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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2321-2328
IMMUNOBIOLOGY
From the Departments of Immunology and Anatomy II, Kumamoto
University School of Medicine, Kumamoto, Japan; the Second Department
of Pathology, Okayama University Medical School, Okayama, Japan; the
Department of Developmental Genetics, Chiba University Graduate School
of Medicine, Chiba, Japan; and the Sir William Dunn School of
Pathology, University of Oxford, Oxford, UK.
Antigen (Ag) immunization induces formation of the germinal center
(GC), with large, rapidly proliferating centroblasts in the dark zone,
and small, nondividing centrocytes in the light zone. We identified a
novel nuclear protein, GANP, that is up-regulated in centrocytes. We
found that GANP was up-regulated in GC B cells of Peyer's patches in
normal mice and in spleens from Ag-immunized mice. GANP-positive cells
appeared in the light zone of the GC, with coexpression of the peanut
agglutinin (PNA) (PNA)-positive B220-positive phenotype.
The expression of GANP was strikingly correlated with GC formation
because Bcl6-deficient mice did not show the up-regulation
of GANP. GANP-positive cells were mostly surrounded by follicular
dendritic cells. Stimulation with anti-µ and anti-CD40 induced
up-regulation of ganp messenger RNA as well as GANP protein in
B220-positive B cells in vitro. GANP is a 210-kd protein localized in
both the cytoplasm and nuclei, with a homologous region to Map80 that
is associated with MCM3, a protein essential for DNA replication.
Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also
up-regulated in the GC area. These results suggest that the
up-regulation of GANP might participate in the development of Ag-driven
B cells in GCs through its interaction with MCM3.
(Blood. 2000;95:2321-2328)
Antigen (Ag) binding to B-cell antigen receptor (BCR)
initiates activation and maturation of Ag-specific B cells in the
peripheral lymphoid organs.1,2 B cells enter the outer
periarterial lymphoid sheath and initiate costimulus-dependent
interactions with specific Th cells and interdigitating dendritic cells
within 48 hours after immunization.3,4 Ag-driven B cells
proliferate in the outer periarterial lymphoid sheath and then undergo
further activation in lymphoid follicles to establish germinal centers (GCs).5-7 Such B cells develop into large centroblasts that
move rapidly through the cell cycle to form the dark zone and further differentiate into centrocytes
(PNA+B220+sIgM+sIgD Several studies have demonstrated various differentiation Ags in GCs as
molecules identified with monoclonal antibodies (mAbs) and their
complementary DNAs (cDNAs) have been cloned.13-15 These molecules appear mostly in cells of the whole GC area; for example, GC
kinase is up-regulated in GC B cells,14 whereas
8-oxoguanine DNA glycosylase is expressed in the dark
zone.15 Ag-driven B cells appear as centroblasts that
proliferate at a very high rate, accompanied by random somatic
mutations in the V-region genes. The affinity maturation of BCRs on
centroblasts potentially generates clones that require subsequent
selection. Such mutated B cells will presumably migrate into the light
zone and differentiate into centrocytes. Studies in several kinds of
gene-targeted mice, including investigations of genes in the tumor
necrosis factor (TNF)- or TNF-receptor families, demonstrated absence
or impairment of GC formation after Ag immunization.16-18
It is possible that the follicular dendritic cell (FDC) network or
immune complexes trapped on FDCs are required for GC formation. FDCs
sequester Ags in a form of immune complexes through the complement
receptor (CR). It is generally assumed that the immune complexes
trapped on the surface of FDCs compete for binding to Ags with the BCR expressed at low levels on the surface of centrocytes. Only centrocytes with highest affinity for the Ags will succeed in binding to Ags on
FDCs. Centrocytes with low affinity will die from apoptosis, whereas
those with the highest affinity will survive. In support of this idea,
histochemical studies found that centrocytes are mostly surrounded by
CR1-positive (CR+) FDCs, with tight contact.3
FDC-mediated inhibition of apoptosis in GC B cells has been suggested,
but the molecular mechanism remains largely unknown.
Studies have shown that RAG-1 and RAG-2 proteins are expressed
selectively in centrocytes in the light zone.19,20 It is known that mature B cells undergo secondary Ig gene rearrangements that
will create a variety of BCR specificity triggered by the binding of
Ags. The GC area probably provides signals leading to receptor editing
through T-cell-dependent activation of B cells, as described by
Papavasiliou et al21 and Han et al.22
Centrocytes expressing RAG proteins will undergo exchange of V-region
segments, which would further generate new combinatory Ab
specificities. The secondary repertoire generated in centrocytes would
be selected, presumably in the microenvironment provided by the FDC network.
Although much information is available on GCs, the molecules involved
in the differentiation and activation of GC B cells remain unknown. To
study such molecules, we prepared mAbs against intracellular components
of a murine B-cell line, WEHI-231. An mAb, 29-15, recognizes a
differentiation Ag whose expression is selectively augmented in
centrocytes of GCs in the spleens of Ag-immunized mice. The
29-15-positive (29-15+) B cells are surrounded by
CR1+ FDCs, suggesting that there is a functional
association between the 29-15 Ag and the FDCs. With use of cDNA
cloning, we determined that the 29-15 Ag is a 210-kd phosphoprotein,
and we named it GANP. GANP shows regional homology to Map80, a protein
that binds to MCM3, which is a protein essential for the initiation of
DNA replication.23 We found that GANP was associated with
MCM3 in B cells and studied the possible relation between molecular
events within the GC and DNA replication.
Mice and cells
Establishment of 29-15 mAb
Antibodies and reagents Reagents were the F(ab')2 fragment of the affinity-purified goat antimouse µ Ab (anti-µ Ab; ICN Pharmaceutical, Costa Mesa, CA), biotin-conjugated PNA (Vector Laboratories, Burlingame, CA), biotin-conjugated anti-CD35 mAb (anti-CR1; PharMingen, San Diego, CA), alkaline phosphatase (ALP)-conjugated goat antirat Ig Ab (ALP-antirat Ig [59 301]; ICN Pharmaceutical), horseradish peroxidase (HRP)-conjugated goat antirat Ig Ab (HRP-antirat Ig; ICN Pharmaceutical), HRP-conjugated streptavidin (HRP-ST; Kirkegaard & Perry Laboratories, Gaithersburg, MD), mouse anti-bromodeoxyuridine (BrdU) mAb (Novocastra Laboratories, Newcastle, UK), ALP-conjugated goat antimouse Ig Ab (ALP-antimouse Ig; Sigma Chemical, St Louis, MI), fluorescein isothiocyanate-conjugated (FITC) mouse antirat mAb (FITC-antirat ; ICN Pharmaceutical),
phycoerythrin (PE)-conjugated anti-B220 mAb (PharMingen), and
ALP-conjugated goat antirabbit Ig Ab (Zymed Laboratories, South San
Francisco, CA). Biotin-conjugated mAbs, such as anti-B220 (RA3-6B2) and
anti- (CS/15), and purified anti-CD40 mAb (LB429) were prepared as
described previously.24 Rabbit antimouse MCM3/P1 Ab was
prepared as described previously.27
Immunohistochemical studies Six-micrometer cryosections of organs were placed on gelatin-coated slides, dried, and fixed in acetone.28,29 After rehydration in PBS, the sections were incubated with the 29-15 mAb, washed, incubated with ALP-antirat Ig, and developed with Vector Blue (Vector Laboratories). Double staining was carried out using biotin-labeled mAbs in combination with HRP-ST and development with 3,3'-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan). BrdU incorporation after labeling in vivo for 1 hour was detected with anti-BrdU mAb in combination with ALP-antimouse Ig and Vector Red (Vector Laboratories).30 Sections from RAG-1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient (Bcl6 /) mice (reconstitution
marrow [RM]) were prepared as described previously.31 For
cell smears, spleen B cells were attached to gelatin-coated slides,
fixed with acetone, and stained by anti-GANP mAb in combination with
ALP-antirat Ig. After development with Vector Red, sections were
lightly counterstained with hematoxylin.
Molecular cloning of a cDNA clone and in situ RNA hybridization on tissue sections A gt-11 cDNA library of WEHI-231 was screened with the 29-15 mAb
in combination with sheep antirat Ig Ab labeled with iodine 125 (Amersham, Buckinghamshire, UK).32 With the initial cDNA clone (280-base pair [bp] insert), we obtained longer cDNA clones and
determined the coding sequence. In situ RNA hybridization was carried
out as described previously.33 Paraffin-embedded sections
mounted on silanized slides were hybridized with
ganp 280-bp riboprobe labeled with digoxigenin. Slides were
washed with TNE buffer (10 mmol/L of Tris-hydrochloric
acid (HCl) [pH 7.6], 500 mmol/L of sodium chloride (NaCl), and 1 mmol/L of EDTA) at 37 °C and with 1 × SSC solution at 50 °C and developed with anti-digoxigenin Ab followed by ALP substrate.
Preparation of the glutathione-S-transferase-cDNA fusion protein The glutathione-S-transferase (GST)-GANP fusion protein was prepared from bacteria harboring a pGEX-4T-1 vector (Pharmacia Biotech, Piscataway, NJ)-based plasmid containing the ganp cDNA encoding amino acids 579 to 1028 with use of glutathione-Sepharose (Pharmacia Biotech) chromatography.32 Another hybridoma secreting the anti-GANP mAb 42-23, used for the immunoprecipitation, was established as described above by immunizing rats with the GST-GANP protein.Immunoprecipitation-Western blot analysis Proteins obtained by subcellular fractionation as described previously34 were immunoprecipitated with the anti-GANP mAb in combination with protein G-Sepharose and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Western blot filter was incubated with the anti-GANP mAb followed by HRP-antirat Ig. An enhanced-chemiluminescence (ECL) detection kit (Amersham) was used for development.Immunostaining of spleen B cells Spleen B cells were fixed with 2.5% paraformaldehyde in PBS and subjected to permeabilization with 70% ethanol. The cells were incubated with the 29-15 mAb in combination with FITC-antirat and
were analyzed using a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA) as described previously.24
Reverse transcriptase-polymerase chain reaction Total RNA (1 µg each) purified from cultured B cells by using Trizol (Gibco BRL, Rockville, MD) was used as a template for cDNA synthesis (100-µL volume) with Superscript (GIBCO BRL). Polymerase chain reaction (PCR) amplification was carried out by using 2 µL of each cDNA solution with Taq Gold (Perkin-Elmer, Foster City, CA) and the primers for ganp or HPRT.20 The ganp transcripts were amplified by 5'-CCGTGGGATGACATCATCAC-3' (the forward primer) and 5'-CATGTCCACCATCTCCAGCA-3' (the reverse primer).In vitro kinase assay and V8 cleavage mapping Kinase reactions were carried out in vitro with immunoprecipitates as described previously.25 Spleen B cells were stimulated in vitro for 48 hours with anti-µ Ab and anti-CD40 mAb.24 After harvesting and washing, cells were lysed with TNE buffer (10 mmol/L of Tris-HCl [pH 7.8], 150 mmol/L of NaCl, 1 mmol/L of EDTA, 1% NP-40, 10 µg/mL of aprotinin, and 1 mmol/L of phenylmethylsulfonyl fluoride [PMSF]), and immunoprecipitated with the anti-GANP mAb. The immunoprecipitates were incubated with -adenosine triphosphate labeled with phosphorous 32 (Amersham) and
radiolabeled proteins, separated by SDS-PAGE, and detected with use of
autoradiography. In addition to the TNE buffer, we used the more harsh
washing buffer, radioimmunoprotein-assay (RIPA) buffer (10 mmol/L of
Tris-HCl [pH 7.4], 150 mmol/L of NaCl, 1 mmol/L of EDTA, 1% NP-40,
0.1% sodium deoxycholate, 0.1% SDS, 10 µg/mL of aprotinin, and 1 mmol/L of PMSF) to detect the target molecule after removing other
coprecipitated molecules. V8 cleavage mapping of the indicated protein
was performed as described previously.25
cDNA transfection and immunoprecipitation The expression vector of influenza hemagglutinin (HA)-tagged mouse MCM3 was described previously.35 An expression vector for FLAG-tagged ganp cDNA was prepared with pCXN2 vector.36 COS7 cells were transfected with 4 µg of each plasmid DNA by LipofectAMINE Plus (GIBCO BRL) according to the manufacturer's instructions. After 48 hours, cells were harvested, washed, and lysed in TNE buffer. The lysates were immunoprecipitated with rabbit anti-HA Ab (Santa Cruz Biotechnology, Santa Cruz, CA) in combination with protein A-Sepharose. After SDS-PAGE and electrotransfer, the blot filter was incubated with mouse anti-FLAG mAb (M2; Stratagene, La Jolla, CA). For ECL detection, we used HRP-conjugated goat antimouse Ig Ab (Zymed) as the secondary Ab.
Expression of the 29-15 Ag in lymphoid organs We prepared an mAb that recognizes a differentiation Ag expressed in peripheral B cells by immunizing rats with the lysate of WEHI-231. Immunohistochemical analysis with the 29-15 mAb on normal lymphoid organs of BALB/c mice did not detect expression of the Ag in the bone marrow, but lymphoid organs such as the thymus, spleen, and lymph nodes were stained weakly (data not shown). Interestingly, expression of the 29-15 Ag was very high in the central area of follicles of the Peyer's patches (Figure 1A). The Ag was expressed in B220-positive (B220+) IgD-negative B cells. In the study with lymphoid follicles from Ag-immunized mice, 29-15+ cells appeared in the GC area of the spleen (Figure 1B) as well as in GCs of the Peyer's patches (Figure 1A). In secondary lymphoid follicles, nearly half of the PNA-positive (PNA+) GC B cells were positive with the 29-15 mAb but were almost all negative with the anti-BrdU mAb. Interestingly, expression of the 29-15 Ag was up-regulated in the centrocyte area at the distal region of the central artery. To confirm this, we used the Bcl6/ RM, which does not show formation of
GCs after Ag immunization, while the extrafollicular responses seem to
be intact.31 We compared the expression of the 29-15 Ag on
follicular regions in Bcl6/ RM and
Bcl6-positive (Bc16+/+) RM. Ag immunization induced the
formation of GCs in Bcl6+/+ RM but not in
Bcl6/RM (Figure 1C). The 29-15 Ag
was up-regulated in PNA+ cells in Bcl6+/+ RM.
Identification of a cDNA clone encoding GANP Ag Using the 29-15 mAb, we isolated a ganp cDNA clone. We studied whether the ganp messenger RNA (mRNA) was up-regulated in the same area detected on sections with the 29-15 mAb. In situ RNA hybridization analysis revealed that the ganp mRNA was expressed abundantly in the central area of GCs of spleens from mice immunized with SRBC but not in spleens (Figure 2A), thymuses, or lymph nodes from nonimmunized mice (data not shown). The ganp mRNA was up-regulated in GC B cells of immunized mice. This expression pattern was quite similar to the results with the 29-15 mAb on similar sections stained with hematoxylin. Further characterization of overlapping cDNA clones revealed a full-length nucleotide sequence (6429 bp). The ganp cDNA encoded a putative polypeptide composed of 1971 amino acids with a predicted molecular size of 210 kd (Figure 2B). GANP showed a regional homology to SAC3, a possible nuclear transcription regulator characterized in temperature-mutant Saccharomyces cerevisiae (Figure 2C),37 and to human Map80 protein.23 GANP has 2 nuclear localization sequences (497HKKK and 1344PMKQKRR), which could potentially support the expression of GANP in the nucleus, as suggested by the PSORT computer program. GANP also has 2 coiled-coil motifs but no zinc-finger, leucine-zipper, or homeo-domain motifs. There are 4 LXXLL motifs that are observed in nuclear transcription coactivator molecules, including CBP/p300 and p/CIP,38,39 but we did not identify the associated molecules through these motifs.
Regulation of GANP expression by BCR- and CD40-mediated signals Anti-GANP mAb 42-23 prepared with the gene product detected a protein band at 210 kd in both the nuclear and cytoplasmic compartments of WEHI-231 (Figure 3A) The GANP in the cytoplasmic fraction might result from the transition from the nucleus in WEHI-231. Because the signals through BCR and CD40 are crucial for activation of GC B cells, we examined whether either or both signals caused up-regulation of GANP expression in vitro. Flow cytometric analysis and cytostaining on the smears (Figure 3B) found that B220+ B cells showed up-regulation of GANP in comparison to the control with irrelevant mAb. Stimulation with both anti-µ Ab and anti-CD40 mAb produced a maximal response, but use of either of these reagents alone resulted in only a marginal response (data not shown). The up-regulation was also detected by the increase in ganp mRNA in B cells stimulated by anti-µ and anti-CD40 coligation in vitro (Figure 3C). Reverse transcriptase (RT-PCR) demonstrated that the amount of ganp mRNA increased at 24 and 48 hours after stimulation in comparison with the control HPRT mRNA.
Association of GANP with MCM3 protein We found a Map80-homologous region (76.3% identity at the amino acid level) in the carboxyl-terminal part of GANP. Map80 is an 80-kd nuclear protein that is involved in the translocation of MCM3, a protein essential for DNA replication, between the cytoplasm and the nuclei.23,27,40-44 Therefore, we examined the interaction between GANP and MCM3 in cells. First, we used COS7 cells transfected with cDNA of HA-MCM3 and FLAG-ganp. GANP coprecipitated with MCM3 in the double cDNA transfectants detected by Western blot analysis (Figure 4A). Next, we used WEHI-231 cells in experiments to determine whether MCM3 would coprecipitate with GANP. Anti-GANP immunoprecipitates also coprecipitated with the 100-kd protein of MCM3 (Figure 4B). These results indicate that GANP is associated with MCM3 in vivo.
We found a novel protein, GANP, that is expressed in GC B cells localized in the light zone of secondary follicles in the spleens of Ag-immunized mice. Although a trace amount of the ganp mRNA was detectable in many kinds of cells under normal conditions (data not shown), GANP appears to be up-regulated in the specified GC area in immunized mice. GANP might be required for maturation of Ag-specific B cells at the centrocyte stage.
We thank Mitsuru Matsumoto (University of Tokushima) and Kensuke Miyake (Saga Medical School) for valuable discussions and useful mAbs, respectively.
Submitted August 9, 1999; accepted November 30, 1999.
Supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture in Japan. K.K. was supported by research fellowships from the Japanese Society for the Promotion of Science for Young Scientists.
Reprints: Nobuo Sakaguchi, Department of Immunology, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto 860-0811, Japan; e-mail: nobusaka{at}kaiju.medic.kumamoto-u.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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 H. Nakajima, T. Tamura, M. Ito, F. Shibata, K. Kuroda, Y. Fukuchi, N. Watanabe, T. Kitamura, Y. Ikeda, and M. Handa SHD1 is a novel cytokine-inducible, negative feedback regulator of STAT5-dependent transcription Blood, January 29, 2009; 113(5): 1027 - 1036. [Abstract] [Full Text] [PDF]
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 K. K. Resendes, B. A. Rasala, and D. J. Forbes Centrin 2 Localizes to the Vertebrate Nuclear Pore and Plays a Role in mRNA and Protein Export Mol. Cell. Biol., March 1, 2008; 28(5): 1755 - 1769. [Abstract] [Full Text] [PDF]
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 Y. Kawatani, H. Igarashi, T. Matsui, K. Kuwahara, S. Fujimura, N. Okamoto, K. Takagi, and N. Sakaguchi Cutting Edge: Double-Stranded DNA Breaks in the IgV Region Gene Were Detected at Lower Frequency in Affinity-Maturation Impeded GANP-/- Mice J. Immunol., November 1, 2005; 175(9): 5615 - 5618. [Abstract] [Full Text] [PDF]
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 Y. Xing, H. Igarashi, X. Wang, and N. Sakaguchi Protein phosphatase subunit G5PR is needed for inhibition of B cell receptor-induced apoptosis J. Exp. Med., September 6, 2005; 202(5): 707 - 719. [Abstract] [Full Text] [PDF]
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 S. Fujimura, Y. Xing, M. Takeya, Y. Yamashita, K. Ohshima, K. Kuwahara, and N. Sakaguchi Increased Expression of Germinal Center-Associated Nuclear Protein RNA-Primase Is Associated with Lymphomagenesis Cancer Res., July 1, 2005; 65(13): 5925 - 5934. [Abstract] [Full Text] [PDF]
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N. Sakaguchi, T. Kimura, S. Matsushita, S. Fujimura, J. Shibata, M. Araki, T. Sakamoto, C. Minoda, and K. Kuwahara Generation of High-Affinity Antibody against T Cell-Dependent Antigen in the Ganp Gene-Transgenic Mouse J. Immunol., April 15, 2005; 174(8): 4485 - 4494. [Abstract] [Full Text] [PDF]
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S.-e- Khuda, M. Yoshida, Y. Xing, T. Shimasaki, M. Takeya, K. Kuwahara, and N. Sakaguchi The Sac3 Homologue shd1 Is Involved in Mitotic Progression in Mammalian Cells J. Biol. Chem., October 29, 2004; 279(44): 46182 - 46190. [Abstract] [Full Text] [PDF]
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Z. K. Mirnics, E. Caudell, Y. Gao, K. Kuwahara, N. Sakaguchi, T. Kurosaki, J. Burnside, K. Mirnics, and S. J. Corey Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center J. Immunol., April 1, 2004; 172(7): 4133 - 4141. [Abstract] [Full Text] [PDF]
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 S. L. Forsburg Eukaryotic MCM Proteins: Beyond Replication Initiation Microbiol. Mol. Biol. Rev., March 1, 2004; 68(1): 109 - 131. [Abstract] [Full Text] [PDF]
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 K. Kuwahara, S. Fujimura, Y. Takahashi, N. Nakagata, T. Takemori, S. Aizawa, and N. Sakaguchi Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses PNAS, January 27, 2004; 101(4): 1010 - 1015. [Abstract] [Full Text] [PDF]
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V. Janssens, J. Jordens, I. Stevens, C. Van Hoof, E. Martens, H. De Smedt, Y. Engelborghs, E. Waelkens, and J. Goris Identification and Functional Analysis of Two Ca2+-binding EF-hand Motifs in the B"/PR72 Subunit of Protein Phosphatase 2A J. Biol. Chem., March 14, 2003; 278(12): 10697 - 10706. [Abstract] [Full Text] [PDF]
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Y. Takei, M. Assenberg, G. Tsujimoto, and R. Laskey The MCM3 Acetylase MCM3AP Inhibits Initiation, but Not Elongation, of DNA Replication via Interaction with MCM3 J. Biol. Chem., November 1, 2002; 277(45): 43121 - 43125. [Abstract] [Full Text] [PDF]
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M. A. EL-Gazzar, K. Maeda, H. Nomiyama, M. Nakao, K. Kuwahara, and N. Sakaguchi PU.1 Is Involved in the Regulation of B Lineage-associated and Developmental Stage-dependent Expression of the Germinal Center-associated DNA Primase GANP J. Biol. Chem., December 14, 2001; 276(51): 48000 - 48008. [Abstract] [Full Text] [PDF]
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K. Kuwahara, S. Tomiyasu, S. Fujimura, K. Nomura, Y. Xing, N. Nishiyama, M. Ogawa, S. Imajoh-Ohmi, S. Izuta, and N. Sakaguchi Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions PNAS, August 28, 2001; 98(18): 10279 - 10283. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
associated protein P1, a murine homolog of yeast MCM3, changes its intracellular distribution during the DNA synthetic period.
EMBO J.
1994;13:4311-4320