720 gccctctggt
gctgcagcag gcctacctca tcatgatcag tgcgctcatg gccctagtgt 780
tcatcaagta cctcccagag tggtccgcgt gggtcatcct gggcgccatc tctgtgtatg
840 atctcgtggc tgtgctgtgt cccaaagggc ctctgagaat gctggtagaa
actgcccagg 900 agagaaatga gcccatattc cctgccctga tatactcatc
tgccatggtg tggacggttg 960 gcatggcgaa gctggacccc tcctctcagg
gtgccctcca gctcccctac gacccggaga 1020 tggaagaaga ctcctatgac
agttttgggg agccttcata ccccgaagtc tttgagcctc 1080 ccttgactgg
ctacccaggg gaggagctgg aggaagagga ggaaaggggc gtgaagcttg 1140
gcctcgggga cttcatcttc tacagtgtgc tggtgggcaa ggcggctgcc acgggcagcg
1200 gggactggaa taccacgctg gcctgcttcg tggccatcct cattggcttg
tgtctgaccc 1260 tcctgctgct tgctgtgttc aagaaggcgc tgcccgccct
ccccatctcc atcacgttcg 1320 ggctcatctt ttacttctcc acggacaacc
tggtgcggcc gttcatggac accctggcct 1380 cccatcagct ctacatctga
gggacatggt gtgccacagg ctgcaagctg cagggaattt 1440 tcattggatg
cagttgtata gttttacact ctagtgccat atatttttaa gacttttctt 1500
tccttaaaaa ataaagtacg tgtttacttg gtgaggagga ggcagaacca gctctttggt
1560 gccagctgtt tcatcaccag actttggctc ccgctttggg gagcgcctcg
cttcacggac 1620 aggaagcaca gcaggtttat ccagatgaac tgagaaggtc
agattagggc ggggagaaga 1680 gcatccggca tgagggctga gatgcgcaaa
gagtgtgctc gggagtggcc cctggcacct 1740 gggtgctctg gctggagagg
aaaagccagt tccctacgag gagtgttccc aatgctttgt 1800 ccatgatgtc
cttgttattt tattgccttt agaaactgag tcctgttc 1848 5 375 DNA Homo
sapiens CDS (1)..(375) 5 atg ctc aca ttc atg gcc tct gac agc gag
gaa gaa gtg tgt gat gag 48 Met Leu Thr Phe Met Ala Ser Asp Ser Glu
Glu Glu Val Cys Asp Glu 1 5 10 15 cgg acg tcc cta atg tcg gcc gag
agc ccc acg ccg cgc tcc tgc cag 96 Arg Thr Ser Leu Met Ser Ala Glu
Ser Pro Thr Pro Arg Ser Cys Gln 20 25 30 gag ggc agg cag ggc cca
gag gat gga gag aac act gcc cag tgg aga 144 Glu Gly Arg Gln Gly Pro
Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg 35 40 45 agc cag gag aac
gag gag gac ggt gag gag gac cct gac cgc tat gtc 192 Ser Gln Glu Asn
Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val 50 55 60 tgt agt
ggg gtt ccc ggg cgg ccg cca ggc ctg gag gaa gag ctg acc 240 Cys Ser
Gly Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr 65 70 75 80
ctc aaa tac gga gcg aag cac gtg atc atg ctg ttt gtg cct gtc act 288
Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr 85
90 95 ctg tgc atg atc gtg gtg gta gcc acc atc aag tct gtg cgc ttc
tac 336 Leu Cys Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe
Tyr 100 105 110 aca gag aag aat gga cag ctt tca tcc atg gct ggt tga
375 Thr Glu Lys Asn Gly Gln Leu Ser Ser Met Ala Gly 115 120 6 124
PRT Homo sapiens 6 Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu
Val Cys Asp Glu 1 5 10 15 Arg Thr Ser Leu Met Ser Ala Glu Ser Pro
Thr Pro Arg Ser Cys Gln 20 25 30 Glu Gly Arg Gln Gly Pro Glu Asp
Gly Glu Asn Thr Ala Gln Trp Arg 35 40 45 Ser Gln Glu Asn Glu Glu
Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val 50 55 60 Cys Ser Gly Val
Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr 65 70 75 80 Leu Lys
Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr 85 90 95
Leu Cys Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr 100
105 110 Thr Glu Lys Asn Gly Gln Leu Ser Ser Met Ala Gly 115 120 7
20 DNA Homo sapiens 7 attcagacct ctctgcggcc 20 8 20 DNA Homo
sapiens 8 gcatggtgtg catccactgg 20 9 20 DNA Homo sapiens 9
ggaccactct gggaggtact 20 10 20 DNA Homo sapiens 10 gctggcacca
aagagctggt 20
* * * * *