U.S. patent application number 11/179624 was filed with the patent office on 2006-02-23 for ganp protein.
This patent application is currently assigned to IMMUNOKICK INCORPORATION. Invention is credited to Kazuhiko Kuwahara, Nobuo Sakaguchi.
Application Number | 20060040372 11/179624 |
Document ID | / |
Family ID | 12763928 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040372 |
Kind Code |
A1 |
Sakaguchi; Nobuo ; et
al. |
February 23, 2006 |
GANP protein
Abstract
The object of the present invention is to provide a novel
protein having a kinase activity and a gene encoding said protein.
According to the present invention, there is provided a GANP
protein which is represented by the arnino acid sequence shown in
SEQ ID No. 1 or No. 3 of the sequence listing, and is involved in
the signal conversion of abnormal B cell differentiation in an
autoimmune state, and has a kinase activity, and a polynucleotide
which encodes said protein.
Inventors: |
Sakaguchi; Nobuo; (Kumamoto,
JP) ; Kuwahara; Kazuhiko; (Kumamoto, JP) |
Correspondence
Address: |
MCDERMOTT WILL & EMERY LLP
600 13TH STREET, N.W.
WASHINGTON
DC
20005-3096
US
|
Assignee: |
IMMUNOKICK INCORPORATION
Kumamoto
JP
|
Family ID: |
12763928 |
Appl. No.: |
11/179624 |
Filed: |
July 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10747133 |
Dec 30, 2003 |
6943237 |
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11179624 |
Jul 13, 2005 |
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09914272 |
Dec 5, 2001 |
6673913 |
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PCT/JP99/04634 |
Aug 27, 1999 |
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10747133 |
Dec 30, 2003 |
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Current U.S.
Class: |
435/194 ;
435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
C07K 16/40 20130101;
C12N 9/1205 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/194 ;
530/388.26; 536/023.2; 435/069.1; 435/320.1; 435/325 |
International
Class: |
C12N 9/12 20060101
C12N009/12; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 16/40 20060101 C07K016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 24, 1999 |
JP |
47035/1999 |
Claims
1-9. (canceled)
10. An antibody which recognizes the protein having the amino acid
sequence shown in SEQ ID NO: 3.
11. An antibody of a variant protein having a kinase activity
substantially similar to an isolated protein having the amino acid
sequence shown in SEQ ID NO: 3, wherein 1-20 amino acids are
deleted, substituted, and/or added.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel protein having a
kinase activity and a gene encoding said protein.
BACKGROUND ART
[0002] Antigen binding to the membrane lgR initiates the activation
and maturation of the antigen-specific B cells in the peripheral
lymphoid organs (Rajewsky, Nature (Lond.)., 381:751-758, 1996;
Sakaguchi et al., Adv. Immunol. 54:337-392, 1993). B cells enter
the outer periarterial lymphoid sheath (PALS) (Rajewsky, Nature
(Lond.)., 381:751-758, 1996) and initiate costimulus-dependent
interactions with specific Th cells and interdigitating dendritic
cells within 48 h after immunization (MacLennan, Annu. Rev.
Immunol. 12:117-139, 1994; Liu et al., Immunol. Rev. 156:111-126,
1997). Antigen-driven B cells proliferate in the outer PALS and
then undergo further activation in the lymphoid follicles to
establish the germinal center (herein sometimes abbreviated as GC)
(Han et al., J. Immunol. 155:556-567, 1995; Jacob et al., J. Exp.
Med. 176:679-687, 1992; Kelsoe, Immunity 4:107-111, 1996). Such B
cells mature into large slg-centroblasts that rapidly move through
the cell cycle to form the dark zone and further mature into
centrocytes that express a unique surface character of
PNA.sup.+B220.sup.+slgM.sup.+slgD.sup.-CD38.sup.- in the light zone
of the GC (Kosco-Vilbois et al., 1997. Immunol. Today 18:225-230,
1997; Kelsoe, Immunol. Today 16:324-326, 1995; Oliver et al., J.
Immunol. 158:1108-1115, 1997).
[0003] Centrocytes presumably undergo the processes of either
apoptosis or affinity maturation of immunoglobulin V regions and
the change process of class switching toward the lgG class antigen.
Some centrocytes survive for a longer period in the lymphoid
compartment as memory B cells. The other centrocytes probably
migrate to the marginal zone of the GC and receive further
antigenic stimulation and costimulatory signals through B cell
activation molecules, such as CD40 and CD38, and receptors for
various B cell stimulatory cytokines (Gray et al., J. Exp. Med.,
180:141-155, 1994; Foy et al., J. Exp. Med., 180:157-163, 1994).
Antigen-specific B cells further stimulated in this area probably
migrate into the interstitial region of the spleen (called red
pulp), where various kinds of other immune-competent cells may
interact with antigen-driven B cells. Histochemical analysis in
several autoimmune mice identified unique antibody-producing cells
in this area which appear as plasma cells or aberrant plasma cells
called Mott cells (Tarlinton et al., Eur. J. Immunol. 22:531-539,
1992; Jiang et al., J. Immunol., 158:992-997, 1997).
[0004] Autoimmunity is a phenomenon in which the impairment of
self/nonself discrimination occurs frequently in the
antigen-specific lymphocytes (Theofilopoulos, Immunol. Today,
16:90-98, 1995). The immune systems of various autoimmune diseases
show the combinatory mechanism involving T cells and B cells
(Theofilopoulos et al., Adv. Immunol., 37:269-290, 1985; Okamoto et
al., J. Exp. Med. 175:71-79, 1992; Reininger et al., J. Exp. Med.,
184:853-861, 1996; Theofilopoulos, et al., Immunol. Rev.
55:179-216, 1981; Watanabe-Fukunaga et al., Nature (Lond.).,
356:314-317, 1992; Takahashi et al., Cell, 76:969-976, 1994;
Shlomchick et al., Nature (Lond.). 328:805-811, 1987).
[0005] NZB and NZW are the strains characterized by multiple
genetic factors generating the severe autoimmune state of SLE as
(NZB.times.NZW)F.sub.1 mice (Theofilopoulos et al., Adv. Immunol.,
37:269-290, 1985; Okamoto et al., J. Exp. Med., 175:71-79, 1992;
Reininger et al., J. Exp. Med., 184:853-861, 1996; Theofilopoulos
et al., Immunol. Rev., 55:179-216, 1981). NZB mice spontaneously
generate the state of autoimmunity with the anti-red blood cell
antibody that causes an autoimmune hemolytic anemia (Okamoto et
al., J. Exp. Med., 175:71-79, 1992). NZW mice show an insidious
autoimmune phenomenon (Reininger et al., J. Exp. Med. 184:853-861,
1996). The SLE state of (NZB.times.NZW)F.sub.1 mice is apparently
caused by multiple genetic factors associated with T and B cells
(Theofilopoulos et al., Immunol. Rev., 55:179-216, 1981). NZB mice
show an apparent abnormality of B cells, but the molecular
mechanism of the abnormal B cell activation in NZB mice remains to
be elucidated.
DISCLOSURE OF THE INVENTION
[0006] To address the issue of which molecules are involved in such
maturation of B cells, the present inventors prepared monoclonal
antibodies against intracellular components of a murine B cell line
WEHI-231, which has the NZB genetic background. A monoclonal
antibody named 29-15 recognizes a differentiation antigen whose
expression is augmented in GC-B cells of peripheral lymphoid
organs. With the 29-15 monoclonal antibody, the present inventors
studied the expression of the antigen in peripheral lymphoid
organs, which characterized the molecule as a differentiation
antigen upregulated in the light zone of the GC from hyperimmunized
mice. In the spleen of NZB mice, lgM-producing plasma cells with
high expression of the GANP antigen appear before the onset of
autoimmunity, which would suggest that this is an important
molecular event for understanding the peripheral immune response
and autoimmunity with autoantibodies.
[0007] The present inventors have studied to identify the
above-mentioned antigen whose expression is selectively increased
in centrocytes of germinal center, and confirmed by in situ RNA
hybridization using an isolated cDNA probe (ganp probe) that the
expression of ganp mRNA is increased in the area stained with 29-15
monoclonal antibody. It was also confirmed that the gene product,
GANP protein, is a protein of 201 kD which is localized in
cytoplasma and nucleus, and is structurally similar with a
transcription regulating factor in yeasts, SAC3. When B cells are
activated with anti-IgM antibody and anti-CD40 antibody, the amount
of kinase which binds to GANP protein increased. These results
suggests that GANP protein may be involved in a signal conversion
of abnormal B cell differentiation in certain autoimmune state. The
present invention has been completed on the basis of these
findings.
[0008] Thus, the present invention provides a GANP protein
represented by the amino acid sequence shown in SEQ ID No.1 or No.3
of the sequence listing. According to the present invention, there
is provided a GANP mutant protein which is consisted of the amino
acid sequence wherein one or more amino acids are deleted, one or
more amino acids are substituted with other amino acid(s), and/or
one or more other amino acids are added in the amino acid sequences
shown in SEQ ID No.1 or No.3 of the sequence listing, and has a
kinase activity similar with that of GANP protein. According to the
present invention, there is provided a polypeptide which contains,
as a partial sequence, a full length amino acid sequence of the
aforementioned GANP protein or the aforementioned GANP mutant
protein.
[0009] According to another aspect, the present invention provides
a polynucleotide which encodes the aforementioned GANP protein or
GANP mutant protein. The typical polynucleotide is DNA encoding
GANP protein derived from mammal, and the DNA of mammal gene is
preferred among them. Examples of most preferred polynucleotide are
represented by the base sequences shown in SEQ ID No. 2 (DNA
sequence encoding GANP protein from mouse) or SEQ ID No. 4 (DNA
sequence encoding GANP protein from human) of the sequence
listing.
[0010] Further, according to the present invention, there is
provided an antisense polynucleotide which is composed of the base
sequence of an antisense chain of the aforementioned
polynucleotide, or derivatives of said antisense polynucleotide.
Furthermore, according to the present invention, there is provided
a polynucleotide or antisense polynucleotide of continuous 12 or
more bases which is a partial sequence of the aforementioned
polynucleotide or the aforementioned antisense polynucleotide, and
a chemically modified polynucleotide or antisense polynucleotide of
the aforementioned polynucleotide or the aforementioned antisense
polynucleotide.
[0011] According to further another aspect, the present invention
provides a method for obtaining DNA of the base sequence shown in
SEQ ID No. 2 or No. 4 of the sequence listing or DNA which is the
homologue from other mammal, wherein the aforementioned
polynucleotide or antisense polynucleotide is used as a probe, and
cDNA which hybridizes to the probe is obtained from mammal cDNA
library. The length of the cDNA is almost the same as that of GANP
gene, and the protein encoded by it has approximately 201 kDa.
Further, according to the present invention, there is provided cDNA
obtained by the aforementioned method and GANP protein encoded by
it.
[0012] According to further another aspect of the present
invention, there is provided an antibody which recognizes GANP
protein or GANP mutant protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a photograph showing detection of 29-15.sup.+
cells in the PP of normal mice. The immunohistochemical analysis
was carried out on PP with the 29-15 mAb and ALP-anti-rat lg
antibody. Positive cells appear in the central area with Vector
Blue ALP substrate, and the strong signal in the surrounding area
is in intestinal villi containing nonspecific endogenous ALP
activity. For two-color staining, the sections are further stained
either with biotin-anti-B220 mAb or biotin-anti-lgD mAb followed by
HRP-streptavidin in combination with DAB.
[0014] FIG. 2 is a photograph showing appearance of 29-15.sup.+
cells in the GC area of SRBC-imnmunized mice. Normal BALB/c mice
were injected four times with SRBC during 12 days and the spleen
sections were stained with hematoxylin or studied by
immunohistochemistry as in FIG. 1. The sections of normal and
SRBC-immunized BALB/c mice are parallel, when compared after
staining with the 29-15 mAb.
[0015] FIG. 3 is a photograph showing appearance of 29-15.sup.+
cells in the GC area of SRBC-immunized mice. The sections of the GC
area are stained with PNA, anti-BrdU, and the 29-15 mAb in
combination with the individual colors as described in the
Materials and Methods. Upper photograph shows hematoxylin staining
of the GC area (GC) and the central artery (CA). Middle photograph
shows three-color staining, indicating the 29-15.sup.+PNA.sup.+
cells. Lower panel shows a schema of 29-15.sup.+PNA.sup.+
cells.
[0016] FIG. 4 is a photograph showing expression of the
GANP.sup.dense+ cells in the red pulp area of autoimmune-prone
mice. Sections were prepared from the spleens of nonimmunized mice
of BALB/c, NOD, NZB, (NZB.times.NZW)F.sub.1, BXSB, and MRL/lpr. All
mice were used 6-8 weeks after birth. The GANP.sup.dense+ cells
stained with the 29-15 mAb appear in the red pulp area of NZB,
(NZB.times.NZW)F.sub.1, MRL/lpr, and BXSB strains.
[0017] FIG. 5 is a photograph showing expression of the
GANP.sup.dense+ cells in the red pulp area of autoimmune-prone
mice. Sections of the LN of popliteal regions were stained with the
29-15 mAb. The GANP.sup.dense+ cells appear in peripheral LN of
older NZB mice (10 month old) and MRL/lpr mice (8 week old).
[0018] FIG. 6 is a photograph showing characterization of the
GANP.sup.dense+ cells in the autoimmune-prone mice. Sections were
prepared with the spleen of nonimmunized NZB mice (8 week old).
Immunohistochemical analysis was performed with the 29-15 mAb in
combination with one of the following reagents: anti-B220, PNA.
[0019] FIG. 7 is a photograph showing characterization of the
GANP.sup.dense+ cells in the autoimmune-prone mice. Sections were
prepared with the spleen of nonimmunized NZB mice (8 week old).
Immunohistochemical analysis was performed with the 29-15 mAb in
combination with one of the following reagents: anti-lgM,
anti-Syndecan-1, or anti-BrdU mAb.
[0020] FIG. 8 is a photograph showing Mott cells that appear in NZB
mice by PAS staining.
[0021] FIG. 9 is a diagram showing a deduced amino acid sequence of
mouse GANP protein in one character notation.
[0022] FIG. 10 is a diagram showing a structure of the GANP
protein. In the figure, S/T rich region: serine/threonine rich
region, SAC3 homology region; SAC3 homology region, nuclear
localizing signal: nuclear localizing signal. Four LXXLL motifs are
present.
[0023] FIG. 11 is a photograph showing a result of in situ RNA
hybridization of the ganp gene. Sections of spleens from
SRBC-immunized, nonimmunized BALB/c, and NZB mice were hybridized
with the ganp anti-sense probe. In the figure, the white pulp area
(WP), red pulp area (RP), and GC area (GC) are indicated. The
GANP.sup.dense+ cells were recognized in the red pulp of NZB
mice.
[0024] FIG. 12 is a diagram showing the results of the analysis by
Western blotting after immunoprecipitation of GANP protein. The
GANP protein was detected as a 210-kD protein expressed in
cytoplasmic and nuclear fractions of WEHI-231 cells.
[0025] FIG. 13 is a diagram showing the results where spleen B
cells from normal BALB/c mice were stimulated with F(ab').sub.2 of
goat anti-lgM Ab (10 .mu.g/ml) and anti-CD40 mAb (10 .mu.g/ml) for
48 hour and stained with the anti-GANP mAb.
[0026] FIG. 14 is a diagram showing the results where in vitro
kinase reaction was carried out with the anti-GANP
immunoprecipitates in the presence of [.gamma.-.sup.32P]-ATP for 10
minutes. Phosphorylation on the proteins were detected by the
autoradiography after SDS-PAGE separation. Phosphorylation of the
GANP is indicated with an arrow (FIG. A), and phosphoamino acid
analysis of phosphorylated GANP protein is also shown (FIG. B).
[0027] FIG. 15 is a diagram of the structure of the mouse GANP
protein. In the figure, the homologous region to SAC3 and Map80,
nuclear localization sequences (NLSs), and coiled-coil regions are
indicated. Four LXXLL motifs are indicated by black.
[0028] FIG. 16 shows a result of RT-PCR assay. The upregulation of
gnap mRNA in anti-.mu.- and anti-CD40-stimulated B calls in vitro
is shown. HPRT was used as a control to confirm the amount of each
template.
[0029] FIG. 17 shows a result of in vitro kinase reaction. The call
lysate was prepared from unstimulated (left) or stimulated (right)
cells and subjected to anti-GNAP immunoprecipitation. In vitro
kinase reaction was carried out with the anti-GNAP (42-23)
immunoprecipitates in the presence of [.gamma..sup.32P]-ATP for 10
minutes. Phosphorylation on the proteins was detected by the
autoradiography after SDS-PAGE separation. An arrow indicates the
position of phosphorylated GNAP.
[0030] FIG. 18 is a scheme showing a physical association between
GNAP and MCM3. The cell lysate from WEHI-231 was immunoprecipitated
with anti-GST, anti-GNAP (42-23), or anti-MCM3 Ab. After separation
by SDS-PAGE, the proteins were electrophoretically transferred to a
membrane and probed with anti-MCM3 Ab.
[0031] FIG. 19 is a scheme showing a physical association between
GNAP and MCM3. Anti-GST, anti-GNAP (42-23) and anti-MCM3
immunoprecipitates from WEHI-231 cell lysates were subjected to in
vitro kinase assay. Normal rabbit serum (NRS) was used as a control
for anti-MCM3 Ab. The samples were separated by 7% SDS-PAGE. The
bands corresponding to GNAP and MCM3 were indicated by arrows in
the left panel. On the right panel, V8 cleavage mapping of 210-kDa
bands showed an identical cleavage pattern. As a control an
irrelevant V8-digested protein was separated in parallel.
[0032] FIG. 20 is a scheme showing a result where double staining
with anti-MCM3 Ab and anti-CR1 mAb, or PNA, was performed. The
expression of MCM3 was upregulated in GC area.
[0033] FIG. 21 is a scheme where a deduced amino acid sequence of
human GANP protein is represented in one character notation.
[0034] FIG. 22 is a photograph showing a result where human ganp
and Map 80 were mapped by FISH method using human chromosome.
PREFERRED EMBODIMENT FOR CARRYING OUT THE INVENTION
[0035] The typical examples of GANP protein of the present
invention are protein represented by the amino acid sequences of
SEQ ID No. 1 and No. 3 of the sequence listing, and are
characterized in that they have a molecular weight of 210 kDa and
have a kinase activity. GANP mutant proteins provided by the
present invention are represented by the amino acid sequences
wherein approximately 1 to several, preferably 1 to 20, more
preferably 1 to 10, most preferably 1 to 5 amino acid residues are
substituted, inserted, and/or deleted in the amino acid sequences
of SEQ ID No. 1 or No. 3, and have a kinase activity which is
substantially similar with GANP protein represented by the amino
acid sequences of SEQ ID No. 1 or No. 2. These GANP mutant proteins
are within the scope of the present invention. The protein
represented by the amino acid sequences of SEQ ID No. 1 or No. 3 of
the sequence listing and homologue thereof are those whose
expression is selectively increased in centrocytes of germinal
center of mammal from which the protein is derived.
[0036] Usually, the active domain of GANP protein or GANP mutant
protein can be readily identified by preparing a polypeptide
wherein amino acid residue(s) are deleted from N-terminal and/or
C-terminal of the full length amino acid sequence, and measuring
the kinase activity of the polypeptide. The polypeptides provided
by the present invention are those comprised of an active domain of
GANP protein and GANP mutant protein and those comprising, as a
partial sequence, a polypeptide comprised of said active domain,
and have a kinase activity which is substantially similar with GANP
protein. Moreover, another polypeptides provided by the present
invention are those comprising, as a partial sequence, a full
length amino acid sequence of GANP protein or GANP mutant protein,
and have a kinase activity which is substantially similar with GANP
protein.
[0037] The polynucleotide provided by the present invention
includes DNA and RNA as well as all of the nucleotides obtained by
chemically modifying DNA or RNA. The term "polynucleotide" used
herein should be most broadly interpreted to include non-naturally
occurring form. The typical examples of the polynucleotide provided
by the present invention are DNA or RNA which encodes the
aforementioned GANP protein or GANP mutant protein. Another example
of the polynucleotide of the present invention is antisense
polynucleotide.
[0038] It is well known for a skilled person in the art that, using
degeneracy of genetic code, at least partial bases of a
polynucleotide can be replaced with another type of bases without
changing the amino acid sequence of the polypeptide which is
produced from the polynucleotide. Therefore, the polynucleotide of
the present invention includes all polynucleotides which encode
GANP protein or GANP mutant protein. As examples of the preferred
gene of the present invention, a gene encoding GANP protein from
mouse is shown in SEQ ID No.2 of the sequence listing, and a gene
encoding GANP protein from human is shown in SEQ ID No.4. The amino
acid sequence of GANP mutant protein can be determined from the
base sequence of a gene encoding said mutant. For example,
sequencing can be carried out by using commercially available
programs (for example, MacVector (registered trademark, Eastman
Chemical), or Genetix (Software Kaihatsu)).
[0039] The scope of the present invention covers antisense
polynucleotides composed of a base sequence of antisense chain of
polynucleotide encoding GANP protein, and derivatives thereof. The
antisense polynucleotides is provided as an embodiment of the
polynucleotide mentioned above, and the term "antisense
polynucleotide" may be herein used to clearly mean that it is a
polynucleotide comprised of base sequence of antisense chain. The
antisense polynucleotide can hybridize to polynucleotide encoding
GANP protein, and if the polynucleotide to which it hybridize is a
polynucleotide of coding region, the biosynthesis of the
polypeptide encoded by the polynucleotide can be inhibited.
[0040] Antisense polynucleotide for inhibiting the biosynthesis of
polypeptide preferably contains 12 or more bases. On the other
hand, an unnecessarily long sequence is not preferred in order to
incorporate full length antisense polynucleotide into cells. When
an antisense polynucleotide is incorporated into cells to inhibit
the biosynthesis of GANP protein, it is preferred to use an
antisense polynucleotide of 12 to 30 bases, preferably 15 to 25
bases, more preferably 18 to 22 bases.
[0041] The antisense polynucleotide of the present invention or
derivatives thereof include all of the form where several
nucleotides composed of base, phosphoric acid and sugar are bound
whether or not they are present in nature. Typical examples include
a naturally occurring antisense DNA and antisense RNA.
Non-naturally occurring polynucleotides include, for example,
polynucleotides of methylphosphonate type and phosphorothioate
type. As to the antisense polynucleotide of the present invention,
various antisense polynucleotide derivatives which are excellent in
binding ability to target DNA or mRNA, tissue selectability, cell
permeation property, nuclease resistance, intercellular stability
and the like, can be obtained by using an antisense technology
available to a skilled person in the art.
[0042] Generally, in view of easiness of hybridization, it is
preferred to design an antisense polynucleotide or derivatives
thereof having a base sequence complementary with base sequence
which forms a loop of RNA. Therefore, as to the antisense
polynucleotide of the present invention and derivatives thereof,
those which hybridize to loop region of RNA are preferred examples.
Moreover, an antisense polynucleotide having a sequence
complementary with a translation initiation codon and neighborhood
thereof, ribosome binding site, capping site, or splicing site can
generally be expected to exhibit high expression inhibition effect.
Therefore, the antisense polynucleotide of the present invention or
derivatives thereof having a sequence complementary with a
translation initiation codon and neighborhood thereof, ribosome
binding site, capping site, and/or splicing site of the gene
encoding GANP protein are preferred example in view of expression
inhibition effect.
[0043] Among the currently generally known polynucleotide
derivatives, the derivatives where at least one of nuclease
resistance, tissue selectability, cell permeation property and
binding ability is enhanced, preferably include derivatives having
a phosphorothioate bond as a skeleton structure. The polynucleotide
of the present invention and derivatives thereof include
derivatives having these function or structure.
[0044] Among the antisense polynucleotide of the present invention,
naturally occurring type of antisense polynucleotide may be
synthesized by using a chemical synthesizer or may be prepared by a
PCR method using a DNA encoding GANP protein as a templeta.
Polynucleotide derivatives such as methylphosphonate type or
phosphorotioate type may usually be prepared by chemical synthesis.
In this case, the procedure can be carried out according to an
instruction attached with the chemical synthesizer, and the
synthesized product thus obtained can be purified by HPLC method
using reverse phase chromatography and the like.
[0045] Polynucleotide which is a polynucleotide encoding GANP
protein of the present invention, antisense polynucleotide thereof,
or a portion thereof (for example, polunucleotide composed of
continuous 12 or more bases) can be used as a probe for screening a
DNA encoding GANP protein from mammalian cDNA library. For such a
purpose, a polynucleotide composed of a sequence of continuous 15
or more bases is particularly preferred. The polynucleotide used as
a probe may be a derivative. Usually, it is recognized that a
sequence having the aforementioned number or more of base is a
specific sequence.
[0046] A DNA of continuous 12 or more bases in the base sequence of
SEQ ID No. 2 or No. 4 of the sequence listing, or a polynucleotide
which hybridizes to said DNA (antisense polynucleotide) can be used
as a probe for screening a DNA encoding GANP protein from cDNA
library or the like.
[0047] Also, a tissue which expresses mRNA from GANP gene can be
detected by performing a Northern Blot hybridization on mRNA
derived from various tissues by using a polynucleotide encoding
GANP protein of the present invention, antisense polynucleotide
thereof, or a polynucleotide of a portion thereof as a probe.
Furthermore, a polynucleotide of 12 or more bases can be used as a
primer for polymerase chain reaction (PCR), and a polynucleotide
encoding GANP protein can be obtained by PCR. Also, the primer can
be appropriately selected to clone any portion of GANP protein.
[0048] As to the cDNA library used in the screening using the
aforementioned probe, one prepared from mRNA can be preferably
used. A group of cDNA selected by random sampling from these cDNA
library may be used as a sample for screening. A commercially
available cDNA library can be used.
[0049] The cDNA which hybridizes to the above-obtained GANP gene is
inserted into a suitable vector (for example, pGEX-4T-1 vector),
and is introduced into a host (for example, E. coli) to prepare a
transformant. The type of the vector and the type of the host are
not particularly limited, and any suitable expression vector may be
selected and used depending on the type of the host. As the host,
bacterium such as E. coli, yeasts, or animal cells can be used. A
method for obtaining a transformant by introducing a recombinant
vector into a suitable host such as E. coli is not particularly
limited, and any method available to a skilled person in the art
may be applied.
[0050] The transformant into which the GANP gene of the present
invention was introduced can be cultured to amplify a gene DNA or
produce a protein, thereby producing GANP protein. The preparation
and culturing of a transformant are described in various
literatures and reports, and many methods have been developed and
have been conventionally used in the art. Therefore, a skilled
person in the art can easily prepare GANP protein on the basis of
the base sequence described herein. The methods for introducing a
gene into cells include calcium chloride method, lipofection
method, protoplast method, and electroporation method.
[0051] Separation and purification of a protein of interest from
the culture can be carried out by using any means available to a
skilled person in the art in combination appropriately. For
example, GANP protein of the present invention can be efficiently
recovered and purified by performing procedures such as
concentration, solubilization, dialysis, various chromatography and
the like. More specifically, selection may be suitably made among
immunoprecipitation, salting out, ultrafilteration, isoelectric
point precipitation, gel filteration, electrophoresis, various
chromatography such as ion exchange chromatography, hydrophobic
chromatography and antibody chromatography, chromatofocusing,
adsorption chromatography, and reverse phase chromatography. By
using a gene encoding GANP mutant protein, GANP mutant protein can
be similarly prepared.
[0052] Also, GANP protein or GANP mutant protein can be prepared as
a fused protein with another polypeptide. Such a fused polypeptide
is within the scope of the present invention. The type of the
polypeptide to be fused is not particularly limited, and includes,
for example, a signal peptide which promotes an extracellular
secretion. The preparation of such a fused protein may be carried
out by using transformant. When a fused protein is used to prepare
GANP protein or GANP mutant protein, a fused protein is treated
with a chemical substance such as bromecyan or an enzyme such as
protease, and the substance of interest which was cut out may be
separated and purified.
[0053] Antibodies which recognize GANP protein or GANP mutant
protein can be prepared by using GANP protein or GANP mutant
protein of the present invention or partial polypeptide thereof.
The antibody of the present invention can be prepared by any means
of a conventional method in the art by immunizing a mammal with
GANP protein or GANP mutation protein. It can be confirmed by
Western blotting, ELISA, immunostaining (for example, measurement
with FACS) or the like that the antibody recognizes GANP protein or
GANP mutation protein of the present invention. As immunogens,
there may used GANP protein or GANP mutant protein as well as a
portion thereof bound to another carrier protein such as calf serum
albumin. A portion of GANP protein or GANP mutation protein
preferably contains 8 or more amino acid residues, and such a
polypeptide may be synthesized by using, for example, a peptide
synthesizer.
[0054] A monoclonal antibody which is produced from hybridoma
prepared by using lymphocytes of immunized animals may be used as
an antibody of the present invention. The process for the
preparation of a monoclonal antibody is well known in the art and
is conventionally used ("Antibodies, A Laboratory Manual" (Cold
Spring Harbor Laboratory Press, 1988), Chapter 6). Moreover, a
fragment of antibody having a antigen-antibody reaction activity
and a chimera antibody may be used as an antibody of the present
invention. GANP protein or GANP mutant protein of the present
invention can be detected by a method using an antibody or a method
using an antibody and an enzyme.
[0055] The present invention is illustrated in detail by the
examples below, but the scope of the present invention is not
limited to the examples below.
EXAMPLE
Example 1
Cloning of Mouse GANP Gene and Analysis of Expression
<Materials and Methods>
(1) Animals and Immunization
[0056] BALB/c mice and Lewis rats were purchased from Seac
Yoshitomi Ltd. (Fukuoka). NZB, NZW, (NZB.times.NZW)F.sub.1 mice (7
week old, female), MRL/lpr mice (8 week old, female), and BXSB mice
(7 week old, male) were obtained from Japan SLC Co. (Shizuoka).
Aged NZB mice (10 month old, female) were kindly gifted from Dr.
Sachiko Hirose (Department of Pathology, Juntendo University School
of Medicine). NOD mice (7 week old, male) were generously provided
from Dr. Junichi. Miyazaki (Department of Nutrition and
Physiological Chemistry, Osaka University Medical School). All
animals were maintained in Center for Animal Resources and
Development in Kumamoto University. BALB/c mice were immunized
multiply with sheep red blood cells (Nippon Bio-Test Laboratories,
Inc., Tokyo). The immunization was performed intravenously with
5-day interval and sections of the thymus, spleen, lymph node (LN),
and Peyer's patches (PP) were prepared for the immunohistochemical
analysis.
(2) Cells and Cell Culture
[0057] Splenic B cells from BALB/c mice were enriched as described
previously (Nomura et al., Immunol. Lett. 45:195-203, 1995). These
cells were cultured in RPMl-1640 medium (Gibco-BRL, Gaithersburg,
Germany) containing 10% heat-inactivated FCS (Dainippon
Pharmaceutical Co., Osaka, Japan), 5 mM L-glutamine (Biowhitteker,
Walkersville, Md., USA), 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, and 50 .mu.M 2-ME at 37.degree. C. in an incubator
with 5% carbon dioxide.
(3) Establishment of the 29-15 monoclonal antibody (Hereinafter
Referred to as "25-15 mAb")
[0058] The mAbs against a murine B cell line WEHI-231, which was
established from a (BALB/c.times.NZB)F.sub.1 mouse with mineral
oil, were prepared by the method described previously (Kuwahara et
al., J. Immunol. 152:2742-2752, 1994). Briefly, the cell lysate of
WEHI-231 with the surface phenotype slgM.sup.+slgD.sup.+B220.sup.+
was prepared with the hypotonic buffer in the absence of detergent
and dialyzed against a phosphate buffered saline (PBS) in
accordance with the method of Sakaguchi et al (Sakaguchi et al.,
EMBO (Eur. Mol. Biol. Organ.) J. 5:2139-2147, 1986). The cell
lysate was immunized into the foot pads of Lewis rats in the
complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit,
Mich., USA) and boosted twice in the incomplete Freund's adjuvant
(IFA) (Difco Laboratories) at day 4 and day 8. After 9 days, the
lymph node of popliteal and inguinal regions were excised and the
lymphoid cell suspension was prepared. Establishment of hybridomas,
selection in the HAT media (Gibco-BRL), and recloning of hybridoma
clones were performed as described previously (Kuwahara et al., J.
Immunol. 152:2742-2752, 1994). The 29-15 mAb was selected to stain
lymphoid cells in the immunohistochemical analysis.
(4) Antibodies and Reagents
[0059] F(ab').sub.2 fragment of the affinity-purified goat
anti-mouse .mu. antibody (ICN Pharmaceutical, Inc., Costa Mesa,
Calif., USA), biotin-conjugated peanut agglutinin (PNA) (Vector
Laboratories, Inc., Burlingame, Calif., USA), biotin-conjugated
anti-CD35mAb (PharMingen, San Diego, Calif.), alkalinephosphatase
(ALP) conjugated goat anti-rat IgAb (#59301, ICN), HRP conjugated
goat anti-rat IgAb (ICN), HRP conjugated streptavidin (Kirkegaard
& Perry Laboratories, Inc., Gaitherburg, Md.), ALP conjugated
goat anti-mouse IgAb (Sigma Chemicals Co., St. Louis, Mo.), FITC
conjugated mouse anti-rat .kappa. mAb (ICN), PE conjugated
anti-B220 mAb (PhaMingen), and ALP conjugated goat anti-rabbit IgAb
(Zymed Laboratories Inc., South San Francisco, Calif.) were
purchased and used. Biotin-conjugated mAbs such as anti-B220
(RA3-6B2), anti-.mu. (AM/3), and anti-.delta. (CS/15) were prepared
in our laboratory. Anti-CD40 mAb (LB429) was established in our
laboratory (Nomura et al., Immunol. Lett. 45:195-203, 1995).
Hybridomas of AM/3 and CS/15 were kindly provided by Dr. Kensuke
Miyake (Department of Immunology, Saga Medical School).
Biotin-conjugated anti-Syndecan-1 was purchased from PharMingen
(San Diego, Calif., USA). Anti-BrdU mAb was obtained from
Novocastra Laboratories, Ltd. (Newcastle, United Kingdom). Rabit
anti-mouse MCM3/P1 Ab is described in the literature (Kimura, H et
al, 1994, EMBO J. 13, 4311-4320).
(5) Immunohistochemistry
[0060] Immunohistochemical staining was performed as described
previously (Ezaki et al., Arch. Histol. Cytol. 58:104-115, 1995;
Yamanouchi et al., Eur. J. Immunol. 28:696-707, 1998). In brief,
the target organs excised from BALB/c, NZB, (NZB.times.NZW)F.sub.1,
NOD, BXSB, and MRL/lpr mice were placed in OCT compound (Miles
Inc., Elkhart, Ind., USA). The 6-.mu.m cryosections placed on the
gelatin-coated slides were air-dried fully. The slides were then
fixed in acetone for 10 minutes, followed by rehydration in PBS for
15 minutes. The slides were incubated with the 29-15 mAb for 60
minutes and were washed with PBS several times. After incubation
with alkaline phosphatase-conjugated goat anti-rat lg antibody
(ALP-anti-rat lg, catalogue #59301, ICN Pharmaceutical, Inc.), the
slides were washed four times with PBS. The slides were developed
using Vector Blue (Vector Laboratories).
[0061] For secondary staining, the slides were incubated with
biotin-labeled mAbs in combination with horseradish peroxidase
(HRP)-conjugated streptavidin (Kirkegaard & Perry Laboratories,
Inc., Gaithersburg, Md., USA). After development with
p-dimehylaminoazobenzene (DAB, Dojindo, Kumamoto), the sections
were fixed lightly with 1% glutaraldehyde solution in PBS. To
detect the cells of active proliferation in vivo, BrdU (Sigma
Chemicals Co., St. Louis, Mo., USA) was injected intravenously 1
hour before obtaining the organs. Cells undergoing DNA synthesis
were detected by staining with anti-BrdU mAb in combination with
ALP-conjugated goat anti-mouse lg Ab (Sigma Chemicals Co.) followed
by development with Vector Red (Matsuno et al., Cell Tissue Res.
257:459-470, 1989). Periodic acid Schiff (PAS) staining was
performed as described previously (Jiang et al., J. Immunol.
158:992-997, 1997). All sections were mounted by Aquatex (E. Merck,
Darmstadt, Germany).
(6) Molecular Cloning of the cDNA Using .lamda.gt11 Vector
[0062] The cDNA libraries constructed with mRNAs from the mouse
spleen, mouse bone marrow, WEHI-231 cells and A20 cells were
screened with the supernatant of the 29-15 mAb after transferring
the fusion protein onto nitrocellulose filters (Schleicher and
Schuell, Darmstadt, Germany) that were presoaked with 20 mM IPTG
(Inui et al., J. Immunol. 154:2714-2723, 1995). The phage plates
were incubated for 4 hours at 42.degree. C. and then the plates
were covered with the filters and further incubated for 4 hours at
37.degree. C. The filters were washed three times with the washing
buffer (PBS containing 0.1% Tween 20), blocked for 1 hour in the
blocking buffer (5% nonfat dry milk in PBS containing 0.1% Tween
20), and then incubated with the 29-15 mAb. Positive signals were
detected by autoradiography using .sup.1251-labeled sheep anti-rat
lg Ab (Amersham, Buckinghamshire, United Kingdom). The initial cDNA
clone contained a 280-bp fragment that is capable of coding a
polypeptide as a fusion protein. With the original 280-bp fragment,
the longer cDNA clones were isolated from another WEHI-231 cDNA
library. The 4.9-kb fragment of the second cDNA clone encodes a
longest open reading frame of 4.5 kb. To further determine the 5'
sequence, the 5'-RACE method was employed. The race kit of
Gibco-BRL was used.
(7) In situ RNA Hybridization on Tissue Sections
[0063] In situ RNA hybridization was carried out as described
previously (Kondo et al., Blood 80:2044-2051, 1992).
Paraffin-embedded sections were mounted on silanized slides. After
the slides were deparaffinized, hybridization with ganp 280-bp
riboprobe labeled by digoxigenin was performed for 16 hours at
50.degree. C. The slides were washed with TNE buffer (10 mM
Tris-HCl [pH 7.6], 500 mM NaCl, 1 mM EDTA) at 37.degree. C. several
times, followed by washing with 2.times. and/or 0.2.times.SSC
solution at 50.degree. C. While using anti-digoxigenin antibody,
the development was performed in the presence of ALP substrate.
(8) Preparation of GST-cDNA Fusion Protein and Another Anti-GANP
mAb
[0064] The ganp cDNA fragment encoding a part of GANP (amino acids
of 679th to 1028th of the amino acid sequence of SEQ ID No. 1 of
the sequence listing) was introduced into a pGEX-4T-1 vector
(Pharmacia Biotech, Piscataway, N.J., USA). The recombinant plasmid
was verified by DNA sequencing of the entire insert and the
junction. The GST-GANP fusion protein was prepared by
glutathione-Sepharose (Pharmacia) column chromatography as
described elsewhere (Inui et al., J. Immunol. 154:2714-2723, 1995).
Anti-GANP mAb, designated 42-23, was established by immunizing the
fusion protein in rats as described above.
(9) Western Blot Analysis
[0065] Protein gel electrophoresis, Western blot transfer, and the
immunodetection of proteins were performed as described previously
(Kuwahara et al., Int. Immunol. 8:1273-1285, 1996). Fifty million
cells were lysed with 1 ml of the TNE lysis buffer (10 mM Tris-HCl
[pH 7.8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.02% NaN.sub.3) and
the immune complex was analyzed on SDS-PAGE (7%). After the
proteins were transferred onto a nitrocellulose filter, the filter
was blocked with PBS-Tween 20 containing 5% nonfat dry milk and
incubated with anti-GANP mAb for 60 minutes. After washing with
PBS-Tween 20 several times, the filter was incubated with
HRP-conjugated goat anti-rat lg (ICN Pharmaceutical, Inc.) for 30
minutes. The development was performed using an ECL detection kit
(Amersham).
(10) Subcellular Fractionation
[0066] Separation of intact nuclei was carried out as described
previously (Schriber et al., Nucleic Acids Res. 17:6419, 1989).
WEHI-231 cells were washed with TBS and the pellets were
resuspended in buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) and incubated for 15
minutes on ice, followed by the addition of NP-40 to a final 1%.
After the centrifugation, the supernatants were recovered as a
cytoplasmic fraction. The pellets were resuspended with the same
buffer and homogenized to obtain the intact nuclei by staining. The
sample was centrifuged and the pellet was resuspended with cold
buffer C (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1
mM DTT, 1 mM PMSF) and centrifuged. The supernatants were frozen at
-80.degree. C. as a nuclear fraction.
(11) In Vitro Kinase Reaction and Phosphoamino Acid Analysis
[0067] Kinase reaction was carried out in vitro with the
immunoprecipitate as described previously (Kuwahara et al., J.
Immunol. 152:2742-2752, 1994). Splenic B cells were purified by the
method described (Nomura et al., Immunol. Lett. 45:195-203, 1995).
The B cells were stimulated in vitro for 48 hours with F(ab').sub.2
fraction of goat anti-lgM Ab and anti-CD40 mAb (LB429) as described
previously (Nomura et al., Immunol. Lett. 45:195-203, 1995). After
harvesting and washing, cells were lysed with TNE lysis buffer and
immunoprecipitated with the anti-GANP mAb (42-23). The
immunoprecipitates were incubated with [.gamma.-.sup.32P]-ATP
(Amersham) and the radiolabeled proteins were analyzed on SDS-PAGE
(7%) and with autoradiography. The band corresponding to GANP was
excised from dried gel. After SDS was removed from the gel, the
homogenized gel was digested by TPCK-trypsin (Sigma Chemicals Co.)
at 37.degree. C. overnight. The samples were subjected to
hydrolysis with 6N HCl and electrophoresed onto TLC (E. Merck).
[0068] V8 cleavage mapping of the indicated proteins was carried
out as described previously (Kuwahara, K., et al, 1994, J. Immunol.
152:2742-2752).
(12) Cytoplasmic Staining
[0069] The cells were fixed with 2.5% paraformaldehyde solution in
PBS followed by permeabilization with 70% ethanol for 1 hour on
ice. The cells were incubated with the 29-15 mAb in combination
with FITC-conjugated mouse anti-rat .kappa. in Ab. Antibody-binding
was analyzed on FACScan flow cytometer (Becton-Dickinson, Mountain
View, Calif., USA).
(13) Immunoprecipitation and Western Blot Analysis
[0070] Proteins obtained in the aforementioned (10) subcellular
fractionation were immunoprecipitated with the anti-GABP mAb in
combination with protein G-Sepharose, and analyzed by SDS-PAGE. The
Western blot filter was incubated with the anti-GANP mAB, followed
by HRP-anti-rat lg. The development was performed using an ECL
detection kit (Amersham).
(14) Reverse Transcriptase-PCR (RT-PCR)
[0071] Total RNA (1 .mu.g each), purified from cultured B cells
using TRISOL (Gibco BRL, Rockville, Md.) was used as a template for
the cDNA synthesis (100 .mu.l volume) with Superscript (Gibco BRL).
PCR amplification was carried out using 2 .mu.l of each cDNA
solution with Taq-Gold (Perkin-Elmer, Foster, Calif.) and the
primers for ganp or HPRT (control) (Han, S., et al, 1996, Science.
274-2092-2097). The ganp transcripts were amplified by
5'-CCGTGGGATGACATCATCAC-3' (the forward primer) (SEQ ID No. 5 of
the sequence listing) and 5'-CATGTCCACCATCTCCAGCA-3' (the reverse
primer) (SEQ ID No. 6 of the sequence listing).
<Results>
(1) Expression of the GANP Antigen in Lymphoid Organs
[0072] An mAb that recognizes a differentiation antigen expressed
in peripheral B cells was prepared by immunizing rats with the
lysate of WEHI-231 cells. Immunohistochemical analysis with the
29-15 mAb on normal lymphoid organs of BALB/c mice did not detect
expression in the bone marrow, but showed the slight expression in
lymphoid organs such as the thymus, spleen, and lymph node. A small
number of cells in the red pulp of the spleen and the deep cortex
of the lymph node strongly express the 29-15 Ag. Interestingly, the
expression was very high in the central area of follicles of the PP
(FIG. 1). The cells were positive with anti-B220 mAb, but not with
anti-lgD mAb. Normal mice show the development of secondary
lymphoid follicles with clear GC in PP because of the continuous
stimulation of various antigenic substances introduced through the
intestinal lumen.
[0073] Repeated immunization with sheep red blood cell (SRBC)
induces the formation of lymphoid follicles in the spleen within 12
days. Antigen immunization induces an appearance of 29-15.sup.+
cells in the GC area of the spleen and lymph node as well as in the
GC of the PP (FIG. 2). The 29-15 antigen appeared upregulated in
cells of the GC. The phenotype of 29-15.sup.+ cells in the
architecture of secondary lymphoid follicles was further analyzed.
Nearly half of PNA.sup.+ GC-B cells are positive with the 29-15
mAb, but they are negative with anti-BrdU mAb (FIG. 3).
Interestingly, the expression of 29-15 Ag is upregulated in the
centrocyte area at the distal region of the entrance from the
central artery. This phenotype is consistent with the criteria of
OC-B cells and supports the name "GANP" for the 29-15 Ag as
described above.
(2) Appearance of GANP.sup.dense+ B Cells in the Red Pulp Area of
Autoimmune-Prone NZB Mice
[0074] Normal mice express few GANP+ B cells in the follicular area
of the spleen without in vivo stimulation but show a few
GANP.sup.dense+ cells which remarkably express GANP protein in the
red pulp area of BALB/c (FIG. 2) and C57BL/6. These cells are large
and obviously different from conventional B cells. In young (8 week
old) NZB mice, however, these GANP.sup.dense+ cells increased
spontaneously in the red pulp area of the spleen without
immunization (FIG. 4). Another autoimmune-prone mouse, NZW, does
not express GANP.sup.dense+ cells in the red pulp at ages of 5 to
12 weeks. A severe-disease combination of (NZB.times.NZW)F.sub.1
shows an intermediate expression of GANP.sup.dense+ cells in the
red pulp.
[0075] Whether the GANP.sup.dense+ cells also appear spontaneously
in the spleen of other autoimmune-prone mice was examined. The
GANP.sup.dense+ cells appear in the spleen of BXSB and MRL/lpr, but
not markedly in NZW and NOD mice at a similar age in the specific
pathogen free condition (SPF). The GANP.sup.dense+ cells become
apparent during aging and appear in the peripheral lymph node of
the aged-NZB mice (10 month old) that have passed the onset of the
disease. The appearance of the GANP.sup.dense+ cells in the lymph
node seems to be mostly in the later stage. Of particular interest,
MRL/lpr shows the appearance of GANP.sup.dense+ cells in the lymph
node at the young stage (8 week old)(FIG. 5). These results
suggested that a genetic factor in autoimmune-prone NZB, BXSB, and
MRL/lpr mice might control the appearance of GANP.sup.dense+ cells
in the red pulp area and the recruitment into the lymph node.
[0076] Two-color analysis showed the phenotype of GANP.sup.dense+
cells in the red pulp area as PNA.sup.-B220.sup.- cells (FIG. 6)
and lgD-CD38.sup.- cells. These cells are positive when stained
with anti-Syndecan-1 mAb, which stains plasma cells selectively.
The GANP.sup.dense+ cells express lgM in cytoplasm (FIG. 7).
Because these cells could be Mott cells (Jiang, Y., S. Hirose, Y.
Hamano, S. Kodera, H. Tsurui, M. Abe, K. Terashima, S. Ishikawa and
T. Shirai. 1997. J. Immunol. 158:992-997.), the section was stained
with PAS staining. The GANP.sup.dense+ cells show PAS.sup.-, as
with the
B220.sup.-Syndecan-1.sup.+PNA.sup.-BrdU.sup.-GANP.sup.dense+ (FIG.
8) and CD40.sup.-CD38.sup.-. These plasma-like cells appear
preferentially in the spleen of NZB mice, but are different from
Mott cells currently reported .
(3) Identification of a cDNA Clone Encoding the GANP Antigen
[0077] Using the 29-15 mAb, we isolated a candidate cDNA clone
(with the insert DNA of 280 bp) from the WEHI-231 cDNA library and
further isolated a longer cDNA clone, named ganp. The full-length
nucleotide sequence (6429 bp) determined from overlapping clones
shows a putative polypeptide composed of 1971 amino acids with a
predicted molecular size of 210-kD (FIG. 9). The amino acid
sequence of GANP protein is shown in SEQ ID No. 1 of the sequence
listing and the base sequence of ganp cDNA is shown in SEQ ID No. 2
of the sequence listing.
[0078] The GANP amino acid sequence shows a regional homology to
SAC3 which is considered to be a nuclear transcription regulation
factor characterized in temperature-mutant Saccharomyces cerevisiae
and human Map 80 protein (Takei, Y et al,. 1998, J. Biol. Chem.
273:22177-22180) (FIG. 10 and FIG. 15; Bauer, A. and R. Koelling.
1996. Yeast 12:965-975). The GANP protein shows mild homologies
within short stretches of the insulin promoter factor (amino acids
996 to 1063) and various transcription factors, including NF-IL-6
(amino acids 388 to 450).
[0079] The GANP gene shows a consensus base sequence for the super
coil motifs, but does not show zinc-finger, leucine-zipper, and
homeo-domain motifs. A serine/threonine-rich region was seen in
N-terminal 100 amino acids, which has slight homology to
nucleoporin, which is known as the nuclear pore complex. GANP has
two possible nuclear localization sequences (.sup.497HKKK and
.sup.1344PMKQKRR), which would potentially support the expression
of the GANP in the nucleus as suggested by the PSORT program.
Moreover, GANP has 2 coiled-coil motifs, but does not have
zinc-finger, leucine-zipper, and homeo-domain motifs. Further,
there were 4 LXXLL motifs which were recognized in nuclear
transcription coactivator molecules including CBP/p300 and p/CIP
(Torchia, J. et al., 1997. Nature (Lond.) 387:677-684; Heery et
al., 1997, Nature (Lond.) 387:733-736), but any association
molecule through these motif has not been identified.
(5) Expression of the ganp Transcripts
[0080] Northern blot analysis detected the 7-kb mRNA as a very weak
signal in comparison to the control .beta.-actin signal, but its
expression was rather ubiquitous in all cell lines, organs, and
tissues tested. In order to examine whether the ganp mRNA is
upregulated in the same areas as detected on sections with the
29-15 mAb, in situ RNA hybridization analysis was carried out. The
ganp mRNA is expressed abundantly in the central area of the GC of
the SRBC-immunized spleen, but not in the nonimmunized spleen (FIG.
11), thymus, and lymph node. The ganp mRNA was upregulated in GC-B
cells of immunized mice. This expression pattern is quite similar
to the results with the 29-15 mAb on the same section based on
staining with hematoxylin. The GC area of the PP also showed
upregulation of the ganp mRNA in nonimmunized BALB/c mice, and the
expression of ganp mRNA is high in plasma-like cells of the red
pulp area of the spleen of nonimmunized NZB mice (FIG. 11). These
results suggests that the ganp gene encodes a molecule recognized
by the 29-15 mAb.
(6) Expression of the GANP in B Cells
[0081] The anti-GANP mAb (42-23) detected a single protein band at
210-kD from both nuclear and cytoplasmic compartments of WEHI-231
cells (FIG. 12). In order to find evidence of the functional
involvement of the GANP in the activation and differentiation of B
lineage cells, B cells from nonimmunized BALB/c mice were
stimulated in vitro with anti-lgM and anti-CD40 in combination, and
as a result, an expression of the GANP protein detected with the
anti-GANP mAb was increased (FIG. 13). An in vitro kinase reaction
with the GANP immunoprecipitates showed an increased kinase
activity assembled with the GANP protein in spleen B cells
stimulated in vitro. Thus, the GANP protein is inducibly
phosphorylated at the serine/threonine residues (FIG. 14). These
results suggest that the GANP might play a role to the activation
of B cells in peripheral immune responses.
[0082] Stimulation with anti-.mu. Ab and anti-CD40 mAb showed
maximal response, but either one of these regents showed only a
marginal response (data not shown). This upregulation was also
detected by the increase of ganp mRNA in B cells stimulated by
anti.mu. and anti-CD40 co-ligation in vitro (FIG. 16). RT-PCR
clearly demonstrated that the amount of ganp mRNA increased at 24
hours and 48 hours after stimulation in comparison with the control
HPRT mRNA.
[0083] Since the 210-kDa GANP has many possible phosphorylation
sites, we examined the induction of phosphorylation by an in vitro
kinase reaction with anti-GANP immunoprecipitates. As shown in FIG.
17, phosphorylation of the 210-kDa protein was found in the
anti-GANP immunoprecipitates from spleen B cells stimulated by
anti-.mu. and anti-CD40 co-ligation. This result indicates that a
kinase activity is maintained even if GANP is precipitated.
(7) Association of GANP with MCM3 Protein
[0084] We found a Map80-homologous region (76.3% identity at amino
acid level) in the carboxyl-terminal part of GANP. Map80 is an
80-kDa nuclear protein that is involved in the translocation of
MCM3 (a protein essential for DNA replication) between the
cytoplasm and the nuclei (Takei, Y. et al, 1998, J. Biol. Chem.
273:22177-22180; Kimura, H. et al, 1994, EMBO J. 13:4311-4320;
Chong, J. P. et al, 1996, Trends Biochem Sci. 21:102-106; and
Romanowski, P et al, 1996, Curr. Biol. 6:1416-1425). Therefore, we
examined the interaction between GANP and MCM3 in WEHI-231. We
detected that anti-GANP immunoprecipitates include MCM3. Because
the phosphorylation states of MCM proteins seem crucial in
regulation of cell cycle progression (Kimura, H. et al, 1994, EMBO
J. 13:4311-4320; Chong, J. P. et al, 1996, Trends Biochem Sci.
21:102-106; and Romanowski, P et al, 1996, Curr. Biol.
6:1416-1425), in vitro kinase assays with anti-MCM3
immunoprecipitates was performed. Immunoprecipitation of MCM3
co-precipitated a phosphorylated protein migrated at 210-kDa, which
is the identical size of GANP (FIG. 19, left panel). These 210-kDa
bands from anti-GANP and anti-MCM3 immunoprecipitates showed an
identical pattern in the V8 cleavage mapping (FIG. 19, right
panel), indicating that GANP and MCM3 are associated in a B cell
line.
[0085] Next, we studied whether MCM3 is upregulated in GC-B cells
by antigen-immunization of mice in vivo. The contiguous sections to
those used above were stained with the anti-MCM3 Ab (FIG. 20). MCM3
is also upregulated in GCs. Double staining clearly demonstrates
the co-localization of both MCM3 and PNA. A part of GC area is
surrounded intensely with FDCs (lymph follicular cells). These
results demonstrate that MCM3 is upregulated in GC-B cells
including centroblasts and the GANP.sup.+ centrocytes that would be
mostly surrounded by FDCs (FIG. 20).
(8) Discussion
[0086] As mentioned above, the present inventors found a novel
protein, GANP, expressed in GC-B cells localized at the light zone
of secondary follicles in the spleen. Although a trace amount of
the ganp mRNA is detectable in many kinds of cells under normal
conditions, the GANP protein appears upregulated in the specified
GC area of immunized mice. A number of studies demonstrated various
differentiation antigens in the GC as molecules recognized with
mAbs or by specific cDNA cloning (Christoph et al., Int. Immunol.
6:1203-1211, 1994; Li et al., Proc. Natl. Acad. Sci. USA.
93:10222-10227, 1996; Kuo et al., J. Exp. Med. 186:1547-1556,
1997). Most molecules appear in GC-B cells of the whole area,
whereas 8-oxoguanine DNA glycosylase is expressed in the dark zone
(Kuo et al., J. Exp. Med. 186:1547-1556, 1997).
[0087] Interestingly, the GANP antigen is selective in the
centrocyte of the light zone. Recent studies have shown that RAG
protein which is necessary for rearrangement of immunoglobulin gene
is selectively expressed in centrocytes at the light zone (Hikida
et al., 1996. Science (Wash. D.C.) 274:2092-2094, 1996; Han et al.,
Science (Wash. D.C.) 274:2094-2097, 1996). Since the GC area
probably provides the site for secondary lg gene rearrangement
occurring during T cell-dependent antibody responses, as described
by Papavasiliou et al. and Han et al. (Papavasiliou et al., Science
(Wash. D.C.), 278:298-301, 1997; Han et al., Science (Wash. D.C.),
278: 301-305, 1997), the GANP protein might be a component
associated with the maturation of antigen-specific B cells at the
centrocyte stage.
[0088] We found that the carboxyl-terminal portion of GANP has a
significant similarity to human Map80, which facilitates the
nuclear transport of MCM3 (Takei, Y et al., 1998, J. Biol.
Chem.273:22177-22180). Immunoprecipitation experiments demonstrated
that GANP also binds to MCM3 in WEHI-231. MCM3 is a member of the
MCM protein family essential for the initiation of DNA replication
(Kimura, H. et al 1994, EMBO J. 13:4311-4320; Blow, J. J. 1993. J.
Cell Biol.122.993-1002; Tye, B. K. 1994. Trends Cell Biol. 4:
160-166; Chong, J. P. et al, 1996, Trends Biochem Sci. 21:102-106;
Romanowski, P et al, 1996, Curr. Biol. 6:1416-1425; and Thommes, P
et al, 1992, Nucl. Acids Res. 20: 1069-1074). The major fractions
of nuclear MCM proteins bind to chromatin at the beginning of the S
phase, but dissociate during replication and accumulate as free
proteins in the nucleosol. The release of MCMs from chromatin is
accompanied by the phosphorylation of several MCM proteins and
their reassociation after mitosis is concomitant with their
dephosphorylation. It was suggested that MCM proteins are no longer
synthesized in growth arrested, differentiating cells and disappear
with kinetics related to their half-life (Musahi, C., et al, 1998,
Exp. Cell. Res. 241, 260-264). The MCM3 protein has recently been
shown to an early target in apoptotic proteolysis (Schwab, B. L. et
al., 1998, Exp. Cell Res. 238:415-421). Schwab, B. L. et al
proposed that active destruction of MCM3 inactivates the MCM
complex and serves to prevent untimely DNA replication events
during the execution of the cell death program. Our results showed
that GC-B cells express high level of MCM3, some of which is
associated with GANP. However, it appears curious that a protein,
upregulated in differentiated cells that arrest the cell cycle,
binds to another protein essential for progression of the S phase.
One possible speculation is that a function of GANP may be
inactivation of MCM3 through its binding. The immunohistochemistry
data are consistent with the following idea; GANP is upregulated in
growth-arrested centrocytes while MCM3 is expressed both in
rapid-cycling centroblasts and still in centrocytes in GCs.
Although the amount of MCM3 would decrease by ceasing the gene
expression and active destruction (Musahl, C., et al, 1998, Exp.
Cell. Res. 241, 260-264; and Schwab, B. L. et al., 1998, Exp. Cell
Res. 238:415-421), inactivation of MCM3, which is still expressed
in centrocytes, through the interaction with GANP could be another
mechanism to prevent DNA replication. In addition, both GANP and
MCM3 become phosphorylated with the co-precipitated kinase (FIG.
19). Since the highly phosphorylated MCM3 is thought to be
inactivated form (Kimura, H. et al, 1994, EMBO J. 13:4311-4320),
the association with GANP may stimulate phosphoryltion of MCM3.
[0089] The GANP protein has a close similarity to the SAC3 (SAC,
suppressor of actin) of yeasts, Saccharomyces cerevisiae, which was
isolated in a genetic screen for suppressors of a
temperature-sensitive mutation (act1-1) in the actin gene (FIG. 10;
Novick et al., Genetics, 121:659-674, 1989). The SAC3 protein is
expressed in the nuclei and is required for normal progression of
mitosis and protection against the loss of chromosomes (Bauer et
al., J. Cell. Sci. 109:1575-1583, 1996). Null mutants of SAC3 grow
very slowly and are larger than wild-type cells. SAC3 participates
in a process that affects both the actin cytoskeleton and mitosis,
which suggest that SAC3 regulates the gene expression of actin or
actin-binding proteins.
[0090] A gene (named LEP-1) that augments the transcription of the
leucine permease activity in Saccharomyces was identical to SAC3
(Stella et al., Yeast 11:460-460, 1995). Although the LEP-1 gene
induces the upregulation of the yeast leucine permease involved in
selective amino acid transport, the amino acid transport in
eukaryotic cells, especially the molecules involved in amino acid
permeation is not known (Mastroberardino et al., Nature (Lond.)
395:288-291, 1998). Although the SAC3/LEP-1 sequence does not show
motifs homologous to a number of transcription factors, the
biological functions determined previously (Bauer et al., J. Cell.
Sci. 109:1575-1583, 1996) suggest its regulatory activity of
various target genes in the nucleus. The mouse GANP does not show
typical consensus motifs for nuclear transcription factors, but has
a common ancestor with SAC3 gene of yeasts and has structural
similarity of possible phosphorylation sites, two nuclear
localization sequences, and two super coil structures that might
interact with other transcription molecules.
[0091] GANP is selectively upregulated in centrocytes of
Ag-immunized spleen. It is also useful as the differentiation
marker to define the centrocyte subset that is closely interacting
with FDCs in GC area. Our study showed that the BCR signal and the
CD40 co-stimulation together cause the upregulation of GANP and
lead to the signal transduction mediated through GANP/MCM3
complex.
[0092] The defective gene in the autosomal recessive genetic
disease autoimmune polyendocrinopathy (APECED) is localized by
linkage analysis to human chromosome 21 (21q22.3), which encodes an
AIRE gene product with a possible transcription regulator (Nagamine
et al., Nature Genet. 17:393-398, 1997). The autoantibody
recognizes the AIRE protein expressed in the adrenal gland and
other gonad-producing tissues. Studies of APECED drew an idea that
the involvement of molecules with nuclear coactivator activity
might be associated with the autoimmunity. Both the AIRE and GANP
proteins do not have typical domains for transcription regulators,
but they have LXXLL motifs as similarly observed in nuclear
transcriptional coactivators.
[0093] A B cell-specific nuclear coactivator (Bob1/OCA-B/OBF1) was
recently characterized as a cell-type-specific regulator of Oct1
and Oct2 (Luo et al., Mol. Cell. Biol. 15:4115-4124, 1995). The
OCA-B targeted mice show the impairment of the GC formation in the
spleen after immunization with T-dependent antigen, which suggests
the functional involvement of B cell maturation in the GC area (Kim
et al., Nature (Lond.) 383:542-547, 1996; Qin et al., EMBO J.
17:5066-5075, 1998). The expression of the GANP protein might be
under the control of the OCA-B cell in centrocytes. The molecular
interaction of the nuclear coactivator molecules would be an
important issue for the understanding of the B cell maturation in
the GC.
[0094] The New Zealand model of SLE has been the experiment subject
of genome linkage studies to map the chromosomal positions of
disease-susceptibility genes. At least 12 non-MHC loci linked with
nephritis and autoantibody production such as on chromosome 4
(designated Nba1), on chromosome 7, and on chromosome 1 (designated
as Nba2; Vyse et al., J. Immunol. 158:5566-5574, 1997) have been
independently mapped. The GANP antigen on large cells is highly
upregulated in the red pulp area of the nonimmunized NZB mice
(FIGS. 4-8). NZB mice contained similar large lgM-producing cells,
named Mott cells, in the red pulp area. Mott cells appear
selectively in NZB and (NZB.times.NZW)F.sub.1 mice, but not in
normal BALB/c or C57BL/6 mice.
[0095] The precursor cells of Mott cells are probably B-1 B cells
(Tarlinton et al., Eur. J. Immunol. 22:531-539, 1992; Jiang et al.,
J. Immunol. 158:992-997, 1997), which suggests a close association
with the autoimmunity of B cells. Mott cells are apparent with the
inclusion body of IgM in the cytoplasm and positive staining with
PAS (Tarlinton et al., Eur. J. Immunol. 22:531-539, 1992; Jiang et
al., J. Immunol. 158:992-997, 1997). Because GANP.sup.dense+ cells
seem to be Mott cells, PAS staining was performed. However,
GANP.sup.dense+ cells in the red pulp area of NZB mice are
PAS.sup.-. The GANP.sup.dense+ lgM-producing cells appear in the
spleen of NZB mice, as do Mott cells, but these cells are
different. The new type of lgM-producing cells could be generated
by the possible activation of an abnormal B cell population related
to one of the chromosomal loci linked to
disease-susceptibility.
[0096] Lyn.sup.-/- mice and CD40L.sup.-/- mice reported from
several laboratories show similar autoimmunities and hyper-lgM
syndrome(s), which have an increased appearance of imnunoblast
cells with the inclusion body in the spleen (Hibbs et al., Cell
83:301-311, 1995; Nishizumi et al., Immunity 3:549-560, 1995; Xu et
al., Immunity 1:423-431, 1994). These observations suggest that the
signal transduction through BCR and CD40 is regulating the
generation of the abnormal antibody-producing plasma cells.
Stimulation of splenic B cells with anti-lgM and anti-CD40
antibodies induces the phosphorylation activity of the GANP
protein. This observation suggests that the GANP protein may be
involved in downstream of the B cell activation site in the GC area
and the abnormal B cell activation in NZB mice might be associated
with the increased expression of GANP protein.
Example 2
Cloning of Human GANP Gene
[0097] On the basis of information of the sequence of rat GANP
gene, human GANP gene was cloned and sequenced. Specifically,
.lamda.11-human heart cDNA library (Clontech) was used, and gsp1-1:
TTTGTCTGGAGGATGATCGC (SEQ ID No.7 of the sequence listing),
gsp1-2:AAAGAGAAAGGGGCCAGGCC (SEQ ID No.8 of the sequence listing)
and gsp1-3:CCAGCTTCTTGTCCAAAAGC (SEQ ID No.9 of the sequence
listing) were used as primers, and 5' RACE System for Rapid
Amplification of cDNA Ends, Version 2.0(Gibco BRL) was used to
carry out the cloning and sequencing by a conventional method.
[0098] The base sequence of the obtained clone was determined. The
base sequence of the obtained human GANP gene is shown in SEQ ID
No.4 of the sequence listing. The amino acid sequence encoded by
this base sequence is shown in SEQ ID No.3 of the sequence listing
and FIG. 21. Human GANP gene shows high homology with mouse GANP
gene, and Human GANP contains Map80 domain of 80 kDa at carboxyl
terminal.
[0099] In in situ RNA hybridization, ganp transcript seems to be
activated at GC region of tonsil. GANP.sup.+ cells express
CD38.sup.+IgD.sup.+ phenotype of memory B cell. These results show
that human GANP is expressed also in GC-B cells of secondary lympho
tissues. Moreover, since human GANP of 1980 amino acids has a
stretch of Map80 homologous region which binds to MCM3 protein in B
cells, it is suggested that GANP is involved in the regulation of
cell cycle in GC-B cells.
[0100] Furthermore, in situ hybridization was carried out by FISH
method with the obtained human GANP gene and human chromosome
specimen. The results are shown in FIG. 22. As is understood from
FIG. 22, the genome fragment containing human GANP gene and Map80
was mapped on 22.3 of the long arm of chromosome 21.
INDUSTRIAL APPLICABILITY
[0101] The protein of the present invention is a novel protein
having a kinase activity, and may be involved in a signal
conversion of abnormal B cell differentiation in an autoimmune
state. Therefore, the protein, polypeptide, polynucleotide,
antisense polynucleotide and antibody of the present invention are
useful for revealing the mechanism of autoimmune.
Sequence CWU 1
1
9 1 1971 PRT Mouse 1 Met His Pro Val Asn Pro Phe Gly Gly Ser Ser
Pro Ser Ala Phe Ala 1 5 10 15 Val Ser Ser Ser Thr Thr Gly Thr Tyr
Gln Thr Lys Ser Pro Phe Arg 20 25 30 Phe Gly Gln Pro Ser Leu Phe
Gly Gln Asn Ser Thr Pro Ser Lys Ser 35 40 45 Leu Ala Phe Ser Gln
Val Pro Ser Phe Ala Thr Pro Ser Gly Gly Ser 50 55 60 His Ser Ser
Ser Leu Pro Ala Phe Gly Leu Thr Gln Thr Ser Ser Val 65 70 75 80 Gly
Leu Phe Ser Ser Leu Glu Ser Thr Pro Ser Phe Ala Ala Thr Ser 85 90
95 Ser Ser Ser Val Pro Gly Asn Thr Ala Phe Ser Phe Lys Ser Thr Ser
100 105 110 Ser Val Gly Val Phe Pro Ser Gly Ala Thr Phe Gly Pro Glu
Thr Gly 115 120 125 Glu Val Ala Gly Ser Gly Phe Arg Lys Thr Glu Phe
Lys Phe Lys Pro 130 135 140 Leu Glu Asn Ala Val Phe Lys Pro Ile Pro
Gly Pro Glu Ser Glu Pro 145 150 155 160 Glu Lys Thr Gln Ser Gln Ile
Ser Ser Gly Phe Phe Thr Phe Ser His 165 170 175 Pro Val Gly Ser Gly
Ser Gly Gly Leu Thr Pro Phe Ser Phe Pro Gln 180 185 190 Val Thr Asn
Ser Ser Val Thr Ser Ser Ser Phe Ile Phe Ser Lys Pro 195 200 205 Val
Thr Ser Asn Thr Pro Ala Phe Ala Ser Pro Leu Ser Asn Gln Asn 210 215
220 Val Glu Glu Glu Lys Arg Val Ser Thr Ser Ala Phe Gly Ser Ser Asn
225 230 235 240 Ser Ser Phe Ser Thr Phe Pro Thr Ala Ser Pro Gly Ser
Leu Gly Glu 245 250 255 Pro Phe Pro Ala Asn Lys Pro Ser Leu Arg Gln
Gly Cys Glu Glu Ala 260 265 270 Ile Ser Gln Val Glu Pro Leu Pro Thr
Leu Met Lys Gly Leu Lys Arg 275 280 285 Lys Glu Asp Gln Asp Arg Ser
Pro Arg Arg His Cys His Glu Ala Ala 290 295 300 Glu Asp Pro Asp Pro
Leu Ser Arg Gly Asp His Pro Pro Asp Lys Arg 305 310 315 320 Pro Val
Arg Leu Asn Arg Pro Arg Gly Gly Thr Leu Phe Gly Arg Thr 325 330 335
Ile Gln Glu Val Phe Lys Ser Asn Lys Glu Ala Gly Arg Leu Gly Ser 340
345 350 Lys Glu Ser Lys Glu Ser Gly Phe Ala Glu Pro Gly Glu Ser Asp
His 355 360 365 Ala Ala Val Pro Gly Gly Ser Gln Ser Thr Met Val Pro
Ser Arg Leu 370 375 380 Pro Ala Val Thr Lys Glu Glu Glu Glu Ser Arg
Asp Glu Lys Glu Asp 385 390 395 400 Ser Leu Arg Gly Lys Ser Val Arg
Gln Ser Lys Arg Arg Glu Glu Trp 405 410 415 Ile Tyr Ser Leu Gly Gly
Val Ser Ser Leu Glu Leu Thr Ala Ile Gln 420 425 430 Cys Lys Asn Ile
Pro Asp Tyr Leu Asn Asp Arg Ala Ile Leu Glu Lys 435 440 445 His Phe
Ser Lys Ile Ala Lys Val Gln Arg Val Phe Thr Arg Arg Ser 450 455 460
Lys Lys Leu Ala Val Ile His Phe Phe Asp His Ala Ser Ala Ala Leu 465
470 475 480 Ala Arg Lys Lys Gly Lys Gly Leu His Lys Asp Val Val Ile
Phe Trp 485 490 495 His Lys Lys Lys Ile Ser Pro Ser Lys Lys Leu Phe
Pro Leu Lys Glu 500 505 510 Lys Leu Gly Glu Ser Glu Ala Ser Gln Gly
Ile Glu Asp Ser Pro Phe 515 520 525 Gln His Ser Pro Leu Ser Lys Pro
Ile Val Arg Pro Ala Ala Gly Ser 530 535 540 Leu Leu Ser Lys Ser Ser
Pro Val Lys Lys Pro Ser Leu Leu Lys Met 545 550 555 560 His Gln Phe
Glu Ala Asp Pro Phe Asp Ser Gly Ser Glu Gly Ser Glu 565 570 575 Gly
Leu Gly Ser Cys Val Ser Ser Leu Ser Thr Leu Ile Gly Thr Val 580 585
590 Ala Asp Thr Ser Glu Glu Lys Tyr Arg Leu Leu Asp Gln Arg Asp Arg
595 600 605 Ile Met Arg Gln Ala Arg Val Lys Arg Thr Asp Leu Asp Lys
Ala Arg 610 615 620 Ala Phe Val Gly Thr Cys Pro Asp Met Cys Pro Glu
Lys Glu Arg Tyr 625 630 635 640 Leu Arg Glu Thr Arg Ser Gln Leu Ser
Val Phe Glu Val Val Pro Gly 645 650 655 Thr Asp Gln Val Asp His Ala
Ala Ala Val Lys Glu Tyr Ser Arg Ser 660 665 670 Ser Ala Asp Gln Glu
Glu Pro Leu Pro His Glu Leu Arg Pro Ser Ala 675 680 685 Val Leu Ser
Arg Thr Met Asp Tyr Leu Val Thr Gln Ile Met Asp Gln 690 695 700 Lys
Glu Gly Ser Leu Arg Asp Trp Tyr Asp Phe Val Trp Asn Arg Thr 705 710
715 720 Arg Gly Ile Arg Lys Asp Ile Thr Gln Gln His Leu Cys Asp Pro
Leu 725 730 735 Thr Val Ser Leu Ile Glu Lys Cys Thr Arg Phe His Ile
His Cys Ala 740 745 750 His Phe Met Cys Glu Glu Pro Met Ser Ser Phe
Asp Ala Lys Ile Asn 755 760 765 Asn Glu Asn Met Thr Lys Cys Leu Gln
Ser Leu Lys Glu Met Tyr Gln 770 775 780 Asp Leu Arg Asn Lys Gly Val
Phe Cys Ala Ser Glu Ala Glu Phe Gln 785 790 795 800 Gly Tyr Asn Val
Leu Leu Asn Leu Asn Lys Gly Asp Ile Leu Arg Glu 805 810 815 Val Gln
Gln Phe His Pro Asp Val Arg Asn Ser Pro Glu Val Asn Phe 820 825 830
Ala Val Gln Ala Phe Ala Ala Leu Asn Ser Asn Asn Phe Val Arg Phe 835
840 845 Phe Lys Leu Val Gln Ser Ala Ser Tyr Leu Asn Ala Cys Leu Leu
His 850 855 860 Cys Tyr Phe Asn Gln Ile Arg Lys Asp Ala Leu Arg Ala
Leu Asn Val 865 870 875 880 Ala Tyr Thr Val Ser Thr Gln Arg Ser Thr
Val Phe Pro Leu Asp Gly 885 890 895 Val Val Arg Met Leu Leu Phe Arg
Asp Ser Glu Glu Ala Thr Asn Phe 900 905 910 Leu Asn Tyr His Gly Leu
Thr Val Ala Asp Gly Cys Val Glu Leu Asn 915 920 925 Arg Ser Ala Phe
Leu Glu Pro Glu Gly Leu Cys Lys Ala Arg Lys Ser 930 935 940 Val Phe
Ile Gly Arg Lys Leu Thr Val Ser Val Gly Glu Val Val Asn 945 950 955
960 Gly Gly Pro Leu Pro Pro Val Pro Arg His Thr Pro Val Cys Ser Phe
965 970 975 Asn Ser Gln Asn Lys Tyr Val Gly Glu Ser Leu Ala Thr Glu
Leu Pro 980 985 990 Ile Ser Thr Gln Arg Ala Gly Gly Asp Pro Ala Gly
Gly Gly Arg Gly 995 1000 1005 Glu Asp Cys Glu Ala Glu Val Asp Leu
Pro Thr Leu Ala Val Leu 1010 1015 1020 Pro Gln Pro Pro Pro Ala Ser
Ser Ala Thr Pro Ala Leu His Val 1025 1030 1035 Gln Pro Leu Ala Pro
Ala Ala Ala Pro Ser Leu Leu Gln Ala Ser 1040 1045 1050 Thr Gln Pro
Glu Val Leu Leu Pro Lys Pro Ala Pro Val Tyr Ser 1055 1060 1065 Asp
Ser Asp Leu Val Gln Val Val Asp Glu Leu Ile Gln Glu Ala 1070 1075
1080 Leu Gln Val Asp Cys Glu Glu Val Ser Ser Ala Gly Ala Ala Tyr
1085 1090 1095 Val Ala Ala Ala Leu Gly Val Ser Asn Ala Ala Val Glu
Asp Leu 1100 1105 1110 Ile Thr Ala Ala Thr Thr Gly Ile Leu Arg His
Val Ala Ala Glu 1115 1120 1125 Glu Val Ser Met Glu Arg Gln Arg Leu
Glu Glu Glu Lys Gln Arg 1130 1135 1140 Ala Glu Glu Glu Arg Leu Lys
Gln Glu Arg Glu Leu Met Leu Thr 1145 1150 1155 Gln Leu Ser Glu Gly
Leu Ala Ala Glu Leu Thr Glu Leu Thr Val 1160 1165 1170 Thr Glu Cys
Val Trp Glu Thr Cys Ser Gln Glu Leu Gln Ser Ala 1175 1180 1185 Val
Lys Ile Asp Gln Lys Val Arg Val Ala Arg Cys Cys Glu Ala 1190 1195
1200 Val Cys Ala His Leu Val Asp Leu Phe Leu Ala Glu Glu Ile Phe
1205 1210 1215 Gln Thr Ala Lys Glu Thr Leu Gln Glu Leu Gln Cys Phe
Cys Lys 1220 1225 1230 Tyr Leu Gln Arg Trp Arg Glu Ala Val Ala Ala
Arg Lys Lys Phe 1235 1240 1245 Arg Arg Gln Met Arg Ala Phe Pro Ala
Ala Pro Cys Cys Val Asp 1250 1255 1260 Val Asn Asp Arg Leu Gln Ala
Leu Val Pro Ser Ala Glu Cys Pro 1265 1270 1275 Ile Thr Glu Glu Asn
Leu Ala Lys Gly Leu Leu Asp Leu Gly His 1280 1285 1290 Ala Gly Lys
Val Gly Val Ser Cys Thr Arg Leu Arg Arg Leu Arg 1295 1300 1305 Asn
Lys Thr Ala His Gln Ile Lys Val Gln His Phe His Gln Gln 1310 1315
1320 Leu Leu Arg Asn Ala Ala Trp Ala Pro Leu Asp Leu Pro Ser Ile
1325 1330 1335 Val Ser Glu His Leu Pro Met Lys Gln Lys Arg Arg Phe
Trp Lys 1340 1345 1350 Leu Val Leu Val Leu Pro Asp Val Glu Glu Gln
Thr Pro Glu Ser 1355 1360 1365 Pro Gly Arg Ile Leu Glu Asn Trp Leu
Lys Val Lys Phe Thr Gly 1370 1375 1380 Asp Asp Ser Met Val Gly Asp
Ile Gly Asp Asn Ala Gly Asp Ile 1385 1390 1395 Gln Thr Leu Ser Val
Phe Asn Thr Leu Ser Ser Lys Gly Asp Gln 1400 1405 1410 Thr Val Ser
Val Asn Val Cys Ile Lys Val Ala His Gly Thr Leu 1415 1420 1425 Ser
Asp Ser Ala Leu Asp Ala Val Glu Thr Gln Lys Asp Leu Leu 1430 1435
1440 Gly Thr Ser Gly Leu Met Leu Leu Leu Pro Pro Lys Val Lys Ser
1445 1450 1455 Glu Glu Val Ala Glu Glu Glu Leu Ser Trp Leu Ser Ala
Leu Leu 1460 1465 1470 Gln Leu Lys Gln Leu Leu Gln Ala Lys Pro Phe
Gln Pro Ala Leu 1475 1480 1485 Pro Leu Val Val Leu Val Pro Ser Ser
Arg Gly Asp Ser Ala Gly 1490 1495 1500 Arg Ala Val Glu Asp Gly Leu
Met Leu Gln Asp Leu Val Ser Ala 1505 1510 1515 Lys Leu Ile Ser Asp
Tyr Ile Val Val Glu Ile Pro Asp Ser Val 1520 1525 1530 Asn Asp Leu
Gln Gly Thr Val Lys Val Ser Gly Ala Val Gln Trp 1535 1540 1545 Leu
Ile Ser Gly Cys Pro Gln Ala Leu Asp Leu Cys Cys Gln Thr 1550 1555
1560 Leu Val Gln Tyr Val Glu Asp Gly Ile Ser Arg Glu Phe Ser Arg
1565 1570 1575 Arg Phe Phe His Asp Arg Arg Glu Arg Arg Leu Ala Ser
Leu Pro 1580 1585 1590 Ser Gln Glu Pro Ser Thr Ile Ile Glu Leu Phe
Asn Ser Val Leu 1595 1600 1605 Gln Phe Leu Ala Ser Val Val Ser Ser
Glu Gln Leu Cys Asp Ile 1610 1615 1620 Ser Trp Pro Val Met Glu Phe
Ala Glu Val Gly Gly Ser Gln Leu 1625 1630 1635 Leu Pro His Leu His
Trp Asn Ser Pro Glu His Leu Ala Trp Leu 1640 1645 1650 Lys Gln Ala
Val Leu Gly Phe Gln Leu Pro Gln Met Asp Leu Pro 1655 1660 1665 Pro
Pro Gly Ala Pro Trp Leu Pro Val Cys Ser Met Val Ile Gln 1670 1675
1680 Tyr Thr Ser Gln Ile Pro Ser Ser Ser Gln Thr Gln Pro Val Leu
1685 1690 1695 Gln Ser Gln Ala Glu Asn Leu Leu Cys Arg Thr Tyr Gln
Lys Trp 1700 1705 1710 Lys Asn Lys Ser Leu Ser Pro Gly Gln Glu Leu
Gly Pro Ser Val 1715 1720 1725 Ala Glu Ile Pro Trp Asp Asp Ile Ile
Thr Leu Cys Ile Asn His 1730 1735 1740 Lys Leu Arg Asp Trp Thr Pro
Pro Arg Leu Pro Val Thr Leu Glu 1745 1750 1755 Ala Leu Ser Glu Asp
Gly Gln Ile Cys Val Tyr Phe Phe Lys Asn 1760 1765 1770 Leu Leu Arg
Lys Tyr His Val Pro Ser Ser Trp Glu Gln Ala Arg 1775 1780 1785 Met
Gln Thr Gln Arg Glu Leu Gln Leu Ser His Gly Arg Ser Gly 1790 1795
1800 Met Arg Ser Ile His Pro Pro Thr Ser Thr Phe Pro Thr Pro Leu
1805 1810 1815 Leu His Val His Gln Lys Gly Lys Lys Lys Glu Glu Ser
Gly Arg 1820 1825 1830 Glu Gly Ser Leu Ser Thr Glu Asp Leu Leu Arg
Gly Ala Ser Ala 1835 1840 1845 Glu Glu Leu Leu Ala Gln Ser Leu Ser
Ser Ser Leu Leu Glu Glu 1850 1855 1860 Lys Glu Glu Asn Lys Arg Phe
Glu Asp Gln Leu Gln Gln Trp Leu 1865 1870 1875 Ser Gln Asp Ser Gln
Ala Phe Thr Glu Ser Thr Arg Leu Pro Leu 1880 1885 1890 Tyr Leu Pro
Gln Thr Leu Val Ser Phe Pro Asp Ser Ile Lys Thr 1895 1900 1905 Gln
Thr Met Val Lys Thr Ser Thr Ser Pro Gln Asn Ser Gly Thr 1910 1915
1920 Gly Lys Gln Leu Arg Phe Ser Glu Ala Ser Gly Ser Ser Leu Thr
1925 1930 1935 Glu Lys Leu Lys Leu Leu Glu Arg Leu Ile Gln Ser Ser
Arg Ala 1940 1945 1950 Glu Glu Ala Ala Ser Glu Leu His Leu Ser Ala
Leu Leu Glu Met 1955 1960 1965 Val Asp Met 1970 2 6429 DNA Mouse 2
gttgcggtgc ggtgggcccg gtagaggctg cacgcagact gtgggcgagc acaagcgctg
60 gcgacagtgg ccgtatctgg cggacttgct cctccctccg cggcctccgc
tgtcccttgt 120 gtctttgccg agttgctgaa ggccttcact agtcttcgct
cgaaggcgtc tgttaaccta 180 gcggccggct tccggagtgt taagcatcgg
ggataaaaag ctattatttc tagaccaggg 240 catcgcaagt tcgagttacc
gggagaaaaa tgagatggtc atcctgagga tgaaggagag 300 cttcccctgg
caacagataa tttaaagagg agagctactt gtgtatagtc catatttatt 360
gccttcagat aattggcttg aagatgcacc cggtgaaccc cttcggaggc agcagcccaa
420 gtgcttttgc ggtatcttcc agcaccacgg gaacatatca gactaaatca
ccatttcgat 480 ttggccagcc ttcccttttt ggacagaaca gcacacccag
caagagcctg gcgttttcac 540 aagtaccaag ctttgcaaca ccctctggag
gaagccattc ttcctccttg ccagcatttg 600 gactcaccca aacctcaagt
gtgggactct tctctagtct cgaatccaca ccttctttcg 660 cagctacttc
gagttcctct gtgcccggca atacggcatt cagctttaag tcaacctcta 720
gcgttggggt tttcccaagt ggcgctactt ttgggccaga aaccggagaa gtagcaggtt
780 ctggctttcg gaagacggaa ttcaagttta aacctctgga aaatgcagtc
ttcaaaccga 840 taccggggcc tgagtcagag ccagaaaaaa cccagagcca
gatttcttct ggatttttta 900 cattttccca tcccgttggt agcgggtctg
gaggcctgac ccctttttct ttcccacagg 960 tgacaaatag ttcggtgact
agctcaagtt ttatcttttc gaaaccagtt actagtaata 1020 ctcctgcctt
tgcctctcct ttgtctaacc aaaatgtaga agaagagaag agggtttcta 1080
cgtcagcgtt tggaagctca aacagtagct tcagtacttt ccccacagcg tcaccaggat
1140 ctttggggga gcccttccca gctaacaaac caagcctccg ccaaggatgt
gaggaagcca 1200 tctcccaggt ggagccactt cccaccctca tgaagggatt
aaagaggaaa gaggaccagg 1260 atcgctcccc gaggagacat tgccacgagg
cagcagaaga ccctgatccc ctgtccaggg 1320 gcgaccatcc cccagataaa
cggccagtcc gcctcaacag accccgggga ggtactttgt 1380 ttggccggac
aatacaggag gtcttcaaaa gcaataaaga ggcaggccgc ctgggcagca 1440
aggaatccaa ggagagtggc tttgcggaac ctggggaaag tgaccacgcg gccgtcccag
1500 gagggagtca gtccaccatg gtaccttccc gccttccagc tgtgactaaa
gaggaagaag 1560 aaagtagaga tgagaaagaa gattctctca ggggaaagtc
tgtgcgccag agtaagcgaa 1620 gggaagagtg gatctacagc ctcgggggcg
tgtcttcttt agagctcaca gccatccagt 1680 gcaagaacat ccccgactac
ctcaacgaca gagccatcct ggagaaacac ttcagcaaaa 1740 tcgctaaagt
ccagcgggtc ttcaccagac gcagcaagaa gctcgccgtg attcattttt 1800
tcgaccacgc atcggcagcc ctggctagga agaaggggaa aggtctgcat aaggacgtgg
1860 ttatcttttg gcacaagaag aaaataagtc ccagcaagaa actctttccc
ctgaaggaga 1920 agcttggtga gagtgaagcc agccagggca tcgaggactc
cccctttcag cactcgcctc 1980 tcagcaagcc catcgtgagg cctgcagccg
gcagcctcct cagcaaaagc tctccagtga 2040 agaagccgag tcttctgaag
atgcaccagt ttgaggcgga tccttttgac tctggatctg 2100 agggctccga
gggccttggt tcttgcgtgt catctcttag caccctgata gggactgtgg 2160
cagacacatc tgaggagaag taccgccttc tggaccagag agaccgcatc atgcggcaag
2220 ctcgagtgaa gaggacggac ctggacaaag ccagggcatt tgttgggact
tgccctgaca 2280 tgtgtcccga gaaggagcgg tacttgaggg agacccggag
ccagctgagc gtgtttgaag 2340 ttgtcccagg gactgaccag gtggaccatg
cagcagccgt gaaggagtac agccggtcct 2400 ctgcagatca ggaggagccc
ctgccacatg agctgagacc ctcagcagtt ctcagcagga 2460 ccatggacta
cctggtgacc cagatcatgg accaaaagga aggcagcctt cgggattggt 2520
atgacttcgt gtggaaccgc acccggggta tacggaagga cataacacag cagcacctct
2580 gtgatcccct gacggtgtct ctgatcgaga agtgtacccg atttcacatt
cactgtgccc 2640 actttatgtg tgaggagcct atgtcttcct ttgatgccaa
gatcaacaat gagaacatga 2700 ccaagtgtct acagagtctg aaggagatgt
accaggacct gaggaacaag ggtgtttttt 2760 gtgccagtga
agcagagttt cagggctaca atgtcctgct taatctcaac aaaggagaca 2820
ttttgagaga agtgcagcag ttccaccctg acgttaggaa ctccccagag gtgaacttcg
2880 ctgtccaggc ttttgctgca ttgaacagca ataattttgt gagatttttc
aaactggttc 2940 agtcagcttc ttacctgaat gcgtgcctgt tacactgtta
ctttaatcag atccgcaagg 3000 atgccctccg ggcactcaat gttgcttata
ctgtaagcac acagcgctct accgtcttcc 3060 ccctggatgg tgtcgtccgc
atgctgctgt tcagagatag tgaagaggcg acaaacttcc 3120 tcaattacca
tggcctcact gtagctgatg gctgtgttga gctgaatcgg tcggcattct 3180
tggaaccgga gggattatgc aaggccagga agtcagtgtt tattggccgg aagctgacgg
3240 tgtcagttgg ggaagttgtg aatggagggc cgttgccccc tgttcctcgc
catacacctg 3300 tgtgcagctt caactcccag aataagtacg ttggagagag
cctggctacg gagctgccca 3360 tcagcactca gagagctggt ggagacccag
caggtggtgg cagaggagag gactgtgagg 3420 cagaggtgga cttgccaaca
ttggcggtcc tcccacagcc gcctcctgca tcctcagcca 3480 cgccggcgct
tcatgtccag ccactggccc cagccgcagc acccagcctt ctccaggcct 3540
ccacgcagcc tgaggtgctg cttccaaagc ctgcgcctgt gtactctgac tcggacctgg
3600 tacaggtggt ggacgagctc atccaggagg ctctgcaagt ggactgtgag
gaagtcagct 3660 ccgctggggc agcctacgta gccgcagctc tgggcgtttc
caatgctgct gtggaggatc 3720 tgattactgc tgcgaccacg ggcattctga
ggcacgttgc cgctgaggaa gtttccatgg 3780 aaaggcagag actagaggaa
gagaagcaac gagctgagga ggaacggttg aagcaagaga 3840 gagaactgat
gttaactcag ctgagcgagg gtctggccgc agagctgaca gaactcacgg 3900
tgacagagtg tgtgtgggaa acctgctctc aggagctaca gagtgcagta aaaatagacc
3960 agaaggtccg tgtggcccgc tgttgtgaag ccgtctgtgc acacctggtg
gatttgtttc 4020 ttgctgagga aattttccag actgcaaaag agacactcca
ggaactccag tgtttctgca 4080 agtatctaca acggtggagg gaggctgttg
cagctcggaa gaaattccgg cgtcagatgc 4140 gggccttccc tgcagcgcca
tgctgtgtgg atgtgaatga ccggctgcag gcactagtgc 4200 ccagcgcaga
gtgccccatt actgaggaga acctggccaa gggtcttttg gacctgggcc 4260
acgcaggcaa agtaggcgtc tcctgtacca ggttgaggcg gcttagaaac aagacagctc
4320 accagataaa ggtccagcac ttccaccagc agctgctgag gaatgctgca
tgggcacctc 4380 tggacctgcc atccattgtg tctgagcacc tccccatgaa
gcagaagcga aggttttgga 4440 aactggtgct ggtgttgcct gatgtggaag
agcagactcc agagagtcct ggcagaatac 4500 tagaaaactg gctaaaggtc
aaattcacag gagatgacag catggtgggt gacataggag 4560 ataatgctgg
tgatatccag accctctcag tctttaatac acttagtagt aaaggggatc 4620
aaacagtttc tgtcaacgtg tgtataaagg tggctcatgg cacccttagt gacagtgccc
4680 ttgatgctgt ggagacccag aaggacctgt tgggaaccag tgggctcatg
ctgctgcttc 4740 ccccgaaagt gaagagtgag gaggtggcag aggaggaact
gtcctggctg tcggctttac 4800 tgcagctcaa gcagcttctg caggccaagc
ccttccagcc tgccctgccg ctggtggtcc 4860 tcgtgcccag ctccagaggg
gactccgcgg ggagggcagt agaggacggt ctgatgttac 4920 aggatttggt
ttcagccaag ctgatttccg attacattgt tgttgagatt cctgactctg 4980
ttaatgattt acaaggcaca gtgaaggttt ctggagcagt ccagtggctg atctccggat
5040 gtcctcaagc cctagacctt tgctgccaga cccttgttca gtatgttgag
gatgggatca 5100 gccgcgagtt cagccgtcgg tttttccacg acaggagaga
gaggcgcctg gctagcctgc 5160 cctcccagga gcctagcacc attattgagt
tgttcaacag tgtgctgcag ttcctggcct 5220 ctgtggtatc ctctgagcag
ctgtgtgaca tctcctggcc tgtcatggaa tttgccgaag 5280 tgggaggcag
ccagctgctt cctcacctgc actggaactc accagagcat ctagcgtggc 5340
tgaaacaagc tgtgcttggg ttccagcttc cacagatgga ccttccaccc ccaggggccc
5400 cctggctccc tgtgtgttcc atggtcattc agtacacctc ccagattccc
agctcaagcc 5460 agacacagcc tgtcctccag tcccaggcgg agaacctgct
gtgcagaaca taccagaagt 5520 ggaagaacaa gagcctctct ccaggccagg
agttggggcc ttctgttgcc gagatcccgt 5580 gggatgacat catcacctta
tgcatcaatc ataagctgag ggactggaca ccccccaggc 5640 tccctgtcac
attagaggcg ctgagtgaag atggtcaaat atgtgtgtat tttttcaaaa 5700
accttttaag aaaataccac gttccctcgt catgggaaca ggccagaatg cagacgcagc
5760 gggaactgca gctgagtcat ggacgttcgg ggatgaggtc catccatcct
cctacaagca 5820 cttttcctac tccattgctt catgtacacc agaaagggaa
gaaaaaggaa gagagtggcc 5880 gagaggggag cctcagtaca gaggacctcc
tgcggggggc ttctgcagaa gagctcctgg 5940 cacagagtct gtccagcagt
cttctggaag agaaggaaga gaacaagagg tttgaagatc 6000 aacttcagca
gtggttatcg caagactcac aggcattcac agagtcaact cggcttcctc 6060
tctacctccc tcagacgcta gtgtcctttc ctgattctat caaaactcag accatggtga
6120 aaacatctac aagtcctcag aattcaggaa caggaaagca gttgaggttc
tcagaggcat 6180 ccggttcatc cctgacggaa aagctgaagc tcctggaaag
gctgatccag agctcaaggg 6240 cggaagaagc agcctccgag ctgcacctct
ctgcactgct ggagatggtg gacatgtagc 6300 tgtctgacgg gagacggatc
tctaattcat aatgctttgt ctgtattcaa ttgtgttata 6360 gatgctgttg
gaaatgtgac tattaattat gcaaataaac tttttgaatc attccaaaaa 6420
aaaaaccat 6429 3 1980 PRT Homo sapiens 3 Met Asn Pro Thr Asn Pro
Phe Ser Gly Gln Gln Pro Ser Ala Phe Ser 1 5 10 15 Ala Ser Ser Ser
Asn Val Gly Thr Leu Pro Ser Lys Pro Pro Phe Arg 20 25 30 Phe Gly
Gln Pro Ser Leu Phe Gly Gln Asn Ser Thr Leu Ser Gly Lys 35 40 45
Ser Ser Gly Phe Ser Gln Val Ser Ser Phe Pro Ala Ser Ser Gly Val 50
55 60 Ser His Ser Ser Ser Val Gln Thr Leu Gly Phe Thr Gln Thr Ser
Ser 65 70 75 80 Val Gly Pro Phe Ser Gly Leu Glu His Thr Ser Thr Phe
Val Ala Thr 85 90 95 Ser Gly Pro Ser Ser Ser Ser Val Leu Gly Asn
Thr Gly Phe Ser Phe 100 105 110 Lys Ser Pro Thr Ser Val Gly Ala Phe
Pro Ser Thr Ser Ala Phe Gly 115 120 125 Gln Glu Ala Gly Glu Ile Val
Asn Ser Gly Phe Gly Lys Thr Glu Phe 130 135 140 Ser Phe Lys Pro Leu
Glu Asn Ala Val Phe Lys Pro Ile Leu Gly Ala 145 150 155 160 Glu Ser
Glu Pro Glu Lys Thr Gln Ser Gln Ile Ala Ser Gly Phe Phe 165 170 175
Thr Phe Ser His Pro Ile Ser Ser Ala Pro Gly Gly Leu Ala Pro Phe 180
185 190 Ser Phe Pro Gln Val Thr Ser Ser Ser Ala Thr Thr Ser Asn Phe
Thr 195 200 205 Phe Ser Lys Pro Val Ser Ser Asn Asn Ser Leu Ser Ala
Phe Thr Pro 210 215 220 Ala Leu Ser Asn Gln Asn Val Glu Glu Glu Lys
Arg Gly Pro Lys Ser 225 230 235 240 Ile Phe Gly Ser Ser Asn Asn Ser
Phe Ser Ser Phe Pro Val Ser Ser 245 250 255 Ala Val Leu Gly Glu Pro
Phe Gln Ala Ser Lys Ala Gly Val Arg Gln 260 265 270 Gly Cys Glu Glu
Ala Val Ser Gln Val Glu Pro Leu Pro Ser Leu Met 275 280 285 Lys Gly
Leu Lys Arg Lys Glu Asp Gln Asp Arg Ser Pro Arg Arg His 290 295 300
Gly His Glu Pro Ala Glu Asp Ser Asp Pro Leu Ser Arg Gly Asp His 305
310 315 320 Pro Pro Asp Lys Arg Pro Val Arg Leu Asn Arg Pro Arg Gly
Gly Thr 325 330 335 Leu Phe Gly Arg Thr Ile Gln Asp Val Phe Lys Ser
Asn Lys Glu Val 340 345 350 Gly Arg Leu Gly Asn Lys Glu Ala Lys Lys
Glu Thr Gly Phe Val Glu 355 360 365 Ser Ala Glu Ser Asp His Met Ala
Ile Pro Gly Gly Asn Gln Ser Val 370 375 380 Leu Ala Pro Ser Arg Ile
Pro Gly Val Asn Lys Glu Glu Glu Thr Glu 385 390 395 400 Ser Arg Glu
Lys Lys Glu Asp Ser Leu Arg Gly Thr Pro Ala Arg Gln 405 410 415 Ser
Asn Arg Ser Glu Ser Thr Asp Ser Leu Gly Gly Leu Ser Pro Ser 420 425
430 Glu Val Thr Ala Ile Gln Cys Lys Asn Ile Pro Asp Tyr Leu Asn Asp
435 440 445 Arg Thr Ile Leu Glu Asn His Phe Gly Lys Ile Ala Lys Val
Gln Arg 450 455 460 Ile Phe Thr Arg Arg Ser Lys Lys Leu Ala Val Val
His Phe Phe Asp 465 470 475 480 His Ala Ser Ala Ala Leu Ala Arg Lys
Lys Gly Lys Ser Leu His Lys 485 490 495 Asp Met Ala Ile Phe Trp His
Arg Lys Lys Ile Ser Pro Asn Lys Lys 500 505 510 Pro Phe Ser Leu Lys
Glu Lys Lys Pro Gly Asp Gly Glu Val Ser Pro 515 520 525 Ser Thr Glu
Asp Ala Pro Phe Gln His Ser Pro Leu Gly Lys Ala Ala 530 535 540 Gly
Arg Thr Gly Ala Ser Ser Leu Leu Asn Lys Ser Ser Pro Val Lys 545 550
555 560 Lys Pro Ser Leu Leu Lys Ala His Gln Phe Glu Gly Asp Ser Phe
Asp 565 570 575 Ser Ala Ser Glu Gly Ser Glu Gly Leu Gly Pro Cys Val
Leu Ser Leu 580 585 590 Ser Thr Leu Ile Gly Thr Val Ala Glu Thr Ser
Lys Glu Lys Tyr Arg 595 600 605 Leu Leu Asp Gln Arg Asp Arg Ile Met
Arg Gln Ala Arg Val Lys Arg 610 615 620 Thr Asp Leu Asp Lys Ala Arg
Thr Phe Val Gly Thr Cys Leu Asp Met 625 630 635 640 Cys Pro Glu Lys
Glu Arg Tyr Met Arg Glu Thr Arg Ser Gln Leu Ser 645 650 655 Val Phe
Glu Val Val Pro Gly Thr Asp Gln Val Asp His Ala Ala Ala 660 665 670
Val Lys Glu Tyr Ser Arg Ser Ser Ala Asp Gln Glu Glu Pro Leu Pro 675
680 685 His Glu Leu Arg Pro Leu Pro Val Leu Ser Arg Thr Met Asp Tyr
Leu 690 695 700 Val Thr Gln Ile Met Asp Gln Lys Glu Gly Ser Leu Arg
Asp Trp Tyr 705 710 715 720 Asp Phe Val Trp Asn Arg Thr Arg Gly Ile
Arg Lys Asp Ile Thr Gln 725 730 735 Gln His Leu Cys Asp Pro Leu Thr
Val Ser Leu Ile Glu Lys Cys Thr 740 745 750 Arg Phe His Ile His Cys
Ala His Phe Met Cys Glu Glu Pro Met Ser 755 760 765 Ser Phe Asp Ala
Lys Ile Asn Asn Glu Asn Met Thr Lys Cys Leu Gln 770 775 780 Ser Leu
Lys Glu Met Tyr Gln Asp Leu Arg Asn Lys Gly Val Phe Cys 785 790 795
800 Ala Ser Glu Ala Glu Phe Gln Gly Tyr Asn Val Leu Leu Ser Leu Asn
805 810 815 Lys Gly Asp Ile Leu Arg Glu Val Gln Gln Phe His Pro Ala
Val Arg 820 825 830 Asn Ser Ser Glu Val Lys Phe Ala Val Gln Ala Phe
Ala Ala Leu Asn 835 840 845 Ser Asn Asn Phe Val Arg Phe Phe Lys Leu
Val Gln Ser Ala Ser Tyr 850 855 860 Leu Asn Ala Cys Leu Leu His Cys
Tyr Phe Ser Gln Ile Arg Lys Asp 865 870 875 880 Ala Leu Arg Ala Leu
Asn Phe Ala Tyr Thr Val Ser Thr Gln Arg Ser 885 890 895 Thr Ile Phe
Pro Leu Asp Gly Val Val Arg Met Leu Leu Phe Arg Asp 900 905 910 Cys
Glu Glu Ala Thr Asp Phe Leu Thr Cys His Gly Leu Thr Val Ser 915 920
925 Asp Gly Cys Val Glu Leu Asn Arg Ser Ala Phe Leu Glu Pro Glu Gly
930 935 940 Leu Ser Lys Thr Arg Lys Ser Val Phe Ile Thr Arg Lys Leu
Thr Val 945 950 955 960 Ser Val Gly Glu Ile Val Asn Gly Gly Pro Leu
Pro Pro Val Pro Arg 965 970 975 His Thr Pro Val Cys Ser Phe Asn Ser
Gln Asn Lys Tyr Ile Gly Glu 980 985 990 Ser Leu Ala Ala Glu Leu Pro
Val Ser Thr Gln Arg Pro Gly Ser Asp 995 1000 1005 Thr Val Gly Gly
Gly Arg Gly Glu Glu Cys Gly Val Glu Pro Asp 1010 1015 1020 Ala Pro
Leu Ser Ser Leu Pro Gln Ser Leu Pro Ala Pro Ala Pro 1025 1030 1035
Ser Pro Val Pro Leu Pro Pro Val Leu Ala Leu Thr Pro Ser Val 1040
1045 1050 Ala Pro Ser Leu Phe Gln Leu Ser Val Gln Pro Glu Pro Pro
Pro 1055 1060 1065 Pro Glu Pro Val Pro Met Tyr Ser Asp Glu Asp Leu
Ala Gln Val 1070 1075 1080 Val Asp Glu Leu Ile Gln Glu Ala Leu Gln
Arg Asp Cys Glu Glu 1085 1090 1095 Val Gly Ser Ala Gly Ala Ala Tyr
Ala Ala Ala Ala Leu Gly Val 1100 1105 1110 Ser Asn Ala Ala Met Glu
Asp Leu Leu Thr Ala Ala Thr Thr Gly 1115 1120 1125 Ile Leu Arg His
Ile Ala Ala Glu Glu Val Ser Lys Glu Arg Glu 1130 1135 1140 Arg Arg
Glu Gln Glu Arg Gln Arg Ala Glu Glu Glu Arg Leu Lys 1145 1150 1155
Gln Glu Arg Glu Leu Val Leu Ser Glu Leu Ser Gln Gly Leu Ala 1160
1165 1170 Val Glu Leu Met Glu Arg Val Met Met Glu Phe Val Arg Glu
Thr 1175 1180 1185 Cys Ser Gln Glu Leu Lys Asn Ala Val Glu Thr Asp
Gln Arg Val 1190 1195 1200 Arg Val Ala Arg Cys Cys Glu Asp Val Cys
Ala His Leu Val Asp 1205 1210 1215 Leu Phe Leu Val Glu Glu Ile Phe
Gln Thr Ala Lys Glu Thr Leu 1220 1225 1230 Gln Glu Leu Gln Cys Phe
Cys Lys Tyr Leu Gln Arg Trp Arg Glu 1235 1240 1245 Ala Val Thr Ala
Arg Lys Lys Leu Arg Arg Gln Met Arg Ala Phe 1250 1255 1260 Pro Ala
Ala Pro Cys Cys Val Asp Val Ser Asp Arg Leu Arg Ala 1265 1270 1275
Leu Ala Pro Ser Ala Glu Cys Pro Ile Ala Glu Glu Asn Leu Ala 1280
1285 1290 Arg Gly Leu Leu Asp Leu Gly His Ala Gly Arg Leu Gly Ile
Ser 1295 1300 1305 Cys Thr Arg Leu Arg Arg Leu Arg Asn Lys Thr Ala
His Gln Met 1310 1315 1320 Lys Val Gln His Phe Tyr Gln Gln Leu Leu
Ser Asp Val Ala Trp 1325 1330 1335 Ala Ser Leu Asp Leu Pro Ser Leu
Val Ala Glu His Leu Pro Gly 1340 1345 1350 Arg Gln Glu His Val Phe
Trp Lys Leu Val Leu Val Leu Pro Asp 1355 1360 1365 Val Glu Glu Gln
Ser Pro Glu Ser Cys Gly Arg Ile Leu Ala Asn 1370 1375 1380 Trp Leu
Lys Val Lys Phe Met Gly Asp Glu Gly Ser Val Asp Asp 1385 1390 1395
Thr Ser Ser Asp Ala Gly Gly Ile Gln Thr Leu Ser Leu Phe Asn 1400
1405 1410 Ser Leu Ser Ser Lys Gly Asp Gln Met Ile Ser Val Asn Val
Cys 1415 1420 1425 Ile Lys Val Ala His Gly Ala Leu Ser Asp Gly Ala
Ile Asp Ala 1430 1435 1440 Val Glu Thr Gln Lys Asp Leu Leu Gly Ala
Ser Gly Leu Met Leu 1445 1450 1455 Leu Leu Pro Pro Lys Met Lys Ser
Glu Asp Met Ala Glu Glu Asp 1460 1465 1470 Val Tyr Trp Leu Ser Ala
Leu Leu Gln Leu Lys Gln Leu Leu Gln 1475 1480 1485 Ala Lys Pro Phe
Gln Pro Ala Leu Pro Leu Val Val Leu Val Pro 1490 1495 1500 Ser Pro
Gly Gly Asp Ala Val Glu Lys Glu Val Glu Asp Gly Leu 1505 1510 1515
Met Leu Gln Asp Leu Val Ser Ala Lys Leu Ile Ser Asp Tyr Thr 1520
1525 1530 Val Thr Glu Ile Pro Asp Thr Ile Asn Asp Leu Gln Gly Ser
Thr 1535 1540 1545 Lys Val Leu Gln Ala Val Gln Trp Leu Val Ser His
Cys Pro His 1550 1555 1560 Ser Leu Asp Leu Cys Cys Gln Thr Leu Ile
Gln Tyr Val Glu Asp 1565 1570 1575 Gly Ile Gly His Glu Phe Ser Gly
Arg Phe Phe His Asp Arg Arg 1580 1585 1590 Glu Arg Arg Leu Gly Gly
Leu Ala Ser Gln Glu Pro Gly Ala Ile 1595 1600 1605 Ile Glu Leu Phe
Asn Ser Val Leu Gln Phe Leu Ala Ser Val Val 1610 1615 1620 Ser Ser
Glu Gln Leu Cys Asp Leu Ser Trp Pro Val Thr Glu Phe 1625 1630 1635
Ala Glu Ala Gly Gly Ser Arg Leu Leu Pro His Leu His Trp Asn 1640
1645 1650 Ala Pro Glu His Leu Ala Trp Leu Lys Gln Ala Val Leu Gly
Phe 1655 1660 1665 Gln Leu Pro Gln Met Asp Leu Pro Pro Leu Gly Ala
Pro Trp Leu 1670 1675 1680 Pro Val Cys Ser Met Val Val Gln Tyr Ala
Ser Gln Ile Pro Ser 1685 1690 1695 Ser Arg Gln Thr Gln Pro Val Leu
Gln Ser Gln Val Glu Asn Leu 1700 1705 1710 Leu His Arg Thr Tyr Cys
Arg Trp Lys Ser Lys Ser Pro Ser Pro 1715 1720 1725 Val His Gly Ala
Gly Pro Ser Val Met Glu Ile Pro Trp Asp Asp 1730 1735 1740 Leu Ile
Ala Leu Cys Ile Asn His Lys Leu Arg Asp Trp Thr Pro 1745 1750 1755
Pro Arg Leu Pro Val Thr Ser Glu Ala Leu Ser Glu Asp Gly Gln 1760
1765 1770 Ile Cys Val Tyr Phe Phe Lys Asn Asp Leu Lys Lys Tyr Asp
Val 1775 1780 1785 Pro Leu Ser Trp Glu Gln Ala Arg Leu Gln Thr Gln
Lys Glu Leu 1790 1795 1800 Gln Leu Arg Glu Gly Arg Leu Ala Ile Lys
Pro Phe His Pro Ser 1805 1810 1815 Ala Asn Asn Phe Pro Ile Pro Leu
Leu
His Met His Arg Asn Trp 1820 1825 1830 Lys Arg Ser Thr Glu Cys Ala
Gln Glu Gly Arg Ile Pro Ser Thr 1835 1840 1845 Glu Asp Leu Met Arg
Gly Ala Ser Ala Glu Glu Leu Leu Ala Gln 1850 1855 1860 Cys Leu Ser
Ser Ser Leu Leu Leu Glu Lys Glu Glu Asn Lys Arg 1865 1870 1875 Phe
Glu Asp Gln Leu Gln Gln Trp Leu Ser Glu Asp Ser Gly Ala 1880 1885
1890 Phe Thr Asp Leu Thr Ser Leu Pro Leu Tyr Leu Pro Gln Thr Leu
1895 1900 1905 Val Ser Leu Ser His Thr Ile Glu Pro Val Met Lys Thr
Ser Val 1910 1915 1920 Thr Thr Ser Pro Gln Ser Asp Met Met Arg Glu
Gln Leu Gln Leu 1925 1930 1935 Ser Glu Ala Thr Gly Thr Cys Leu Gly
Glu Arg Leu Lys His Leu 1940 1945 1950 Glu Arg Leu Ile Arg Ser Ser
Arg Glu Glu Glu Val Ala Ser Glu 1955 1960 1965 Leu His Leu Ser Ala
Leu Leu Asp Met Val Asp Ile 1970 1975 1980 4 6114 DNA Homo sapiens
4 gtaatactta attaccttct aataattgga gcagaagatg aacccaacta atcctttcag
60 tgggcagcag cctagtgctt tttcggcgtc ttctagtaat gtaggaacac
ttccatctaa 120 gccgccattt cgatttggtc aaccttctct ttttggacaa
aacagtacct tatctgggaa 180 gagctcggga ttttcacagg tatccagctt
tccagcgtct tctggagtaa gtcattcctc 240 ttcagtgcaa acattagggt
tcacccaaac ctcaagtgtt ggaccctttt ctggacttga 300 gcacacttcc
acctttgtgg ctacctctgg gccttcaagt tcatctgtgc tgggaaacac 360
aggatttagt tttaaatcac ccaccagtgt tggggctttc ccaagcactt ctgcttttgg
420 acaagaagct ggagaaatag tgaactctgg ttttgggaaa acagaattca
gctttaaacc 480 tctggaaaat gcagtgttca aaccaatact gggggctgaa
tctgagccag agaaaaccca 540 gagccaaatt gcttctgggt tttttacatt
ttcccaccca attagtagtg cacctggagg 600 cctggcccct ttctcttttc
ctcaagtaac aagtagttca gctaccactt caaattttac 660 cttttcaaaa
cctgttagta gtaataattc attatctgcc tttacccctg ctttgtcaaa 720
ccaaaatgta gaggaagaga agagaggacc taagtcaata tttggaagtt ctaataatag
780 cttcagtagc ttccctgtat catctgcggt tttgggcgaa cctttccagg
ctagcaaagc 840 aggtgtcagg caggggtgtg aagaagctgt ttcccaggtg
gaaccacttc ccagcctaat 900 gaaaggactg aaaaggaagg aggaccagga
tcgctcccca aggagacatg gccacgagcc 960 agcagaagat tcggatcctc
tgtcccgggg cgatcatcct ccagacaaac gacctgtccg 1020 cctgaatcga
ccccggggag gtactttatt tggtcggacg atacaggatg ttttcaaaag 1080
caataaggaa gtaggtcgtc tgggcaacaa ggaggccaaa aaggaaactg gctttgttga
1140 gtctgcagaa agtgaccaca tggctatccc aggagggaat cagtctgtcc
tggcaccttc 1200 ccggattcca ggtgtgaata aagaggaaga aactgaaagt
agagagaaga aagaagattc 1260 tctaagagga actccggcgc gtcagagtaa
cagaagcgag agcacagaca gtcttggggg 1320 cttgtctccc tctgaagtca
cagccatcca gtgcaagaac atccctgact acctcaacga 1380 caggaccatt
ctggagaacc attttggcaa aattgctaaa gtgcagcgca tctttaccag 1440
gcgcagcaaa aagcttgcag tggtacattt ctttgatcat gcatctgcag ccctggctag
1500 aaagaagggg aaaagtttgc ataaagacat ggctatcttt tggcacagga
agaaaataag 1560 ccccaataag aaaccctttt ccctgaagga gaagaaacca
ggtgacggtg aagtcagccc 1620 gagcacagag gatgcaccct ttcagcactc
tcctcttggc aaggccgcag ggaggactgg 1680 tgctagcagc ctcctgaata
aaagctctcc agtgaagaag ccaagtcttc taaaggccca 1740 ccaattcgag
ggagactctt ttgactcagc ctccgagggc tccgagggcc tcgggccatg 1800
tgtgctctcc ctcagtaccc tgataggcac tgtggctgag acatccaagg agaagtaccg
1860 cctgcttgac cagagagaca ggatcatgcg gcaagctcgg gtgaagagaa
ccgatctgga 1920 caaagcgagg acttttgttg gcacctgcct ggatatgtgt
cctgagaagg agaggtacat 1980 gcgggagacc cgtagccagc tgagcgtgtt
cgaagtggtc ccagggactg accaggtgga 2040 ccacgcagca gctgtgaaag
agtacagtcg gtcctcggcg gatcaggagg agcccctgcc 2100 ccacgagctg
cggcccttgc cagtgctcag caggaccatg gactacctgg tgacccagat 2160
catggaccag aaggagggca gcctgcggga ttggtatgac ttcgtgtgga accgcacgcg
2220 tggcatacgg aaggatatca cgcagcagca cctctgtgac cccctgacgg
tgtccctgat 2280 tgagaagtgc acccggtttc acatccactg tgcccacttc
atgtgtgagg agcccatgtc 2340 ctcctttgat gccaagatca ataatgagaa
catgaccaag tgcctgcaga gcctgaagga 2400 gatgtaccag gacctgagaa
acaagggtgt cttctgtgcc agcgaagcgg agttccaggg 2460 ctacaatgtt
ctgctcagtc tcaacaaggg agacatccta agagaagtac aacagttcca 2520
tcctgctgtt agaaactcat ctgaggtgaa atttgctgtt caggcttttg ctgcattgaa
2580 cagtaataat tttgtgagat ttttcaaact ggtccagtca gcttcttacc
tgaacgcttg 2640 tcttttacac tgttacttca gtcagatccg caaggatgct
ctccgggcgc tcaactttgc 2700 gtacacggtg agcacacagc gatctaccat
ctttcccctg gatggtgtgg tgcgcatgct 2760 gctgttcaga gactgtgaag
aggccaccga cttcctcacc tgccacggcc tcaccgtttc 2820 cgacggctgt
gtggagctga accggtctgc attcctggaa ccagagggat tatccaagac 2880
caggaagtcg gtgtttatta ctaggaagct gacggtgtca gtcggggaaa ttgtgaacgg
2940 agggccattg ccccccgtcc ctcgtcacac ccctgtgtgc agcttcaact
cccagaacaa 3000 gtacatcggg gagagcctgg ccgcggagct gcccgtcagc
acccagagac ccggctccga 3060 cacagtgggc ggagggagag gagaggagtg
tggtgtagag ccggatgcac ccctgtccag 3120 tctcccacag tctctaccag
cccctgcgcc ctcaccagtg cctctgcctc ctgtcctggc 3180 actgaccccg
tctgtggcgc ccagcctctt ccagctgtct gtgcagcctg aaccaccgcc 3240
tccagagccc gtgcccatgt actctgacga ggacctggcg caggtggtgg acgagctcat
3300 ccaggaggcc ctgcagaggg actgtgagga agttggctct gcgggtgctg
cctacgcagc 3360 tgccgccctg ggtgtttcta atgctgctat ggaggatttg
ttaacagctg caaccacggg 3420 cattttgagg cacattgcag ctgaagaagt
gtctaaggaa agagagcgaa gggagcagga 3480 gaggcagcgg gctgaagagg
aaaggttgaa acaagagaga gagctggtgt taagtgagct 3540 gagccagggc
ctggccgtgg agctgatgga acgcgtgatg atggagtttg tgagggaaac 3600
ctgctcccag gagttgaaga atgcagtaga gacagaccag agggtccgtg tggcccgttg
3660 ctgtgaggat gtctgtgccc acttagtgga cttgtttctc gtggaggaaa
tcttccagac 3720 tgcaaaggag accctccagg agcttcagtg cttctgcaag
tatctacagc ggtggaggga 3780 agctgtcaca gcccgcaaga aactgaggcg
ccaaatgcgg gctttccctg ctgcgccctg 3840 ctgcgtggac gtgagcgacc
ggctgagggc gctggcgccc agcgcagagt gccccattgc 3900 tgaagagaac
ctggccaggg gcctcctgga cctgggccat gcagggagat tgggcatctc 3960
ttgcaccagg ttaaggcggc tcagaaacaa gacagctcac cagatgaagg ttcagcactt
4020 ctaccagcag ctgctgagtg atgtggcatg ggcgtctctg gacctgccat
ccctcgtggc 4080 tgagcacctc cctgggaggc aggagcatgt gttttggaag
ctggtgctgg tgttgccgga 4140 tgtagaggag cagtccccag agagttgtgg
cagaattcta gcaaattggt taaaagtcaa 4200 gttcatggga gatgaaggct
cagtggatga cacatccagc gatgctggtg ggattcagac 4260 gctttcgctt
ttcaactcac ttagcagcaa aggggatcag atgatttctg ttaacgtgtg 4320
tataaaggtg gcccatggcg ccctcagtga tggtgccatt gatgctgtgg agacacagaa
4380 ggacctcctg ggagccagtg ggctcatgct gctgcttccc cccaaaatga
agagtgagga 4440 catggcagag gaggacgtgt actggctgtc ggccttgctg
cagctcaagc agctcctgca 4500 ggctaagccc ttccagcctg cgcttcctct
ggtggttctt gtgcctagcc caggagggga 4560 cgccgttgag aaggaagtag
aagatggtct gatgctacag gacttggttt cagctaagct 4620 gatttcagat
tacactgtta ccgagatccc tgataccatt aatgatctac aaggttcaac 4680
taaggttttg caagcagtgc agtggctggt ttcccactgc ccccattccc ttgacctctg
4740 ctgccagact ctcattcagt acgtcgaaga cgggattggc catgagttta
gtggccgctt 4800 tttccatgac agaagagaga ggcgtctggg cggtcttgct
tctcaggagc ctggcgccat 4860 cattgagctg tttaacagtg tgctgcagtt
cctggcttct gtggtgtcct ctgaacagct 4920 gtgtgacctg tcctggcctg
tcactgagtt tgctgaggca gggggcagcc ggctgcttcc 4980 tcacctgcac
tggaatgccc cagagcacct ggcctggctg aagcaggctg tgctcgggtt 5040
ccagcttccg cagatggacc ttccacccct gggggccccc tggctccccg tgtgctccat
5100 ggttgtccag tacgcctccc agatccccag ctcacgccag acacagcctg
tcctccagtc 5160 ccaggtggag aacctgctcc acagaaccta ctgtaggtgg
aagagcaaga gtccctcccc 5220 agtccatggg gcaggcccct cggtcatgga
gatcccatgg gatgatctta tcgccttgtg 5280 tatcaaccac aagctgagag
actggacgcc cccccggctt cctgttacat cagaggcgct 5340 gagtgaagat
ggtcagatat gtgtgtattt ttttaaaaac gatttgaaaa aatatgatgt 5400
tcctttgtcg tgggaacaag ccaggttgca gacgcagaag gagctacagc tgagagaggg
5460 acgtttggca ataaagcctt ttcatccttc tgcaaacaat tttcccatac
cattgcttca 5520 catgcaccgt aactggaaga ggagcacaga gtgtgctcaa
gaggggagga ttcccagcac 5580 agaggatctg atgcgaggag cttctgctga
ggagctcttg gcgcagtgtt tgtcgagcag 5640 tctgctgctg gagaaagaag
agaacaagag gtttgaagat cagcttcagc aatggttgtc 5700 tgaagactca
ggagcattta cggatttaac ttcccttccc ctctatcttc ctcagactct 5760
agtgtctctt tctcacacta ttgaacctgt gatgaaaaca tctgtaacta ctagcccaca
5820 gagtgacatg atgagggagc aactgcagct gtcagaggcg acaggaacgt
gtctaggcga 5880 acgactaaag cacctggaaa ggctgatccg gagttcaagg
gaagaggaag ttgcctctga 5940 gctccatctc tctgcgctgc tagacatggt
ggacatttga gcagcctgac ctgtggggag 6000 ggggtctctc ccgaagagtt
tctgttttta ctcaaaataa tgttattctc agatgcttga 6060 tgcactgttg
gaaatgtgat taatttaatc atgcagataa accatttaaa tgtc 6114 5 20 DNA
Artificial Sequence Forward Primer chemically synthesized 5
ccgtgggatg acatcatcac 20 6 20 DNA Artificial Sequence Reverse
Primer chemically synthesized 6 catgtccacc atctccagca 20 7 20 DNA
Artificial Sequence Primer chemically synthesized 7 tttgtctgga
ggatgatcgc 20 8 20 DNA Artificial Sequence Primer chemically
synthesized 8 aaagagaaag gggccaggcc 20 9 20 DNA Artificial Sequence
Primer chemically synthesized 9 ccagcttctt gtccaaaagc 20
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