SAKAI Atsushi
| Faculty Division of Natural Sciences Research Group of Biological Sciences | Professor |
| Faculty of Science | Dean, Faculty of Science |
Last Updated :2025/05/10
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SakaiName (Kana)
Atsushi
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Research History
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Teaching Experience
■Ⅱ．研究活動実績
Published Papers
- Refereed, Aquatic Botany, Elsevier BV, An obligate-halophytic mangrove, Rhizophora mucronate, does not require Na+ for the uptake of nutrient ions in their roots, Hiromi Kanai; Atsushi Sakai, Feb. 2021, 169, 103328, 103328, Scientific journal, 10.1016/j.aquabot.2020.103328
- Refereed, CYTOLOGIA, International Society of Cytology, C3-Like Photosynthetic Properties of Senescing Maize Leaves Are Accompanied by Preferential Senescence of Mesophyll Cells, Saya Shiogai; Satoshi Tamotsu; Atsushi Sakai, 25 Dec. 2018, 83, 4, 387, 391, Scientific journal, 10.1508/cytologia.83.387
- Refereed, Marine Biology, Springer Science and Business Media LLC, Phototaxis of sacoglossan sea slugs with different photosynthetic abilities: a test of the ‘crawling leaves’ hypothesis, Ayaka Miyamoto; Atsushi Sakai; Rie Nakano; Yoichi Yusa, Jun. 2015, 162, 6, 1343, 1349, Scientific journal, 10.1007/s00227-015-2673-1
- Refereed, CYTOLOGIA, International Society of Cytology, Cytological Studies on Proliferation, Differentiation, and Death of BY-2 Cultured Tobacco Cells, Atsushi Sakai; Mari Takusagawa; Asuka Nio; Yu Sawai, A procedure for the simultaneous isolation of mitochondrial and plastid nucleoids was first established in BY-2 cells. Biochemical analysis suggested the presence of nucleosome-like repetitive structural units in both of the plant organelle nucleoids. The isolated organelle nucleoids were also used for establishment of in vitro transcription/DNA synthesis systems, with which regulation of organelle genomes during proliferation and differentiation was investigated. The results revealed that transient and synchronous activation of DNA synthesis occurred in mitochondria and plastids in the initial phase of cell proliferation, which was caused by a transient activation of dually-targeted organelle DNA polymerase genes. Another series of investigations revealed a drastic difference in the transcription activity between BY-2 proplastids and leaf chloroplasts, which was brought about by differential use of bacterial- and phage-type plastid RNA polymerases. To investigate regulation of plastid gene expression further, a procedure to induce amyloplast formation in BY-2 cells was established. Various changes observed during this process were collectively similar to those observed during root cap cell differentiation. BY-2 cells were also used to develop a novel model system for programmed cell death during hypersensitive reaction (HR), in which we have succeeded in inducing programmed cell death with 100% efficiency by application of an elicitor. This system revealed that the HR cells activated anti-microbial defense mechanism by themselves and transmitted signal(s) to establish acquired resistance in neighboring cells, while executing cell-death program. Thus, BY-2 cells have taught us a lot about proliferation, differentiation, and death of plant cells., Jun. 2015, 80, 2, 133, 141, Scientific journal, 10.1508/cytologia.80.133
- Refereed, MARINE BIOLOGY, SPRINGER, Relative importance and interactive effects of photosynthesis and food in two solar-powered sea slugs, Ayana Akimoto; Yayoi M. Hirano; Atsushi Sakai; Yoichi Yusa, Sacoglossans use chloroplasts taken from algal food for photosynthesis (kleptoplasty), but the adaptive significance of this phenomenon remains unclear. Two con-generic sacoglossans (Elysia trisinuata and E. atroviridis) were collected in 2009-2011 from Shirahama (33.69A degrees N, 135.34A degrees E) and Mukaishima (34.37A degrees N, 133.22A degrees E), Japan, respectively. They were individually maintained for 16 days under four experimental conditions (combination of light/dark and with/without food), and their survival rate and relative (=final/initial) weights were measured. Both light and food had positive effects on the survival in E. trisinuata, whereas no positive effects of light or food on survival were detected in E. atroviridis. Both light and food had positive effects on relative weights in both species, but light had smaller effects than food. A significant interaction term between light and food was detected in E. trisinuata (but not in E. atroviridis) in that only the presence of both resulted in weight gains. This result suggests that E. trisinuata can obtain sufficient additional energy from photosynthesis for sustaining growth when fresh chloroplasts are continuously supplied from algal food. In addition, fluorescence yield measurements showed that unfed individuals of both E. trisinuata and E. atroviridis lost photosynthetic activity soon (< 4 and 4-8 days, respectively). In conclusion, photosynthesis may function to obtain supplementary nutrition for sustaining growth when food is available in sacoglossans with short-term functional kleptoplasty., May 2014, 161, 5, 1095, 1102, Scientific journal, 10.1007/s00227-014-2402-1
- Refereed, International Journal of Plant & Soil Science, Sciencedomain International, Effects of Salinity on the Growth and Survival of the Seedlings of Mangrove, Rhizophora stylosa, Hiromi Kanai; Mitsuki Tajima; Atsushi Sakai, 10 Jan. 2014, 3, 7, 879, 893, Scientific journal, 10.9734/ijpss/2014/9812
- Refereed, Cell Biology International, Wiley, Histone H3 is absent from organelle nucleoids in BY-2 cultured tobacco cells, Mari Takusagawa; Satoshi Tamotsu; Atsushi Sakai, Jul. 2013, 37, 7, 748, 754, Scientific journal, 10.1002/cbin.10091
- Refereed, Journal of the Marine Biological Association of the United Kingdom, Cambridge University Press (CUP), Effects of photosynthesis on the survival and weight retention of two kleptoplastic sacoglossan opisthobranchs, Shoko Yamamoto; Yayoi M. Hirano; Yoshiaki J. Hirano; Cynthia D. Trowbridge; Ayana Akimoto; Atsushi Sakai; Yoichi Yusa, Many sacoglossan sea slugs utilize chloroplasts ingested from food algae for photosynthesis (functional kleptoplasty), and the extent and duration of kleptoplast retention differs greatly among sacoglossan species. Although most recent studies focus on the genetic, microscopic, or physiological mechanisms responsible for this unique phenomenon, its effects on the life history traits of sacoglossans have not been fully explored. To study the effects of light conditions on survival and weight retention, adult individuals of two sacoglossan species,
Elysia trisinuata andPlakobranchus ocellatus (‘black type'), were reared under light conditions (a 14-hour light: 10-hour dark photoperiod with an irradiance level of 28 µmol m−2s−1) or complete darkness for 21 days. There was no significant difference in the survival rate between the light and dark treatments forE. trisinuata , and its wet weight relative to the initial weight was smaller in the light than in the dark. However, both the survival and relative weights were greater in the light than dark forP. ocellatus . Based on the fluorescent yield measurement using pulse-amplitude-modulated fluorometry, the retention duration of functional chloroplasts was longer (>17 days) forP. ocellatus than E. trisinuata (<4 days). These results indicate thatP. ocellatus benefits from photosynthesis for survival and growth, whereasE. trisinuata does not under starved conditions. This interspecific difference is likely related to the period of functional chloroplast retention., Feb. 2013, 93, 1, 209, 215, Scientific journal, 10.1017/s0025315412000628 - Refereed, CYTOLOGIA, UNIV TOKYO CYTOLOGIA, Theca Cell Layer Formation in Mouse Ovarian Follicle Culture in vitro, Saori Itami; Keiko Yasuda; Satoshi Tamotsu; Atsushi Sakai, Sep. 2012, 77, 3, 288, 288, Scientific journal
- Refereed, PLANT MORPHOLOGY, The Japanese Society of Plant Morphology, The maize coleoptiles do not perform typical C4 photosynthesis: investigation with special reference to anatomy, photosynthetic property, and gene expression, Yasuko Fukaya; Naoko Yoshioka; Hitoshi Sakakibara; Carlos S. Andreo; Tadahiko Mae; Keiko Yasuda; Atsushi Sakai, 2012, 24, 1, 111, 121, Scientific journal, 10.5685/plmorphol.24.111
- Refereed, Reproductive Biology and Endocrinology, Springer Science and Business Media LLC, Co-culturing of follicles with interstitial cells in collagen gel reproduce follicular development accompanied with theca cell layer formation, Saori Itami; Keiko Yasuda; Yuka Yoshida; Chiyuki Matsui; Sachie Hashiura; Atsushi Sakai; Satoshi Tamotsu, Dec. 2011, 9, 1, 159, 159, Scientific journal, 10.1186/1477-7827-9-159
- Refereed, Protoplasma, Springer Science and Business Media LLC, Effects of chloroplast dysfunction on mitochondria: white sectors in variegated leaves have higher mitochondrial DNA levels and lower dark respiration rates than green sectors, Haruka Toshoji; Tomomi Katsumata; Mari Takusagawa; Yoichi Yusa; Atsushi Sakai, Oct. 2011, 249, 3, 805, 817, Scientific journal, 10.1007/s00709-011-0325-y
- Refereed, Biology of Reproduction, Oxford University Press (OUP), The Roles of THY1 and Integrin Beta3 in Cell Adhesion During Theca Cell Layer Formation and the Effect of Follicle-Stimulating Hormone on THY1 and Integrin Beta3 Localization in Mouse Ovarian Follicles, Saori Itami; Satoshi Tamotsu; Atsushi Sakai; Keiko Yasuda, 01 May 2011, 84, 5, 986, 995, Scientific journal, 10.1095/biolreprod.110.087429
- Refereed, Journal of Chemical Ecology, Springer Science and Business Media LLC, 1,8-Cineole Inhibits Both Proliferation and Elongation of BY-2 Cultured Tobacco Cells, Hiroko Yoshimura; Yu Sawai; Satoshi Tamotsu; Atsushi Sakai, Feb. 2011, 37, 3, 320, 328, Scientific journal, 10.1007/s10886-011-9919-2
- Refereed, CYTOLOGIA, International Society of Cytology, Effects of Growth Phase and Cell Density on Cryptogein-induced Programmed Cell Death in Suspension-cultured Tobacco BY-2 Cells: Development of a Model System for 100% Efficient Hypersensitive Cell Death, Yu Sawai; Satoshi Tamotsu; Kazuyuki Kuchitsu; Atsushi Sakai, Dec. 2010, 75, 4, 389, 396, Scientific journal, 10.1508/cytologia.75.389
- Refereed, PLANT MORPHOLOGY, The Japanese Society of Plant Morphology, Effects of chloroplast dysfunction in a subpopulation of leaf mesophyll cells on photosynthetic and respiratory activities of a whole leaf: A study using variegated leaves of Hedera helix L., Naoko Yoshioka; Yuki Imanishi; Keiko Yasuda; Atsushi Sakai, A variegated leaf of Hedera helix is composed of green and white mesophyll cells with and without photosynthetic capacity, respectively. We measured the photosynthesis and dark respiration rates, as well as CO2 compensation points, of H. helix leaves with various extents of variegation. The photosynthetic rate (on an area basis) of the variegated leaves increased almost linearly according to the increase in the proportion of green area to total leaf area. In contrast, dark respiration rate was nearly constant irrespective of the extent of leaf variegation. These results suggest that chloroplast dysfunction in white mesophyll cells did not drastically affect photosynthetic activity of green mesophyll cells, or respiratory rates of both green and white mesophyll cells. CO2 compensation point was elevated when the proportion of green area became extremely low, indicating that the proportion of non-photosynthetic cells within a photosynthetic organ could affect its CO2 compensation point., Dec. 2009, 21, 1, 87, 91, Scientific journal, 10.5685/plmorphol.21.87
- Refereed, CYTOLOGIA, International Society of Cytology, Organization of Mitochondrial-Nucleoids in BY-2 Cultured Tobacco Cells, Mari Takusagawa; Tomomi Hayashi; Hiroyoshi Takano; Atsushi Sakai, Sep. 2009, 74, 3, 329, 341, Scientific journal, 10.1508/cytologia.74.329
- Refereed, Plant and Cell Physiology, Oxford University Press (OUP), NtPolI-like1 and NtPolI-like2, Bacterial DNA Polymerase I Homologs Isolated from BY-2 Cultured Tobacco Cells, Encode DNA Polymerases Engaged in DNA Replication in Both Plastids and Mitochondria, Y. Ono; A. Sakai; K. Takechi; S. Takio; M. Takusagawa; H. Takano, 17 Oct. 2007, 48, 12, 1679, 1692, Scientific journal, 10.1093/pcp/pcm140
- Refereed, Journal of Hattori Botanical Laboratory, Profiling of Bryophyte gene expression by hybridization of an Arabidopsis cDNA array with Bryophyte cDNA., SUNG JIN CHUNG; KATSUAKI TAKECHI; ATSUSHI SAKAI; KANJI ONO; HIROYOSHI TAKANO, Jan. 2006, 99, 99, 233, 244, Scientific journal
- Refereed, Journal of Hattori Botanical Laboratory, Detection of genes differentially expressed in cultured cells of Marchantia polymorpha, but not in Arabidopsis thaliana, using an Arabidopsis cDNA microarray., Sung Jin Chung; Katsuaki Takechi; Atsushi Sakai; Kanji Ono; Hiroyoshi Takano, Jan. 2006, 99, 99, 245, 247, Scientific journal
- Refereed, Journal of Chemical Ecology, Springer Science and Business Media LLC, Allelopathic Effects of Volatile Monoterpenoids Produced by Salvia leucophylla: Inhibition of Cell Proliferation and DNA Synthesis in the Root Apical Meristem of Brassica campestris Seedlings, Nami Nishida; Satoshi Tamotsu; Noriko Nagata; Chieko Saito; Atsushi Sakai, May 2005, 31, 5, 1187, 1203, Scientific journal, 10.1007/s10886-005-4256-y
- Refereed, Zoological Science, Zoological Society of Japan, Changes in the Distribution of Tenascin and Fibronectin in the Mouse Ovary During Folliculogenesis, Atresia, Corpus Luteum Formation and Luteolysis, Keiko Yasuda; Emi Hagiwara; Akiko Takeuchi; Chinatsu Mukai; Chiyuki Matsui; Atsushi Sakai; Satoshi Tamotsu, Tenascin and fibronectin are components of the extracellular matrices that oppose and promote adhesion, respectively. Using immunohistochemical techniques, we studied the distribution of tenascin and fibronectin in the mouse ovary, in which dynamic reconstruction and degeneration occur during folliculogenesis, atresia, ovulation, corpus luteum formation and luteolysis. In growing follicles, tenascin was only detected in the theca externa layer, while fibronectin was detected in the theca externa layer, theca interna layer and basement membrane. During follicular atresia, granulosa cells, which are surrounded by the basement membrane, began to die through apoptosis. In atretic follicles, tenascin was detected in the basement membrane and theca externa layer. Distribution of fibronectin in atretic follicles was similar to that in healthy growing follicles, except that granulosa cells were slightly immunopositive for fibronectin. In young corpus luteum, luteal cells exhibit high 3 β-hydroxysteroid dehydrogenase (3 β-HSD) activity, an enzyme indispensable for progesterone production. Tenascin was barely detected in young luteal cells. 3 β-HSD activity in luteal cells declines with corpus luteum age, and in older corpus luteum there is an increase in apoptotic death of luteal cells. Tenascin was intensely immunopositive in old luteal cells. In contrast, fibronectin immunostaining in luteal cells was relatively constant during corpus luteum formation and luteolysis. Our observations suggest that tenascin is critical in controlling the degenerative changes of tissues in mouse ovaries. Moreover, in all circumstances observed in this study, tenascin always co-localized with fibronectin, suggesting fibronectin is indispensable for the function of tenascin., Feb. 2005, 22, 2, 237, 245, Scientific journal, 10.2108/zsj.22.237
- Refereed, International Review of Cytology, Organelle nuclei in higher plants: Structure, composition, function, and evolution, Atsushi Sakai; Hiroyoshi Takano; Tsuneyoshi Kuroiwa, Plant cells have two distinct types of energy-converting organelles: plastids and mitochondria. These organelles have their own DNAs and are regarded as descendants of endosymbiotic prokaryotes. The organelle DNAs associate with various proteins to form compact DNA-protein complexes, which are referred to as organelle nuclei or nucleoids. Various functions of organelle genomes, such as DNA replication and transcription, are performed within these compact structures. Fluorescence microscopy using the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole has played a pivotal role in establishing the concept of "organelle nuclei." This fluorochrome has also facilitated the isolation of morphologically intact organelle nuclei, which is indispensable for understanding their structure and composition. Moreover, development of an in vitro transcriptionDNA synthesis system using isolated organelle nuclei has provided us with a means of measuring and analyzing the function of organelle nuclei. In addition to these morphological and biochemical approaches, genomics has also had a great impact on our ability to investigate the components of organelle nuclei. These analyses have revealed that organelle nuclei are not a vestige of the bacterial counterpart, but rather are a complex system established through extensive interaction between organelle and cell nuclear genomes during evolution. Extensive diversion or exchange during evolution is predicted to have occurred for several important structural proteins, such as major DNA-compacting proteins, and functional proteins, such as RNA and DNA polymerases, resulting in complex mechanisms to control the function of organelle genomes. Thus, organelle nuclei represent the most dynamic front of interaction between the three genomes (cell nuclear, plastid, and mitochondrial) constituting eukaryotic plant cells. © 2004 Elsevier Inc., Jan. 2004, 238, 59, 118, In book, 10.1016/S0074-7696(04)38002-2
- Refereed, Molecular Biology of the Cell, American Society for Cell Biology (ASCB), Glom Is a Novel Mitochondrial DNA Packaging Protein inPhysarum polycephalumand Causes Intense Chromatin Condensation without Suppressing DNA Functions, Narie Sasaki; Haruko Kuroiwa; Chikako Nishitani; Hiroyoshi Takano; Tetsuya Higashiyama; Tamaki Kobayashi; Yuki Shirai; Atsushi Sakai; Shigeyuki Kawano; Kimiko Murakami-Murofushi; Tsuneyoshi Kuroiwa, Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions., Dec. 2003, 14, 12, 4758, 4769, Scientific journal, 10.1091/mbc.e03-02-0099
- Refereed, Journal of Experimental Botany, Oxford University Press (OUP), Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents, Yutaka Miyazawa; Naoko Nakajima; Tomoko Abe; Atsushi Sakai; Shozo Fujioka; Shigeyuki Kawano; Tsuneyoshi Kuroiwa; Shigeo Yoshida, 29 Oct. 2003, 54, 393, 2669, 2678, Scientific journal, 10.1093/jxb/erg312
- Refereed, Plant and Cell Physiology, Oxford University Press (OUP), Effects of Antibiotics that Inhibit the Bacterial Peptidoglycan Synthesis Pathway on Moss Chloroplast Division, Nami Katayama; Hiroyoshi Takano; Motoji Sugiyama; Susumu Takio; Atsushi Sakai; Kan Tanaka; Haruko Kuroiwa; Kanji Ono, 15 Jul. 2003, 44, 7, 776, 781, Scientific journal, 10.1093/pcp/pcg096
- Refereed, Journal of Experimental Botany, Oxford University Press (OUP), Isolation and expression of a novel starch-storing cell-specific gene containing the KH RNA binding domain from tobacco-cultured cells BY-2, Yutaka Miyazawa; Atsushi Sakai; Sachihiro Matsunaga; Tadao Asami; Shigeyuki Kawano; Tsuneyoshi Kuroiwa; Shigeo Yoshida, 01 Dec. 2002, 53, 379, 2451, 2452, Scientific journal, 10.1093/jxb/erf111
- Refereed, PLANT MORPHOLOGY, The Japanese Society of Plant Morphology, Activation of organelle DNA synthesis during the initial phase of proliferation of BY-2 cultured tobacco cells after medium renewal, Saori Okamura; Takeshi Suzuki; Yutaka Miyazawa; Tsuneyoshi Kuroiwa; Atsushi Sakai, Dec. 2002, 14, 1, 16, 28, Scientific journal, 10.5685/plmorphol.14.16
- Refereed, Sexual Plant Reproduction, Springer Science and Business Media LLC, Angiosperm species that produce sperm cell pairs or generative cells with polarized distribution of DNA-containing organelles, Chieko Saito; Noriko Nagata; Atsushi Sakai; Kimie Mori; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, Dec. 2002, 15, 4, 167, 178, Scientific journal, 10.1007/s00497-002-0152-6
- Refereed, Genome, RAPD isolation of a Y chromosome specific ORF in a dioecious plant, Silene latifolia., Nakao S; Matsunaga S; Sakai A; Kuroiwa T; Kawano S, Apr. 2002, 45, 413, 420
- Refereed, CYTOLOGIA, International Society of Cytology, Ampicillin Inhibits Chloroplast Division in Cultured Cells of the Liverwort Marchantia polymorpha., Eiichirou Tounou; Susumu Takio; Atsushi Sakai; Kanji Ono; Hiroyoshi Takano, 2002, 67, 4, 429, 434, Scientific journal, 10.1508/cytologia.67.429
- Refereed, International Review of Cytology, Elsevier, Three-Dimensional Progression of Programmed Death in the Rice Coleoptile, Noriko Inada; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, 2002, 221, 260e, In book, 10.1016/s0074-7696(02)18014-4
- Refereed, Journal of Plant Research, Springer Science and Business Media LLC, In vitro Transcription/DNA Synthesis Using Isolated Organelle-nuclei: Application to the Analysis of the Mechanisms that Regulate Organelle Genome Function, Atsushi Sakai, Jun. 2001, 114, 2, 199, 211, Scientific journal, 10.1007/pl00013984
- Refereed, PROTOPLASMA, SPRINGER-VERLAG WIEN, Behavior of plastid nucleoids during male gametogenesis in Plumbago auriculata, C Saito; N Nagata; A Sakai; H Kuroiwa; T Kuroiwa, We characterized the behavior of plastid (pt) and mitochondrial(mt) nucleoids during male gametogenesis in Plumbago auriculata in three dimensions. The behavior of pt-nucleoids and mt-nucleoids differed throughout male gametogenesis. Pt-nucleoids were distributed in a characteristic manner in three stages: in the early microspore, pt-nucleoids assemble around cell nucleus: in the mid-generative cell, pt-nucleoids gather at the internal side of the pollen: in the late-generative cell, pt-nucleoids aggregation turns its pole to the external side of the pollen. We also studied organelle nucleoids in the egg and the central cell by a method in which semi thick sections of resin-embedded anthers and ovaries were observed by confocal laser scanning microscopy. The number of pt-nucleoids in the sperm cell did not differ significantly from that in the egg. These results suggest that the behavior of DNA-containing organelles is regulated strictly during male gametogenesis in P. auriculata, and that a biparental inheritance of plastids in the Plumbago embryo is more favored than was previously thought., 2001, 216, 3-4, 143, 154, Scientific journal
- Refereed, Journal of Plant Physiology, Elsevier BV, Differential regulation of starch synthesis gene expression during amyloplast development in cultured tobacco BY-2 cells, Yutaka Miyazawa; Atsushi Sakai; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, Jan. 2001, 158, 8, 1077, 1084, Scientific journal, 10.1078/0176-1617-00246
- Refereed, CYTOLOGIA, International Society of Cytology, Organellar Protein Synthesis Controls Amyloplast Formation Independent of Starch Synthesis Gene Expression., Yutaka Miyazawa; Atsushi Sakai; Shigeyuki Kawano; Tsuneyoshi Kuroiwal, The transfer of stationary-phase cultured tobacco (Nicotiana tabacum L.) BY-2 cells into auxin-depleted culture medium induces amyloplast formation. To investigate the timing and requirement for de novo protein synthesis in organelles during amyloplast development and the enhancement of starch synthesis gene expression, we added chloramphenicol, at various times, to cells grown in amyloplast-inducing medium. Changes in cell growth, starch accumulation, and the mRNA levels of the ADP-glucose pyrophosphorylase small subunit (AgpS) gene Were monitored. Chloramphnicol inhibited starch accumulation, but had no significant effect on cell growth, irrespective of the time of addition. RNA gel-blot analyses revealed that chloramphenicol treatment did not reduce the accumulation of mRNA from the AgpS gene, irrespective of addition time. These results suggest that organellar protein synthesis affects starch accumulation independently of starch synthesis gene expression. The necessity of organellar gene expression for starch accumulation is also discussed., Dec. 2000, 65, 4, 435, 442, Scientific journal, 10.1508/cytologia.65.435
- Refereed, PLANT MORPHOLOGY, The Japanese Society of Plant Morphology, Isolation of chloroplasts and chloroplast-nuclei(nucleoids)from Chlamydomonas reinhardtii, Atsushi Sakai; Lena Suzuki; Osami Misumi; Tetsuya Higashiyama; Tsuneyoshi Kuroiwa, Dec. 2000, 12, 1, 2, 9, Scientific journal, 10.5685/plmorphol.12.2
- Refereed, MOLECULAR AND GENERAL GENETICS, SPRINGER-VERLAG, A putative mitochondrial ftsZ gene is present in the unicellular primitive red alga Cyanidioschyzon merolae, M Takahara; H Takahashi; S Matsunaga; S Miyagishima; H Takano; A Sakai; S Kawano; T Kuroiwa, Two ftsZ homologues were isolated from the unicellular primitive red alga Cyanidioschyzon merolae (CmftsZ1 and CmftsZ2). Phylogenetic analysis revealed that CmftsZ1 is most closely related to the ftsZ genes of alpha -Proteobacteria, suggesting that it is a mitochondrial-type ftsZ gene, whereas CmftsZ2 is most closely related to the ftsZ genes of cyanobacteria, suggesting that it is a plastid-type ftsZ gene. Southern analysis indicates that CmftsZ1 and CmftsZ2 are both single-copy genes located on chromosome XIV in the C. merolae genome. Northern analysis revealed that both CmftsZ1 and CmftsZ2 are transcribed, and accumulate specifically before cell and organelle division. The results: of Western analysis suggest that CmFtsZ1 is localized in mitochondria., Nov. 2000, 264, 4, 452, 460, Scientific journal
- Refereed, Molecular Genetics and Genomics, Springer Science and Business Media LLC, A putative mitochondrial ftsZ gene is present in the unicellular primitive red alga Cyanidioschyzon merolae, M. Takahara; H. Takahashi; S. Matsunaga; S. Miyagishima; H. Takano; A. Sakai; S. Kawano; T. Kuroiwa, Nov. 2000, 264, 4, 452, 460, Scientific journal, 10.1007/s004380000307
- Refereed, Sexual Plant Reproduction, Springer Science and Business Media LLC, Unequal distribution of DNA-containing organelles in generative and sperm cells of Erythrina crista-galli (Fabaceae), C. Saito; N. Nagata; A. Sakai; K. Mori; H. Kuroiwa; T. Kuroiwa, 14 Apr. 2000, 12, 5, 296, 301, Scientific journal, 10.1007/s004970050198
- Refereed, Protoplasma, Springer Science and Business Media LLC, Unique positioning of mitochondria in developing microspores and pollen grains inPharbitis nil: mitochondria cover the nuclear surface at specific developmental stages, N. Nagata; C. Saito; A. Sakai; H. Kuroiwa; T. Kuroiwa, Mar. 2000, 213, 1-2, 74, 82, Scientific journal, 10.1007/bf01280507
- Refereed, Current Genetics, Springer Science and Business Media LLC, Isolation, characterization, and chromosomal mapping of an ftsZ gene from the unicellular primitive red alga Cyanidium caldarium RK-1, Manabu Takahara; Hidenori Takahashi; Sachihiro Matsunaga; Atsushi Sakai; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, 23 Feb. 2000, 37, 2, 143, 151, Scientific journal, 10.1007/s002940050021
- Refereed, Protoplasma, Senescence in the nongreening region of the rice (Oryza sativa) coleoptile, Inada N; Sakai A; KuroiwaH; Kuroiwa T, 2000, 214, 180, 193, Scientific journal
- Refereed, Plant Physiology, Oxford University Press (OUP), Auxin and Cytokinin Have Opposite Effects on Amyloplast Development and the Expression of Starch Synthesis Genes in Cultured Bright Yellow-2 Tobacco Cells, Yutaka Miyazawa; Atsushi Sakai; Shin-ya Miyagishima; Hiroyoshi Takano; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, 01 Oct. 1999, 121, 2, 461, 470, Scientific journal, 10.1104/pp.121.2.461
- Refereed, Planta, Springer Science and Business Media LLC, The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance, Noriko Nagata; Chieko Saito; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, 19 Jul. 1999, 209, 1, 53, 65, Scientific journal, 10.1007/s004250050606
- Refereed, European Journal of Cell Biology, Elsevier BV, Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development, Noriko Nagata; Chieko Saitoa; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, Apr. 1999, 78, 4, 241, 248, Scientific journal, 10.1016/s0171-9335(99)80057-0
- Refereed, Plant Cell Reports, Springer Science and Business Media LLC, Amyloplast formation in cultured tobacco cells. III Determination of the timing of gene expression necessary for starch accumulation, A. Sakai; Y. Miyazawa; C. Saito; N. Nagata; H. Takano; H.-Y. Hirano; T. Kuroiwa, 09 Mar. 1999, 18, 7-8, 589, 594, Scientific journal, 10.1007/s002990050627
- Refereed, Journal of Plant Physiology, Elsevier BV, Plastid Gene Expression during Amyloplast Formation in Cultured Tobacco Cells, Atsushi Sakai; Yutaka Miyazawa; Takeshi Suzuki; Narie Sasaki; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, Jan. 1999, 154, 1, 71, 78, Scientific journal, 10.1016/s0176-1617(99)80320-4
- Refereed, Plant Science, Elsevier BV, Comparative analysis of DNA synthesis activity in plastid-nuclei and mitochondrial-nuclei simultaneously isolated from cultured tobacco cells, Atsushi Sakai; Takeshi Suzuki; Noriko Nagata; Narie Sasaki; Yutaka Miyazawa; Chieko Saito; Noriko Inada; Yoshiki Nishimura; Tsuneyoshi Kuroiwa, Jan. 1999, 140, 1, 9, 19, Scientific journal, 10.1016/s0168-9452(98)00207-6
- Refereed, Plant and Cell Physiology, Oxford University Press (OUP), Semi-Automatic Laser Beam Microdissection of the Y Chromosome and Analysis of Y Chromosome DNA in a Dioecious Plant, Silene latifolia, S. Matsunaga; S. Kawano; T. Michimoto; T. Higashiyama; S. Nakao; A. Sakai; T. Kuroiwa, Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes., 01 Jan. 1999, 40, 1, 60, 68, Scientific journal, 10.1093/oxfordjournals.pcp.a029475
- Refereed, Biotechnic & Histochemistry, Informa UK Limited, Improved Sensitivity for High Resolution in Situ Hybridization Using Resin Extraction of Methyl Methacrylate Embedded Material, Chieko Saito; Makoto Hay Ashi; Atsushi Sakai; Makoto Fujie; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, Jan. 1999, 74, 1, 40, 48, Scientific journal, 10.3109/10520299909066476
- Refereed, PROTOPLASMA, SPRINGER-VERLAG WIEN, Senescence program in rice (Oryza sativa L.) leaves: analysis of the blade of the second leaf at the tissue and cellular levels, N Inada; A Sakai; H Kuroiwa; T Kuroiwa, Previously, we showed that all greening mesophyll cells in the coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) follow the identical program of senescence, which features the early degradation of chloroplast DNA (cpDNA) and subsequent nuclear condensation and disorganization. Following the coleoptile study, we analyzed the senescence-associated changes in the blade of the second leaf of rice at the tissue and cellular levels. Under the experimental conditions, the second leaf started to elongate rapidly 2 days after sowing and emerged on day 3. The blade of the second leaf completed its growth on day 4, although the sheath continued to grow until day 7. The amount of soluble protein and chlorophyll (Chl) per blade reached a maximum on day 7, and then declined. When blades were divided into three parts (the tip, mid-region, and base), levels of both soluble protein and Chl in the tip segment peaked earlier and decreased at a faster rate than in the other parts, demonstrating a longitudinal gradient of senescence from the tip to the base of the blade. In cross sections through the center of the tip and base segments, all the mesophyll cells senesced synchronously. They passed through the following steps in order: (i) degradation of cpDNA, (ii) decrease in the size of the chloroplast with degeneration of the chloroplast inner membranes, and (iii) condensation and disorganization of the nuclei. Although some differences were shown between the coleoptile and the second leaf in the timing and rate of each event, the order of those senescence-related events was conserved, suggesting an identical program of senescence exists in rice leaves., 1999, 207, 3-4, 222, 232, Scientific journal
- Refereed, Plant and Cell Physiology, Oxford University Press (OUP), Two Types of ftsZ Genes Isolated from the Unicellular Primitive Red Alga Galdieria sulphuraria, M. Takahara; H. Takahashi; S. Matsunaga; A. Sakai; S. Kawano; T. Kuroiwa, FtsZ plays a crucial role in bacterial cell division, and may be involved in plastid division in eukaryotes. To investigate the evolution of the dividing apparatus from prokaryotes to eukaryotes, the ftsZ genes were isolated from the unicellular primitive red alga Galdieria sulphuraria. Two ftsZ genes (GsftsZ1 and GsftsZ2) were isolated. This suggests that duplication and divergence of the ftsZ gene occurred in an early stage of plant evolution. A comparison of the FtsZs of G.sulphuraria and other organisms shows that FtsZ is highly and universally conserved among prokaryotes, primitive eukaryotic algae, and higher plants. The GsftsZ2 gene seems to contain an intron. Southern hybridization analysis of the G.sulphuraria chromosomes separated by CHEF revealed that each ftsZ gene and its flanking region may be duplicated., 01 Jan. 1999, 40, 8, 784, 791, Scientific journal, 10.1093/oxfordjournals.pcp.a029606
- Refereed, PROTOPLASMA, SPRINGER WIEN, Isolation and phenotypic characterization of Chlamydomonas reinhardtii mutants defective in chloroplast DNA segregation, O Misumi; L Suzuki; Y Nishimura; A Sakai; S Kawano; H Kuroiwa; T Kuroiwa, Each wild-type Chlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we named moc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed that moc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in the moc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2-8 copies) of cpDNA, However, most individual moc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of the moc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure of moc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells and moc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novel moc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA., 1999, 209, 3-4, 273, 282, Scientific journal
- Refereed, Planta, Springer Science and Business Media LLC, Three-dimensional analysis of the senescence program in rice ( Oryza sativa L.) coleoptiles, Noriko Inada; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, 07 Oct. 1998, 206, 4, 585, 597, Scientific journal, 10.1007/s004250050436
- Refereed, Plant and Cell Physiology, Oxford University Press (OUP), Transcriptional Activities of the Chloroplast-Nuclei and Proplastid-Nuclei Isolated from Tobacco Exhibit Different Sensitivities to Tagetitoxin: Implication of the Presence of Distinct RNA Polymerases, A. Sakai; C. Saito; N. Inada; T. Kuroiwa, We examined the effects of tagetitoxin, a potent inhibitor of RNA polymerases from chloroplasts and Escherichia coli, on the transcriptional activities of chloroplast- and proplastid-nuclei(nucleoids) isolated from mature tobacco(Nicotiana tabacum L.) leaves and cultured tobacco cells(line BY-2), respectively. Transcription by the isolated chloroplast-nuclei was effectively inhibited by tagetitoxin(95-99% reduction at 10 μM tagetitoxin), but transcription by the isolated proplastid-nuclei was only partially inhibited(40-50% reduction) by this compound. Southern hybridization experiments revealed that the transcription of various plastid genes (psbA, atpA, rpoB, psaA/B, atpB, rbcL, petB, rpl16, and rrn23) was sensitive to tagetitoxin in the isolated chloroplast-nuclei, whereas the transcription of the same genes was relatively resistant to this compound in the isolated proplastid-nuclei. These results suggest that; (i)distinct RNA polymerase activities with different sensitivities to tagetitoxin are present in plastids, (ii)a tagetitoxin-sensitive RNA polymerase is the major RNA polymerase in chloroplasts whereas a tagetitoxin-insensitive enzyme is major in proplastids, and (iii)both RNA polymerases can transcribe various plastid genes., 01 Sep. 1998, 39, 9, 928, 934, Scientific journal, 10.1093/oxfordjournals.pcp.a029456
- Refereed, CYTOLOGIA, International Society of Cytology, A High Density of rRNA in the Generative Cells and Sperm Cells of Pollen Grains of Five Angiosperm Species., Chieko Saito; Makoto Fujie; Atsushi Sakai; Noriko Nagata; Sachihiro Matsunaga; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, To examine whether cytoplasmic rRNA are present at high density in the cytoplasm of generative and sperm cells (generative/sperm cells) in pollen grains of various plant species, in situ hybridization of 18S/25S rRNA was performed in thin sections of samples embedded in Technovit 7100 resin. The five plant species studied were Lilium longiflorum, Pharbitis nil, Silene latifolia, Pelargonium zonale and Plumbago auriculata. The timing and mode of pollen mitosis II (PM II) and the distribution of organelle nucleoids in generative/sperm cells differ in these species. In situ hybridization revealed that the density of rRNA was the same as or higher in generative/sperm cells than in vegetative cells, suggesting that the presence of rRNA in the generative/sperm cells at high density may be a general characteristic of mature pollen grains of various plant species., Sep. 1998, 63, 3, 293, 300, Scientific journal, 10.1508/cytologia.63.293
- Refereed, Plant and Cell Physiology, Oxford University Press (OUP), Comparative Analysis of Plastid Gene Expression in Tobacco Chloroplasts and Proplastids: Relationship between Transcription and Transcript Accumulation, A. Sakai; T. Suzuki; Y. Miyazawa; S. Kawano; T. Nagata; T. Kuroiwa, We compared the modes of plastid gene expression between chloroplasts and proplastids in tobacco (Nicotiana tabacum L.). Chloroplast-nuclei (chloroplast-nucleoids) and proplastid-nuclei (proplastid-nucleoids) were isolated from mature leaves and cultured cells (line BY-2), respectively, and their transcriptional activities in vitro were compared. Overall transcriptional activity of the isolated chloroplast-nuclei (250 pmol UTP (μg DNA)^<-1>h^<-1>) was approximately 25 times that of the isolated proplastid-nuclei (9.7 pmol UTP (μg DNA)^<-1>h^<-1>). This difference in overall transcriptional activities was accompanied by intensive modulation of the relative transcriptional activities of individual genes. Differences in the transcriptional activities of nine plastid genes (psbA, atpA, ropB, psaA/B, atpB, rbcL, petB, rpl16, and rrn23) between the isolated chloroplast- and proplastid-nuclei correlated moderately with differences in the amounts of corresponding transcripts accumulating in leaves and cultured cells. This suggests that transcriptional regulation is responsible, to a considerable extent, for the differential accumulation of plastid transcripts. Possible mechanisms underlying the differential transcription in proplastids and chloroplasts are discussed in relation to the existence of multiple RNA polymerases and structural changes involving plastid-nuclei., 01 Jun. 1998, 39, 6, 581, 589, Scientific journal, 10.1093/oxfordjournals.pcp.a029408
- Refereed, Journal of Phycology, Wiley, Isolation, characterization, and chromosome mapping of an actin gene from the primitive green alga, Nannochloris bacillaris (Chlorophyceae), Sayaka Arai; Hidenori Takahashi; Hiroyoshi Takano; Atsushi Sakai; Shigeyuki Kawano, Jun. 1998, 34, 3, 477, 485, Scientific journal, 10.1046/j.1529-8817.1998.340477.x
- Planta, Springer Science and Business Media LLC, Three-dimensional analysis of the senescence program in rice ( Oryza sativa L.) coleoptiles, Noriko Inada; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, 18 May 1998, 205, 2, 153, 164, Scientific journal, 10.1007/s004250050307
- Refereed, Plant Science, Elsevier BV, Simultaneous isolation of cell-nuclei, plastid-nuclei and mitochondrial-nuclei from cultured tobacco cells; comparative analysis of their transcriptional activities in vitro, Atsushi Sakai; Takeshi Suzuki; Yutaka Miyazawa; Tsuneyoshi Kuroiwa, Apr. 1998, 133, 1, 17, 31, Scientific journal, 10.1016/s0168-9452(98)00018-1
- Refereed, Journal of Plant Research, Springer Science and Business Media LLC, Detection and quantification of rRNA by high-resolutionin situ hybridization in pollen grains, Chieko Saito; Makoto Fujie; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, Mar. 1998, 111, 1, 45, 52, Scientific journal, 10.1007/bf02507148
- Refereed, International Review of Cytology, Elsevier, The Division Apparatus of Plastids and Mitochondria, Tsuneyoshi Kuroiwa; Haruko Kuroiwa; Atsushi Sakai; Hidenori Takahashi; Kyoko Toda; Ryuuichi Itoh, 1998, 1, 41, In book, 10.1016/s0074-7696(08)60415-5
- Refereed, PROTOPLASMA, SPRINGER-VERLAG WIEN, DNA synthesis in isolated mitochondrial nucleoids from plasmodia of Physarum polycephalum, N Sasaki; A Sakai; S Kawano; H Kuroiwa; T Kuroiwa, A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mt-nucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia of Physarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl2 for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC., 1998, 203, 3-4, 221, 231, Scientific journal
- Refereed, Phycological Research, Wiley, Isolation of organellar DNA from Codium fragile (Codiaceae, Codiales, Ulvophyceae), Masaya Satoh; Atsushi Sakai; Shizue Hamazaki; Yuki Takashima; Tsuneyoshi Kuroiwa, Dec. 1997, 45, 4, 213, 216, Scientific journal, 10.1111/j.1440-1835.1997.tb00078.x
- Refereed, CYTOLOGIA, International Society of Cytology, Amyloplast Formation in Cultured-Tobacco Cells II: Effects of Transcription/Translation Inhibitors on Accumulation of Starch., Atsushi Sakai; Yutaka Miyazawa; Kumiko Yashiro; Takeshi Suzuki; Shigeyuki Kawano, Sep. 1997, 62, 3, 295, 301, Scientific journal, 10.1508/cytologia.62.295
- Refereed, CYTOLOGIA, International Society of Cytology, Changes in the Extent of the Condensation of Nuclear Chromatin and the Localization of RNA During Pollen Development in Nicotiana tabacum, Chieko Saito; Makoto Fujie; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, We examined the differentiation of the generative and the vegetative cells of tobacco (Nicotiana tabacum SR-1) by a newly developed cytological method using fluorescence microscopy after embedding of samples in Technovit 7100 resin. The extent of condensation of the chromatin in generative and vegetative nuclei was measured semi-quantitatively in sections of uniform thickness by microphotometry. The extent of chromatin condensation in the generative nucleus was about 5 to 10 times as high as the vegetative nucleus throughout pollen maturation which lasted about 5 days. Such a difference was clear immediately after PM I, indicating that the differentiation of the two cells had already started as soon as PM I has finished. Maturing generative cells had almost as much condensed chromatin as chromosomes of PM I. We also examined whether the generative cell has the potential for gene expression. The localization of RNA was visualized by staining with acridine orange. The results revealed the cytoplasm of the generative cell contained RNA and the density of RNA was as high as in the vegetative cell. This result suggests that the generative cell has the potential for gene expression in spite of the highly condensed chromatin in the nucleus. Furthermore, the generative nucleus in the mature pollen retained small nucleoli. Their presence implies that rRNA is synthesized consistently in generative cells., Jun. 1997, 62, 2, 121, 132, Scientific journal, 10.1508/cytologia.62.121
- Refereed, Journal of Plant Research, Springer Science and Business Media LLC, 1,8-Cineole inhibits root growth and DNA synthesis in the root apical meristem ofBrassica campestris L., Ritsuko Koitabashi; Takeshi Suzuki; Tamotsu Kawazu; Atsushi Sakai; Haruko Kuroiwa; Tsuneyoshi Kuroiwa, Mar. 1997, 110, 1, 1, 6, Scientific journal, 10.1007/bf02506836
- Refereed, PROTOPLASMA, SPRINGER WIEN, Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells of Pelargonium zonale during pollen development, N Nagata; Sodmergen; C Saito; A Sakai; H Kuroiwa; T Kuroiwa, In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen of Pelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4',6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells of P. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of prastids in P. zonale., 1997, 197, 3-4, 217, 229, Scientific journal
- Refereed, The Plant Journal, Wiley, Isolation and developmental expression of male reproductive organ-specific genes in a dioecious campion, Melandrium album (Silene latifolia), Sachihiro Matsunaga; Shigeyuki Kawano; Hiroyoshi Takano; Hidenobu Uchida; Atsushi Sakai; Tsuneyoshi Kuroiwa, Oct. 1996, 10, 4, 679, 689, Scientific journal, 10.1046/j.1365-313x.1996.10040679.x
- Refereed, CYTOLOGIA, International Society of Cytology, Cytological Analysis of the Mature Pollen of Actinidia deliciosa (Kiwifruit)., Sachihiro Matsunaga; Atsushi Sakai; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, Mature pollen grains from the male and the female flowers of Actinidia deliciosa (kiwifruit) were examined by DAPI (4', 6-diamidino-2-phenylindole) -fluorescence microscopy. The generative nucleus and the vegetative nucleus were observed in the male pollen grain while neither nucleus was observed in the female pollen grain. In the generative cell of the pollen grains from the male flower, many organelle nuclei were observed. The DNA content of the generative and the vegetative nuclei was measured fluorimetrically by propidium iodide staining with a video-intensified microscope photon counting system. The DNA content of the generative nucleus was about twice as large as that of the vegetative nucleus. This suggests that the generative nucleus has 2C DNA and is arrested in the G2 phase., Sep. 1996, 61, 3, 337, 341, Scientific journal, 10.1508/cytologia.61.337
- Refereed, CYTOLOGIA, International Society of Cytology, Organelle DNA Synthesis before Cell Nuclear Replication is Essential for Subsequent Cell Propagation., Takeshi Suzuki; Atsushi Sakai; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, Elevation of intra-cellular organelle DNA levels due to preferential synthesis of organelle DNAs before cell multiplication is generally observed in meristematic cells of higher plants, such as those in apical meristems and cultured cells. We analyzed the physiological significance of this phenomena using cultured tobacco cell BY-2 as a model system for cell multiplication in plants. Cultured tobacco cell BY-2 multiplied approximately 50-fold in a week in normal culture medium. However, when nalidixic acid, which inhibits prokaryotic DNA gyrase, was added to the culture, synthesis of organelle DNA was selectively inhibited and cell multiplication was arrested. This indicates that the synthesis of organelle DNAs is a prerequisite for cell multiplication. To quantitatively clarify the effect of the elevation of organelle DNA levels on the capacity for cell proliferation, cells with various organelle DNA levels were prepared by preculturing cells for various lengths of time in culture medium which contained aphidicolin, a specific inhibitor of eukaryotic DNA polymerase a, and then transferring them to culture medium which contained nalidixic acid. Their growth rates in the absence of further organelle DNA synthesis were monitored. A correlation was observed between intra-cellular organelle DNA levels and the capacity of the cells to proliferate, indicating that intra-cellular organelle DNA levels limited the capacity for cell proliferation., Jun. 1996, 61, 2, 235, 245, Scientific journal, 10.1508/cytologia.61.235
- Refereed, Plant Cell Reports, Springer Science and Business Media LLC, Amyloplast formation in cultured tobacco cells; effects of plant hormones on multiplication, size, and starch content, Atsushi Sakai; Kumiko Yashiro; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, Apr. 1996, 15, 8, 601, 605, Scientific journal, 10.1007/bf00232461
- Refereed, Genes & Genetic Systems, Genetics Society of Japan, Variability of mitochondrial subgenomic molecules in the meristematic cells of higher plants., Takeshi Suzuki; Shigeyuki Kawano; Atsushi Sakai; Atsushi Hirai; Tsuneyoshi Kuroiwa, MtDNAs from BY-2 cells and rice root were analyzed by random amplified polymorphic DNA (RAPD) assay and Southern hybridization analysis. A number of differences were observed in the RAPD patterns amplified from mtDNAs sampled at different phases of the BY-2 cell culture. RAPD fragments also varied with the template DNAs derived from various areas of rice root tip. When a RAPD fragment was hybridized to restriction fragments of whole DNAs, isolated from the distal area of the apical meristem and differentiated elongation zone of a root, two distinct stoichiometric differences were observed in the hybridization signals. This suggests that the organization of mt-genome in prototypic cells in the root apical meristem differs from that found in the differentiated cells.
, 1996, 71, 5, 329, 333, Scientific journal, 10.1266/ggs.71.329 - Refereed, Journal of Experimental Botany, Oxford University Press (OUP), Localization of organelle DNA synthesis within the root apical meristem of rice, Takeshi Suzuki; Narie Sasaki; Atsushi Sakai; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, Jan. 1995, 46, 1, 19, 25, Scientific journal, 10.1093/jxb/46.1.19
- Refereed, JOURNAL OF CELL SCIENCE, COMPANY OF BIOLOGISTS LTD, Preferential mitochondrial and plastid DNA synthesis before multiple cell divisions in Nicotiana tabacum., T. Suzuki; S. Kawano; A. Sakai; M. Fujie; H. Kuroiwa; H. Nakamura; T. Kuroiwa, Organelle DNA synthesis in root meristem and cultured cell line BY-2, both derived from Nicotiana tabacum cv. Bright Yellow 2, was examined by immunofluorescence microscopy of Technovit sections with antibody against 5-bromodeoxyuridine (BrdU) and co-fluorescent staining with 4',6-diamidino-2-phenylindole (DAPI) and quantitative Southern hybridization. In the root meristem, the mitochondrial DNAs (mtDNAs) were synthesized in a specific region near to the quiescent center, where a low frequency of DNA synthesis of cell nuclei was observed. The mitochondrial nuclei (nucleoids) changed morphologically from long ellipsoids with a high frequency of DNA synthesis, in the region just above the quiescent center, to granules with a low frequency of DNA synthesis, as cell distance from the quiescent center increased. Similar patterns were observed in the cultured tobacco cell line (BY-2), in which large amounts of preferential synthesis of DNA of both mitochondria and plastids occurred prior to cell nuclear DNA synthesis just after stationary phase cells were transferred to fresh medium. Granular mitochondria which vigorously synthesized mtDNA were observed in both lag phase and logarithmic growth phase cells. However, long, ellipsoidal mitochondria which showed a low frequency of mtDNA synthesis were observed in stationary phase cells. Morphological changes of plastids were more conspicuous than those of mitochondria. After the medium was renewed, spherical plastids became extremely elongated and string-like, for 24 h, but were divided into small pieces after the third day. Vigorous synthesis of plastid DNA (ptDNA) occurred during this period of plastids elongation., Nov. 1992, 103, 831, 837, Scientific journal
- Refereed, Plant Physiology, Oxford University Press (OUP), Conversion of Proplastids to Amyloplasts in Tobacco Cultured Cells Is Accompanied by Changes in the Transcriptional Activities of Plastid Genes, Atsushi Sakai; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, 01 Oct. 1992, 100, 2, 1062, 1066, Scientific journal, 10.1104/pp.100.2.1062
- Refereed, Plant and Cell Physiology, Oxford University Press (OUP), Transcriptional Activity of Morphologically Intact Proplastid-Nuclei (Nucleoids) Isolated from Tobacco Cultured Cells, Atsushi Sakai; Hirofumi Yamashita; Yasuyuki Nemoto; Shigeyuki Kawano; Tsuneyoshi Kuroiwa, The transcriptional activity of morphologically intact proplastid-nuclei (synonymous with proplastid-nucleoids) isolated from cultured tobacco cells (line BY-2) was examined by an assay of transcription in vitro and Southern hybridization. The isolated proplastid-nuclei incorporated about 7 pmol of UTP into RNA per microgram of DNA during a 60-min incubation. This incorporation of UTP was identified as synthesis of RNA that was dependent on DNA-dependent RNA polymerase. Heparin, a potent inhibitor of initiation of transcription, had no inhibitory effect, suggesting that the transcriptional activity in the isolated proplastid-nuclei is primarily due to chain elongation of nascent transcripts. The compactly organized, chromatin-like structure of the proplastid-nuclei was maintained during the assay of transcription in vitro under suitable conditions. Southern hybridization studies revealed that the pattern of transcripts generated in vitro by the isolated proplastid-nuclei were similar to that of transcripts produced in a lysed proplastid system and that of RNA accumulated in the living cells. Therefore, the system for the assay of transcription in vitro using isolated proplastid-nuclei seems to provide a suitable method for analyzing the relationship between the transcriptional regulation of plastid genes and the structural changes in plastid-nuclei., Sep. 1991, 32, 6, 835, 843, Scientific journal, 10.1093/oxfordjournals.pcp.a078151
- Refereed, PROTOPLASMA, SPRINGER-VERLAG WIEN, Dispersion of the structural organization of proplastid-nuclei in vitro changes the transcriptional activity of plastid genes.(共著), A SAKAI; S KAWANO; T KUROIWA, We have developed a novel assay system for analyzing the relationship between the structure and the transcriptional activity of the plastid-nuclei (plastid-nucleoids). The organization of morphologically intact proplastid-nuclei, isolated from tobacco cultured cells (line BY-2), was dispersed by treatment with NaCl at various concentrations and their transcriptional activities were examined by an assay of transcription in vitro and Southern hybridization. Disturbance of the structural organization of the proplastid-nuclei caused changes in both absolute and relative transcriptional activities of plastid genes, a result that suggests that the transcriptional activity of plastid genes may actually be regulated by structural changes in the plastid-nuclei., Jun. 1991, 163, 2-3, 203, 206, Scientific journal
MISC
- Not Refereed, Plant Organelles News Letter, プラスチド核（核様体）とDNA合成, SAKAI Atsushi, 2008, 7
- Not Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Studies on the plant organelle DNA polymerases using BY-2 cultured tobacco cells, A Sakai; Y Ono; K Takechi; S Takio; H Takano, 2005, 46, S67, S67, Summary international conference
- Not Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Isolation of a gene for organelle DNA polymerase from tabacco, Y Ono; K Takechi; S Takio; A Sakai; H Takano, 2004, 45, S59, S59, Summary international conference
- Zoological science, Zoological Society of Japan, CHENGES IN THE DISTRIBUTION OF TENASCIN AND FIBRONECTIN IN MURINE OVARY(Endocrinology,Abstracts of papers presented at the 75^
Annual Meeting of the Zoological Society of Japan) :, Yasuda Keiko; Hagiwara Emi; Takeuchi Akiko; Mukai Chinatsu; Matsui Chiyuki; Sakai Atsushi; Tamotsu Satoshi, 2004, 21, 12, 1336, 1336 Zoological science, Zoological Society of Japan, ANALYSIS OF VITELLOGENIN RELATED PROTEINS INDUCED BY ENDOCRINE DISRUPTING CHEMICALS(Endocrinology,Abstracts of papers presented at the 75^Annual Meeting of the Zoological Society of Japan) :, Kobayashi Kayo; Sakai Atsushi; Tamotsu Satoshi; Oishi Tadashi, 2004, 21, 12, 1343, 1343 VITELLOGENIN AND VITELLOGENIN RELATED PROTEINS IN THE LIVER AND TESTIS OF ESTRADIOL TREATED MALE MEDAKA (ORYZIAS LATIPES) : WESTERN BLOTTING ANALYSIS, KOBAYASHI Kayo; SAKAI Atsushi; TAMOTSU Satoshi; OISHI Tadashi, 08 Aug. 2003, 18, 66, 66Zoological science, Zoological Society of Japan, VITELLOGENIN RELATED PROTEINS IN THE LIVER AND TESTIS OF 17β-ESTRADIOL EXPOSED MALE MEDAKA (ORYZIAS LATIPES)(Endocrinology,Abstracts of papers presented at the 74^Annual Meeting of the Zoological Society of Japan) :, Kobayashi Kayo; Sakai Atsushi; Tamotsu Satoshi; Oishi Tadashi, 2003, 20, 12, 1608, 1608 The Japanese Society for Chemical Regulation of Plants, ISOLATION OF GENES SPECIFICALLY EXPRESSED DURING AMYLOPLAST DEVELOPMENT IN TOBACCO CULTURED CELLS BY-2., MiyazaWa Yutaka; Sakai Atsushi; Matsunaga Sachihiro; Kawano Shigeyuki; Kuroiwa Tsuneyoshi; Yoshida Shigeo, In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4- dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine. Thus, this simple in vitro amyloplast-inducing system provides an opportunity to analyze and compare heterotrophic cells in meristematic and starch-storing phases. In order to analyze the mechanisms controlling starch-storing cell differentiation, we started to isolate genes specifically expressed during amyloplast differentiation. Using differential display method, we cloned six cDNA fragments whose transcripts accumulate at high levels during starch-storing cell differentiation. Three clones had homology to known genes;i.e. clone 26, 30, and 60 had significant identities with cellulose synthase, glutamate dehydrogenase, and malate dehydrogenase, respectively. Other three clones (clone 10, 22, and 25) had no significant identities with well-characterized genes, while sequence similarity search using BLAST algorithm suggested that they are homologous to Arabidopsis genes of unknown functions. Analyses on these genes are now in progress., 09 Oct. 2001, 36, 55, 56Plant and cell physiology, Japanese Society of Plant Physiologists, Analyses on the mode of starch synthesis gene expression in tobacco cultured cells BY-2 :, MIYAZAWA Yutaka; SAKAI Atsushi; KAWANO Shigeyuki; KUROIWA Tsuneyoshi, 2000, 41, s218Plant and cell physiology, Japanese Society of Plant Physiologists, ANALYSIS ON THE ROLES OF MULTIPLE RNA POLYMERASES IN NON-PHOTOSYNTHETIC PLASTIDS :, NIO Asuka; SAKAI Atsushi; KUROIWA Tsuneyoshi, 2000, 41, s222Plant and cell physiology, Japanese Society of Plant Physiologists, CHANGES IN THE SECRETED PROTEINS DURING AMYLOPLAST FORMATION IN BY-2 CULTURED TOBACCO CELLS :, NISHIO Kayo; NIO Asuka; SAKAI Atsushi; SAITO Chieko; MIYAZAWA Yutaka; KUROIWA Tsuneyoshi, 2000, 41, s207QUANTITATIVE ANALYSES ON THE ORGANELLE DNA SYNTHESIS DURING PROLIFERATION OF CULTURED TOBACCO CELLS., SAKAI Atsushi; INADA Noriko; SAITO Chieko; MIYAZAWA Yutaka; KUROIWA Tsuneyoshi, Mar. 1999, 40, s20, s20Oct. 1996, 60, 157, 157ISOLATION OF CELL-NUCLEI, PLASTID-NUCLEI, AND MITOCHONDRIAL-NUCLEI FROM CULTURED TOBACCO CELLS, SAKAI Atsushi; SASAKI Narie; KUROIWA Tsuneyoshi, Mar. 1996, 37, 102, 102Not Refereed, 色素体の細胞科学-人類存続の科学へ-(共著), SAKAI Atsushi, 1996, 22-35Not Refereed, Research Journal of Food and Agriculture, New Images on the Proliferation of Plant Cells ; Regulation by organelles., SAKAI Atsushi, 1995, 18, 4, 18-22Not Refereed, Function and Differentiation of Plastids., SAKAI Atsushi, 1995, 110-123Not Refereed, Plant Cell Technology, Plastid Differentiation from the viewpoint of Plastid-nuclei, SAKAI Atsushi, 1993, 5, 5, 366Not Refereed, Metals and Technology "KINZOKU", Plant cells and genes., SAKAI Atsushi, 1992, 62, 8Not Refereed, 葉緑体の遺伝子発現-植物はどのようにして色素体を作動させるか-(共著翻訳), SAKAI Atsushi, 1990, 2, 臨時増刊, 321-335Books etc
- 植物学の百科事典, SAKAI Atsushi, 2016, Not Refereed
- 生物学辞典, SAKAI Atsushi, Dec. 2010, Not Refereed
- 「大台ケ原の自然誌―森の中のシカをめぐる生物間相互作用」, SAKAI Atsushi, 2009, 第11章 シカの影響の有無によるササの形態と光合成能力の変化, Not Refereed
- 生物学大辞典, SAKAI Atsushi, 2007, Not Refereed
- プラントミメティックスー−植物に学ぶー−, SAKAI Atsushi, 2006, pp. 279 - 285, 葉緑体と色素体-色素体七変化-, Not Refereed
- 光合成事典, SAKAI Atsushi, 2003, Not Refereed
- 細胞生物学事典, SAKAI Atsushi, 2002, Not Refereed
- 分子生物学(共著), SAKAI Atsushi, 1999, 27-35頁, Not Refereed
- 分子細胞生物学辞典, SAKAI Atsushi, 1997, Not Refereed
- オルガネラ形成の遺伝子発現機構(共著), SAKAI Atsushi, 1994, 214-231頁, Not Refereed
Presentations
- 15 Sep. 2024, 14 Sep. 2024 - 16 Sep. 2024
- 13 Sep. 2024, 13 Sep. 2024 - 13 Sep. 2024
- 13 Sep. 2024, 13 Sep. 2024 - 13 Sep. 2024
- Oral presentation, 08 Sep. 2023, 07 Sep. 2023 - 09 Sep. 2023
- Oral presentation, 08 Sep. 2023, 07 Sep. 2023 - 09 Sep. 2023
- Oral presentation, 08 Sep. 2023, 07 Sep. 2023 - 09 Sep. 2023
- Oral presentation, 07 Sep. 2023, 07 Sep. 2023 - 09 Sep. 2023
- Oral presentation, 07 Sep. 2023, 07 Sep. 2023 - 09 Sep. 2023
- Poster presentation, 06 Sep. 2023, 06 Sep. 2023 - 06 Sep. 2023
- Poster presentation, 06 Sep. 2023, 06 Sep. 2023 - 06 Sep. 2023
- 17 Sep. 2022, 15 Sep. 2022 - 20 Sep. 2022
- Poster presentation, 16 Sep. 2022, 16 Sep. 2022 - 16 Sep. 2022
- Poster presentation, 16 Sep. 2022, 16 Sep. 2022 - 16 Sep. 2022
- Poster presentation, 16 Sep. 2022, 16 Sep. 2022 - 16 Sep. 2022
- Poster presentation, 16 Sep. 2022, 16 Sep. 2022 - 16 Sep. 2022
- Oral presentation, 19 Sep. 2021, 16 Sep. 2021 - 20 Sep. 2021
- Atsushi Sakai; Yukiko Ushikoshi; Akiko Sasaki, Reevaluation of allelopathic potential of goldenrod (Solidago altissima) naturalized in Japan., 21 Mar. 2020, 19 Mar. 2020 - 21 Mar. 2020
- SAKAI Atsushi, マングローブ植物ヤエヤマヒルギのイオン吸収速度に対するNa+の影響 ～modified root-split法を用いた解析～, Sep. 2018, False
- SAKAI Atsushi, トウモロコシ第一葉老化過程における葉緑体核様体の挙動と光合成特性の変化, Sep. 2018, False
- SAKAI Atsushi, タバコ培養細胞BY-2の細胞死誘導過程におけるDNA断片化へのタンパク質合成阻害の影響, Sep. 2017, False
- SAKAI Atsushi, 地下部からの滲出による成長阻害物質の放出を検出する実験法の検討, Sep. 2017, False
- SAKAI Atsushi, セイタカアワダチソウの生葉・枯葉由来成長阻害物質の同定に向けた解析, Sep. 2016, False
- SAKAI Atsushi, タバコ培養細胞BY-2の3種類の細胞死誘導過程におけるDNA断片化プロセスの検討, Sep. 2016, False
- SAKAI Atsushi, ヒマラヤスギの根からの成長阻害物質放出について, Sep. 2016, False
- SAKAI Atsushi, ヒマラヤスギ周囲の裸地形成における枯葉の役割, Sep. 2015, False
- SAKAI Atsushi, 単細胞緑藻Chlamydomonas reinhardtii 高温耐性株の解析, Sep. 2015, False
- SAKAI Atsushi, セイタカアワダチソウの枯葉由来成長阻害物質の同定に向けて, Sep. 2015, False
- SAKAI Atsushi, 低温下における葉の赤色化は葉温を上昇させるか？, Sep. 2015, False
- SAKAI Atsushi, タバコ培養細胞BY-2の細胞密度が過敏感細胞死誘導に及ぼす影響 ―過敏感細胞から発信されるシグナル物質の関与の可能性―, Sep. 2015, False
- SAKAI Atsushi, ヒマラヤスギ周囲の裸地形成には複数の要因が関与する, Sep. 2014, False
- SAKAI Atsushi, 接触形態形成が窒素含量の変化を通して光合成に及ぼす影響, Sep. 2014, False
- SAKAI Atsushi, タバコ培養細胞BY-2の細胞学：細胞の増殖・分化と死, Sep. 2014, False
- SAKAI Atsushi, 接触形態形成と光合成の接点, Dec. 2013, False
- SAKAI Atsushi, 栄養環境の違いが接触形態形成、窒素含量、光合成能力に及ぼす影響, Sep. 2013, False
- SAKAI Atsushi, 機械的ストレスに対するシロイヌナズナの応答：サイズ、窒素含量、および光合成機能の変化, Sep. 2013, False
- SAKAI Atsushi, 盗葉緑体が光合成ウミウシの走光性に与える影響, Mar. 2013, False
- SAKAI Atsushi, ヒマラヤスギ周囲の裸地は他感作用によるものか？II. 他感物質の供給経路と季節変化, Sep. 2012
- SAKAI Atsushi, ヤエヤマヒルギ幼植物体の生存・成長・呼吸・光合成に対するNaClの影響, Sep. 2012
- SAKAI Atsushi, オルガネラ核様体におけるMicrococcal nucleaseによる分解からDNAを保護する構造の比較, Sep. 2012
- SAKAI Atsushi, 接触形態形成が窒素含量の変化を介して光合成能力に影響を及ぼす現象について：刺激耐性の異なる種間での比較, Sep. 2012, False
- SAKAI Atsushi, ヒマラヤスギ周囲の裸地は他感作用によるものか？, Sep. 2011
- SAKAI Atsushi, 接触形態形成による植物体サイズの変化は植物体内窒素濃度の変化を介して光合成能力の変化をもたらす, Sep. 2011, False
- SAKAI Atsushi, タバコ培養細胞におけるＰＣＤと非ＰＣＤに伴う核ＤＮＡの断片化, Sep. 2011, False
Research Projects
- Grant-in-Aid for Scientific Research (C), 01 Apr. 2012 - 31 Mar. 2015, 24570049, Effects of mechanical-stress induced changes in plant body size on photosynthetic function: mechanisms and biological significance, SAKAI Atsushi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Nara Women's University, 4160000, 3200000, 960000, Plants change their body sizes in response to mechanical stress (MS) such as trampling. During the process of decrease/increase of plant body size in response to the application/elimination of MS, nitrogen within the plant body might be concentrated/diluted accordingly, which, in turn, result in lower/higher photosynthetic potentials. Such a “chain reaction” appeared to be more popular among perennial clonal plants and relatively rare among plants with high MS sensitivity.
Under MS, photosynthetic production of a plant/stand as a whole declines as a result of miniaturization. However, increase in photosynthetic potential per area resulting from nitrogen enrichment should partly compensate for the reductions in the total photosynthetic productivity. Moreover, the MS-induced miniaturization was found to increase survivability of plants dramatically under certain circumstances., kaken - Grant-in-Aid for Scientific Research (C), 2009 - 2011, 21610013, Research of the Environmental Design and the Educational Meaning on the Play Area and Garden in the Kindergarten, HAMADA Sumio; MUTO Takashi; SETO Akiko; NISHIMURA Takuo; MOTOYAMA Masako; AMAGASE Masahiro; SUZUKI Koshi; ASAO Takeshi; SAKAI Atsushi; HORIKOSHI Norika; HIGASHIMURA Tomoko, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Nara Women's University, 4680000, 3600000, 1080000, The play area and garden in the kindergarten has functions as educational facilities and as social capitals. In this research, we investigated and inquiried the history of the kindergarten and play area, the feature of the gardening design in the present, and the using for children's plays and activities, and we clarified the environmental design and the educational meaning of the play area and garden, kaken
- Grant-in-Aid for Scientific Research (B), 2006 - 2008, 18380090, Effects of deer browsing and debarking on life history and population dynamics of insects through the changes of morphology and quality of host plants, SHIBATA Eiichi; SATO Hiroaki; SAKAI Atsushi; HINO Teruaki, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Nagoya University, 17640000, 15000000, 2640000, ニホンジカによる樹木剥皮と林床植生の採食が激しい大台ヶ原と奈良公園において、シカをめぐる生物間相互作用を明らかにした。大台ヶ原では、シカの採食はササの小型化をもたらすが、それは接触刺激によることが示唆された。またゴールを形成するタマバエの間接効果を明らかにした。奈良公園では、防御形質と考えられるイラクサ刺毛密度の進化にシカの被食が淘汰圧として作用していることが示唆された。, kaken
- Grant-in-Aid for Scientific Research on Priority Areas, 2004 - 2008, 16085208, Analysis on multiplication of plastids and organelle gene expression in plant function, TAKANO Hiroyoshi; TAKIO Susumu; SAKAI Atsushi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Kumamoto University, 65000000, 65000000, 植物の高次機能とオルガネラとの関係を探るため、色素体分裂機構とオルガネラDNA複製機構の研究を行った。色素体の分裂機構に関しては、コケ植物でペプチドグリカン(PG)合成系が葉緑体の分裂に関与することを明らかにした。一方種子植物では、PG系の遺伝子が色素体遺伝子発現に関与していた。オルガネラDNA複製機構については、タバコより単離したDNA合成酵素がミトコンドリアと色素体の両オルガネラDNAを複製する主たる酵素であることを見いだした。, kaken
- 萌芽研究, 2005 - 2006, 17657019, 大型植食動物による被食圧に対する植物の反応, 酒井 敦; 佐藤 宏明; 柴田 叡弌, 日本学術振興会, 科学研究費助成事業, 奈良女子大学, 3500000, 3500000, 本研究では、大台ケ原山においてニホンジカによる被食圧下で分布を拡大しつつあるミヤコザサを材料に、植物の耐被食戦略を検討した。前年度までの研究により、「シカによる被食圧下にあるササは接触刺激を認識して小型化し、その結果植物体各部の窒素濃度の増大・光合成装置の増加により光合成能力が向上し、その結果として小型化(=受光面積の縮小)に伴う光合成物質生産の低下が補償される」、という可能性が示唆された。18年度はこの「接触形態形成に端を発する連鎖反応による補償光合成」の可能性をさらに検討した。
1.小型化の意義:シカによる被食圧下にあるササ群落内に、プロテクトケージを用いて様々な高さのササのパッチを作製した後ケージを撤去し、シカの食害による葉の損失速度を測定した。その結果、周囲よりわずかに背が高くなると葉の損失が急増することから、小型化は集中的な食害の回避に必須であることが分かった。
2.稈密度:大台ケ原山正木峠周辺のササ群落の当年稈密度は、防鹿柵外で約3000本/m^2、防鹿柵内(シカから5年にわたり保護)では約1200本/m^2であった。プロテクトケージを、時期をずらして設置したところ、当年稈の密度は前年の初夏(6月から7月頃)に決定されることが示唆された。柵外における稈密度の増大は、食害回避のための個々の稈の小型化のもとで群落レベルでの物質生産速度を維持することに貢献していると推察される。
3.シカによる被食圧のかわりに接触刺激のみを与えてササを栽培した場合にも、小型化と光合成能力の増大が誘導された。しかし、窒素含量の増加は認められなかった。この原因としては、生育環境(野外では強光条件下、栽培実験では弱光・貧栄養条件下)、あるいは変化の方向性(野外実験では大型化に伴う変化、栽培実験では小型化に伴う変化)の影響が考えられた。現在、これらの点を検討するための野外実験および栽培実験を開始している。, kaken - 基盤研究(C), 2004 - 2004, 16637001, 植物モデル細胞の高度利用と関連情報の統合に関する調査研究, 松岡 健; 長田 敏行; 馳澤 盛一郎; 江坂 宗春; 園部 誠司; 酒井 敦, 日本学術振興会, 科学研究費助成事業, 独立行政法人理化学研究所, 3100000, 3100000, 本企画調査は、日本で作成され、世界的に最も広く基礎研究に利用されている植物培養細胞株であるBY-2細胞に関する研究知識を統合し、その利用価値を格段に高めることを目的として、そのためにどのような枠組みを作るべきかを調査し、可能であればその枠組みの構築を図ることを目的としている。そのために、BY-2細胞を利用して研究している研究者間の交流と、主要な研究者における枠組みに関する討議が必要となると考えられた。そこでまず、分担者がそれぞれの該当するフィールドで、この細胞を用いて研究を行っている主な研究者をリストアップした。ついで、これらの外国の研究者を交えて、研究交流とこの細胞に関する国際的な研究協力体制構築のための立ち上げを模索するために、国際シンポジウムを開催することとし、本科学研究費の研究グループ、理化学研究所植物科学研究センター、BY-2研究会の3者の共催として、平成16年9月に理化学研究所横浜研究所を会場として国際会議を開催した。この会議には、海外の6カ国からの招待講演者15名を含む32名の口頭発表と、20件のポスター発表が行われ、合計152名が参加した。また、この会議の折に、本研究のメンバーと海外の代表的な研究者5名で、今後の共同研究体制に関する打合せを行った。結果的には、今後の国際共同研究を企画するための情報を交換するためのホームページを代表者が年度内に立ち上げること、そのホームページを利用しながら、国際的な協調体制のための予算申請についての討論を行ってゆくこと、及び情報の蓄積場所と内容についての国際的な分担等を合意した。そこで、この合意を会議の最後のセッションで参加者にアナウンスした。また、このためのホームページを代表者が立ち上げた。, kaken
- Grant-in-Aid for Scientific Research (C), 2000 - 2002, 12640634, Hormone-induced differentiation of BY-2 cultured tobacco cells ; development of a model system for differentiation of root-cap cells., SAKAI Atsushi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Nara Women's University, 4800000, 4800000, In conventional medium containing auxin (2,4-D, 0.2mg1^<-1>), BY-2 cultured tobacco cells proliferate actively, forming simple cell clusters composed of 8 to 16 small cylindrical cells connected in tandem, and the plastids within the cells remain undifferentiated throughout the culture period. By contrast, in a modified medium that contain cytokinin (BA, 1mg1^<-1>) instead of auxin, BY-2 cells become large without active cell proliferation, and the plastids within the cells differentiate into amyloplasts. In addition to these characteristics (inactive cell proliferation, cell enlargement, amyloplast development), we found that the behavior of BY-2 cells in the auxin-depleted medium resembled to that of root-cap cells in that (i) they easily exfoliate with each other, (ii) exhibited short lifetime, and (iii) showed higher secretion activity. Four proteins specifically secreted by root-cap cell-like BY-2 cells were detected, and one of them (named SPCT44) was identified as secreted peroxidase by N-terminal peptide sequencing of the purified protein. For other three proteins, we could not determine N-terminal peptide sequence because N-terminal was blocked. Their identification by determining internal peptide sequence is now on progress. To analyze changes in the gene expression during differentiation of normal BY-2 cells to root-cap like ones, we performed ddRT-PCR and microarray analyses (collaboration with RIKEN), using mRNAs purified from BY-2 cells cultured 12 h in either conventional medium or modified medium, and identified several genes whose expression is activated according to differentiation of root-cap like cells. The results are opened in the BY-2 EST database by RIKEN (http://mrg.psc.riken.go.jp/strc/). I'd like to further analyze the similarity between root-cap cells and the root-cap cell-like BY-2 cells in the modified medium, by using molecular markers obtained through this research project., kaken
- 特定領域研究(A), 2000 - 2000, 12025220, 光合成機能の獲得に伴う色素体及びミトコンドリアの転写複製活性の変化とその制御, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 奈良女子大学, 2600000, 2600000, トウモロコシは典型的なC_4植物であるが、葉位あるいは葉の発達・老化段階に依ってはC_3的な光合成特性を示すと報告されている。トウモロコシ芽生えの各葉について、クロロフィル含量が最大になる時期のCO_2補償点を測定した結果、第一葉、第二葉、第三葉いずれもC_4植物に特徴的な低い値を示したのに対し、幼葉鞘は極めて高い値を示した。組織切片観察の結果、幼葉鞘にはC_4植物に特徴的なクランツ構造がなく、C_4光合成に必要なC_4型PEPCのmRNA及びタンパク質も検出されなかったことから、幼葉鞘はC_3型の光合成を行っていることが示唆された。次に第一葉の成熟/老化過程における光合成特性の変化を解析し、CO_2補償点は葉の成熟につれ低下し、老化に伴って高くなることを明らかにした。組織切片観察の結果から、維管束鞘細胞よりも葉肉細胞の方が早く葉緑体が退化すること、またwestern解析の結果から、維管束鞘細胞で働くRubiscoよりも葉肉細胞で働くC_4型PEPCの方が早く分解/消失することが示された。これらの結果から、トウモロコシの老化葉がC_3的な光合成特性を示すのは、CO_2を濃縮する役割を果たしている葉肉細胞が維管束鞘細胞よりも早く老化し、機能を失うためと推測された。また、RT-PCRによりパクテリアのDNA polymerase Iとの類似性が認められるアラビドプシス遺伝子の単離を行い、この遺伝子産物が葉緑体へ移行するためのトランジットペプチドをもち、DNA polymerase IのN末端側にあるエキソヌクレアーゼ活性部分のみをコードしていることを明らかにした。, kaken
- 特定領域研究(A), 1999 - 1999, 11151205, 光合成機能の獲得に伴う色素体およびにミトコンドリアの転写・複製活性の変化とその制御, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 奈良女子大学, 3600000, 3600000, 本研究は、単離色素体核・ミトコンドリア核を用いたin vitro転写/DNA合成体を活用し、特にDNAおよびRNAポリメラーゼに注目しつつ、(1)これらオルガネラのDNA複製/転写系が互いにどのような影響を及ぼしつつ進化してきたか、(2)植物細胞の増殖・分化過程(特に光合成機能獲得過程)において両オルガネラの複製/転写機能がどのような機能により統合的に制御されているか解明することを目的とする.今年度の主要成果は以下のとおりである.
1.対数増殖期のタバコ培養細胞BY-2、根冠型細胞への分化を誘導したBY-2、タバコ植物体の成熟葉から単離した原色素体核、アミロプラスト核、緑葉体核を用いたin virto転写系を用いた解析の結果、光合成型色素体における転写にはtagetitoxin感受性型RNAポリメラーゼが働いているのに対し、非光合成型色素体においてはtagetitoxin非感受性型RNAポリメラーゼが主力となっていることが明らかになった。しかしながら、詳細な解析の結果、幾つかの光合成関連遺伝子(psbA等)やtRNA遺伝子(trnQ,trn fM等)は非光合成型色素体においてもtagetitoxin感受性PNAポリメラーゼにより選択的に転写されていることが明らかになった。
2.対数増殖期にあるタバコ培養細胞BY-2から単離した原色素体核とミトコンドリア核の2MNaCl抽出液をアフィニティー(heparin)、陽/陰イオン交換、ゲルろ過、ならびに疎水性などの各種クロマトグラフィーにかけ、色素体およびミトコンドリアのDNAポリメラーゼの挙動を調べた結果、いずれの場合も両オルガネラのDNAポリメラーゼはよく似た挙動を示すことを明らかにした。, kaken - 奨励研究(A), 1998 - 1999, 10740366, 色素体RNAポリメラーゼの多様性と役割分担に関する生理・生化学的研究, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 2300000, 2300000, 本研究は、単離色素体核を用いたin vitro転写系を活用し、特にRNAポリメラーゼの多様性と役割分担に注目しつつ、植物細胞の増殖・分化過程(特に光合成機能獲得過程)において色素体の転写機能がどのような機構により統合的に制御されているか解明することを目的とする。色素体では、少なくとも2種類(大腸菌型とT7ファージ型)のRNAポリメラーゼが働いていることが明らかになっており、大腸菌型は転写阻害剤tagetitoxinに対し感受性、T7ファージ型はtagetitoxin非感受性と推測される。対数増殖期のタバコ培養細胞BY-2、根冠型細胞への分化を誘導したBY-2、タバコ植物体の成熟葉から、それぞれ原色素体核、アミロプラスト核、葉緑体核を単離し、それら単離色素体核を用いたin vitro転写反応を行い、tagetitoxinの阻害効果を比較することにより、これら3タイプの色素体におけるtagetitoxin感受性/非感受性RNAポリメラーゼの相対的な活性の比較を行った。その結果、非光合成型色素体(原色素体およびアミロプラスト)の転写装置はtagetitoxinに対する感受性が低く(阻害率20〜50%)、光合成型色素体(葉緑体)の転写装置は感受性が高い(阻害率95〜98%)ことが判明した。本研究の結果は、非光合成型色素体における色素体遺伝子の転写にはT7ファージ型が、光合成型色素体における転写には大腸菌型が主力となっていることを示唆する。, kaken
- 特定領域研究(A), 1998 - 1998, 10170208, 光合成能の獲得に伴う色素体およびミトコンドリアの転写-複製活性の変化とその制御機構, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 2100000, 2100000, 本研究は、単離色素体核・ミトコンドリア核を用いたin vitro転写/DNA合成系を活用し、特にDNAおよびRNAポリメラーゼに注目しつつ、(1)これらオルガネラのDNA複製/転写系が互いにどのような影響を及ぼしつつ進化してきたか、(2)植物細胞の増殖・分化過程(特に光合成機能獲得過程)において両オルガネラの複製/転写機能がどのような機構により統合的に制御されているか解明することを目的とする.今年度は、色素体およびミトコンドリアのDNAポリメラーゼの性質の比較ならびに細胞増殖・分化過程におけるオルガネラDNA合成活性の変化について重点的に解析を行った.主要成果は以下のとおりである.
1. タバコ培養細胞BY-2から単離した原色素体核およびミトコンドリア核はDNA合成活性を保持していた.ゲル内アッセイの結果、両オルガネラ核はいずれも見かけの分子量約116kDaのDNAポリメラーゼを含んでおり、各種クロマトグラフィーにおける挙動も含めて両DNAポリメラーゼの性質は酷似していることが確認された.この結果は、DNAポリメラーゼもT7ファージ型の色素体/ミトコンドリアRNAポリメラーゼのように、オルガネラ間での共通化という方向へ進化してきた可能性を示唆する.
2. DAPI蛍光顕微鏡観察、定量的サザンハイブリダイゼーション、チミジンのアナログであるBrdUの取り込み、ならびに単離オルガネラ核のin vitro DNA合成活性の測定等の手法を用いた解析の結果、タバコ培養細胞BY-2の増殖過程において、色素体とミトコンドリアのDNA合成活性は植え継ぎ後3〜6時間でほぼ同時に活性化されるが、活性が低下するタイミングは若干異なることが示された.アサガオ花粉およびイネ子葉鞘における観察の結果も、色素体とミトコンドリアのDNAの増加・減少は、互いに独立に制御され得ることを示した., kaken - 特定領域研究(A), 1998 - 1998, 10158203, 雌雄異株植物を用いた性染色体と生殖器官特異的遺伝子群に関する分子細胞学的解析, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 2800000, 2800000, 本研究の目的は、性染色体をもちXY型の性決定を行う雌雄異株植物メランドリウム(ヒロハノマンテマ、Silenelatifolia)を用いて生殖器官形成に関わる遺伝子群を単離・同定すると同時に、高等植物の性染色体にコードされる遺伝子群が生殖器官形成に果たす役割を明らかにすることである.特に、雄にしか存在しないY染色体は性決定において重要な役割を担っていると考えられるので、本研究では雄性生殖器官特異的に発現される遺伝子群およびY染色体特異的遺伝子群に解析を集中した.今年度の成果は以下のとおりである.
(1) レーザーマイクロダイセクションを用いて単離されたY染色体由来の高度反復配列のひとつ、RMY1(repetitive sequence in microdissected Y chromosome)は、チェコのグループがマイクロダイセクションによりメランドリウムX染色体から単離したサプテロメア様配列X43.1に高いホモロジーを示した.FISH解析の結果、RMY1は常染色体を含む多数の染色体末端部分に局在を示したが、性染色体間では局在様式が異なっていた(X染色体は両端、Y染色体は一方の末端).この配列は性染色体の異型化を考察する上で有用な指標になると考えられる.
(2) 10merのプライマーを用いて雌雄ゲノムDNAを鋳型とするRAPD解析を行い、雄DNAから特異的に増幅されてくるDNA断片を得た,ゲノミックサザンハイブリダイゼーションにより雄特異性を検討し、最も雄特異性が高いと思われたクローンについてSTS化を行い、雄特異的に単一のバンドが増幅されることを示した.inverse PCRにより、その周辺領域の取得を試みたところ、逆転写酵素様のタンパク質をコードし得るORFを含む約9.6kbpのクローンを得た.この配列はY染色体DNAのマーカーとして有用であるとともに、Y染色体特異的遺伝子の有力な候補である., kaken - 重点領域研究, 1997 - 1997, 09274205, 光合成能の獲得に伴う色素体及びミトコンドリアのDNA複製活性の変化とその制御機構, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 1400000, 1400000, 本研究の目的は、色素体およびミトコンドリアのDNAおよびRNAポリメラーゼについてその多様性と役割分担を明らかにし、植物細胞進化過程におけるオルガネラゲノム間のクロストークについて考察することである。今年度の成果は、以下の通りである。
1.タバコ培養細胞BY-2から細胞核、原色素体核、ミトコンドリア核を高純度で単離し、in vitro転写反応を行わせる系を確立した。単離原色素体核のin vitro転写活性は、葉緑体RNAポリメラーゼの阻害剤であるtagetitoxinによって部分的にしか阻害されず、原色素体では葉緑体と性質の異なるRNAポリメラーゼが主カを占めている可能性が示唆された。
2.タバコ培養細胞BY-2とタバコ成熟葉における色素体遺伝子の発現様式を転写および転写産物蓄積のレベルで比較し、転写と転写産物蓄積との関係を検討した。単離葉緑体核のin vitro転写活性は単離原色素体核の約25倍(鋳型DNA1μgあたり)に達する。転写活性化の程度と転写産物量の増大との間には一定の直線的な関係が成り立ち、色素体転写産物蓄積量の決定において転写レベルでの制御が重要であることが示された。また、BY-2における色素体遺伝子発現の様相は、色素体ゲノムからrpoB(大腸菌型RNAポリメラーゼのβサブユニット遺伝子)を欠失させたタバコ(Alisonら、1996)における色素体遺伝子発現の様式に類似しており、BY-2の原色素体においては色素体コードの(大腸菌型)RNAポリメラーゼの役割が相対的に小さいと推測された。
3.BY-2から単離した原色素体核のin vitro転写活性はtagetitoxinに比較的耐性であるのに対し、タバコ葉肉細胞から単離した葉緑体核の転写活性は感受性が高いことを明らかにし、分化にともなう色素体転写装置の性質の変化を示した。
4、BY-2から単離した原色素体核およびミトコンドリア核に存在するDNAポリメラーゼの性質(鋳型選択性等)についてゲル内DNAポリメラーゼアッセイ法および単離核のin vitroDNA合成系を用いて検討し、その類似性を示した。, kaken - 重点領域研究, 1997 - 1997, 09262204, 雌雄異株植物を用いた性染色体と生殖器官特異的遺伝子群に関する分子細胞学的解析, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 1200000, 1200000, 本研究では、性染色体をもちXY型の性決定を行うナデシコ科の雌雄異株植物メランドリウム(Silene latifolia)を材料に用いて、雄性生殖器官特異的に発現する遺伝子と雄ゲノム特異的なY染色体との関連について解析を行っている。今年度の成果は以下の通り。
1.ディファレンシャルスクリーニングにより単離した4種類の雄性生殖器官特異的(Male Reproductive Organ Specific)遺伝子(MROS1-4)のうち、MROS3のゲノミッククローンをinverse-PCRを用いて単離した。約4kbpと3kbpの2種類のクローンが得られた(MROS3AおよびMROS3Bと命名)。両者のORF部分は93.3%の高い相同性を示すが、上流域・下流域は有意な相同性を示さない。従来得られていたcDNAに対応するMROS3Aの転写領域中の3非翻訳領域には、162bpの非自律性トランスポゾン様の挿入配列が認められた。単離したゲノミッククローンをブローブとするマルチカラーFISHを行った結果、MROS3AおよびMROS3B遺伝子は常染色体の末端部にマップされ、性染色体上には存在していないことが明らかになった。
2.メランドリウム根端分裂組織を材料に用いて染色体標本を作製し、UVレーザービームマイクロダイセクションによりY染色体を直接単離した。回収したY染色体1本(0.5pg)をトボイソメラーゼI処理後DOP-PCRにかけ、鎖長200から300bpのY染色体由来DNA断片を増幅した。増幅されたDNA断片の全体をブローブとしてFISHを行うと、雌のゲノムDNAをブローブの200倍量サブレッサーとして加えてあるにも関わらず、Y染色体特異的なシグナルは検出されず、全ての染色体にシグナルが見られた。このことから、メランドリウムのY染色体はその大部分が他の常染色体やX染色体と共通のDNA配列からなり、特異的な反復配列は蓄積していないと考えられる。また、Y染色体由来DNAのライブラリーからY染色体の末端部分由来である可能性の高いクローンが一つ得られたので、現在解析している。, kaken - 奨励研究(A), 1996 - 1996, 08740612, 細胞増殖及び色素体分化にともなう植物ミトコンドリアゲノムの機能的分化, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 1000000, 1000000, タバコ培養細胞BY-2からミトコンドリア核を単離する手法を確立した。この際、細胞核と色素体核も同時に単離することができる。対数増殖期の細胞約10^9個を出発材料とした場合、ミトコンドリア核の収率は5%で、収量は80μgDNAであった。サザンハイブリダイゼーションにより細胞核DNAおよび色素体DNAの混入率を測定した結果、単離ミトコンドリア核の純度は98%であった。分化にともなうミトコンドリアゲノムの機能変化を明らかにするために、タバコ緑葉からミトコンドリア核を単離する方法の開発も行っているが、収量の点でなお改善が必要である。BY-2から単離したミトコンドリア核を適当な条件下でインキュベートすると、[5,6-^3H]UTPを高分子画分に取り込む活性を示す(7pmol/μgDNA/hr)。この活性はDNAおよびタンパク質に依存したRNA合成であり、アクチノマイシンDやEB、N-ethylmaleimide、ヘパリンにより阻害されるが、α-アマニチン、リファンピシン、タゲチトキシン、ナリジキシン酸、及びノボビオシンでは阻害されない。in vitroで合成されたRNAは、ミトコンドリアDNAと特異的にハイブリダイズした。単離ミトコンドリア核はまた、[5-^3H]dCTPの取り込み活性も示す(48pmol/μgDNA/hr)。この活性はDNAおよびタンパク質に依存したDNA合成であり、アクチノマイシンDやEB、N-ethylmaleimide、ddCTP、ヘパリンにより阻害されるが、アフィデイコリン、ナリジキシン酸、ノボビオシンでは阻害されない。in vitroで合成されたDNAの制限酵素解析の結果、DNA合成はミトコンドリアDNAの全領域にわたって起きていることが明らかになった。また、ミトコンドリア核構成タンパク質をSDS-PAGE後、ゲル内DNAポリメラーゼアッセイにかけたところ、分子量約116kDaのDNAポリメラーゼが検出された。本研究の結果、単離ミトコンドリア核のDNA合成反応の至適条件や各種阻害剤に対する応答、DNAポリメラーゼの分子量などは、単離色素体核のそれらと極めて類似していることが明らかになった。この色素体及びミトコンドリアのDNA合成装置の類似性は、両オルガネラのDNAの合成が、細胞増殖初期にそろって活性化される機構を説明するものかもしれない。, kaken
- 奨励研究(A), 1995 - 1995, 07740613, 色素体ゲノム機能を制御する色素体核構築タンパク質の同定と単離, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 1100000, 1100000, タバコ培養細胞BY-2の色素体は、オーキシン(0.2mg/1の2,4-D)を含む通常の培地中では未分化名原色素体の状態を保つが、オーキシンのかわりにサイトカイニン(1mg/1のBA)を含む改変培地中では大量のデンプンを蓄積し、アミロプラストへと分化する。一方、タバコ植物体の葉肉細胞中には葉緑体が存在する。これら3種の色素体からそれぞれの色素体核(DNA-タンパク質複合体) を単離し、色素体ゲノムの転写・複製活性を比較した。原色素体の場合、細胞の増殖初期過程で一過的にDNA合成が活性化されるが、転写活性はほぼ一定のレベルを保つ。アミロプラスト分化過程では、DNA合成、転写ともに活性は低下する。成熟した葉緑体では原色素体に比べDNA合成活性は約1/4に低下しているが、転写活性は約15倍に上昇していた。このようにそれぞれ特徴的な機能を示す原色素体核、アミロプラスト核、葉緑体核を構成するタンパク質のうち、DNA結合性タンパク質をサウスウェスタン法により比較した。原色素体核およびアミロプラスト核に共通する主要なDNA結合性タンパク質は78, 64,および50kDaである。その他に原色素体核およびアミロプラスト核に特異的なDNA結合性タンパク質も検出されている。また、葉緑体核の主要なDNA 結合性タンパク質は120, 70, 50,および26kDaであった。これらのDNA結合タンパク質の殆どは3M Nacl/5M尿素処理により可溶化されるが、葉緑体核特異的70-および26-kDaタンパク質は一部不溶性画分に残留する。これら難溶性のDNA結合タンパク質は葉緑体核と膜系との結合に関与している可能性がある。また、単離原色素体核を用いてゲル内アッセイ法により色素体ポリメラーゼ(116 kDa)を検出した。現在、このDNAポリメラーゼ量の変化と色素体ゲノムの複製活性との間に相関関係が見られるかどうか検討している。, kaken
- 奨励研究(A), 1994 - 1994, 06740590, 色素体分化過程における色素体ゲノムの機能変化とその制御機構に関する研究, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 900000, 900000, 最初に、色素体ゲノムの機能解析に用いる単離色素体核の性格付けを行った。単離色素体核は高次構造がよく保たれ、転写・DNA合成活性も高いうえ、可溶性のDNA/RNAポリメラーゼやヌクレアーゼの活性をほとんど含まない等、転写・複製活性の測定に適している。本研究では、単離色素体核のin vitro転写・DNA合成系を用いて、タバコ培養細胞のBY-2の原色素体とアミロプラスト、およびタバコ葉肉細胞の葉緑体の三者について色素体ゲノムの転写・複製機能を比較した。通常の培養条件におけるBY-2の増殖過程では、原色素体ゲノムの複製は細胞増殖の極初期(植え継ぎ後24時間以内)に一過的に約5倍に活性化され、原色素体DNA量は著しく増加する。一方、転写活性は培養過程を通じてほぼ一定である。また、アミロプラスト分化を誘導したBY-2では、色素体のDNA複製はあまり活性化されず、転写活性も誘導開始後24-48時間で1/3から1/4に低下する。しかし、転写産物量は減少しないことから、アミプラストでは転写産物の寿命が極端に長いと考えられる。この時、psbA遺伝子の転写活性は例外的に比較的高く保たれ、そのmRNAは増加を続けることがわかった。一方、葉肉細胞の葉緑体とBY-2の原色素体の比較では、単離葉緑体核の転写活性は単離原色素体核の約15倍の値を示したが、DMA合成活性は約1/3と低かった。転写活性の増大の程度は個々の遺伝子ごとに異なり、転写の活性化と転写産物量の増大の間には正の相関がみられた。この結果は、葉緑体分化にともなう色素体遺伝子の発現制御には、転写レベルでの制御が重要であることを示唆する。単離色素体核のDNAタンパク質をサウスウェスタン法により調べたところ、それぞれの色素体核に特徴的なDNA結合タンパク質が検出された。DNA結合タンパク質の違いが色素体核の構造と転写・複製機能に及ぼす影響を探るため、単離色素体核をDNAとタンパク質に解体した後、再構成する系を現在開発中である。, kaken
- 重点領域研究, 1994 - 1994, 06269206, タバコ培養細胞系におけるアミロプラストの誘導と細胞肥大のホルモンによる制御, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 2800000, 2800000, タバコ培養細胞BY-2は、合成オーキシンである2,4-D(0.2mg/l)を含む通常の培地中では活発に細胞増殖を行い、その色素体は培養の全期間を通じて未分化な原色素体の状態を保つ。一方、2,4-Dを含まず、合成サイトカイニンであるBA(1mg/l)を添加した改変培地中では細胞はほとんど増殖せずに著しく大型化し、色素体は大量のデンプンを蓄積してアミロプラストに変換する(Sakai et al.1992)。今年度は、BY-2細胞の植物ホルモンに対する応答を詳細に解析し、アミロプラストの分化誘導に関しては-2,4D,+BAの条件が最良であることを確認した。しかし、ホルモンを全く含まない(-2,4-D,-BA)培地中でもデンプンの蓄積が認められることから、BY-2のアミロプラスト分化誘導では2,4-Dの除去が第一の要因であり、BAは2,4-Dの除去によって生じる変化を促進していると考えられる。それぞれのホルモンに対する応答を調べた結果、2,4-Dは細胞増殖を促進し、デンプン蓄積を抑制すること、BAは逆に細胞増殖を抑制し、デンプン蓄積を促進すること、そしてこれらの反応はホルモン濃度依存的であることが確認された。2,4-DとBA以外のオーキシンやサイトカイニンも類似の作用を示すことから、これらはオーキシン、サイトカイニンに共通の効果であると考えられる。各種培養条件を比較すると、細胞増殖率の低下とデンプン蓄積量の増大は平行関係にあった。しかし、真核型ポリメラーゼαの阻害剤であるアフィディコリンを添加して細胞の増殖を強制的に停止させても、デンプンの蓄積量は培地の種類ごとに明らかに異なっていた。したがって、デンプンの蓄積は増殖率の低下によって二次的にもたらされるのではなく、ホルモンにより独自の制御を受けていると考えられる。また、単離色素体核を用いたin vitroアッセイ系によりアミロプラスト分化過程における色素体ゲノムの機能変化を調べた結果、転写・複製活性ともに不活発であることが明らかになった。, kaken
- 奨励研究(A), 1993 - 1993, 05740476, アミロプラストの分化過程における色素体遺伝子の発現制御とその分子機構, 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 1000000, 1000000, タバコ培養細胞BY-2は、オーキシン(2,4-D;0.2mg/l)を含む通常培地中では未分化な原色素体をもつが、オーキシンを含まずサイトカイニン(BA;1mg/l)を添加した改変培地に植え継ぐと、細胞内の色素体は大量のデンプンを蓄積し、約48時間でアミロプラストへと分化する。このアミロプラスト分化誘導系を用いて、アミロプラストの分化過程における色素体遺伝子の発現制御様式を解析した。アミロプラスト分化過程では細胞数はほとんど増加せず、細胞当たりの色素体数、色素体DNAコピー数も分化過程を通じてほぼ一定であった。この過程における色素体遺伝子の転写活性の変化を調べるため、経時的に色素体核を単離し、そのin vitro転写活性を測定した。単位DNAあたりの転写活性は誘導開始6時間後に一過的に上昇した後急速に低下し、48時間後には誘導開始時の25%程度に減少した。9種類の代表的な色素体遺伝子の転写活性の変動をハイブリダイゼーションにより調べたところ、atpA,atpB,rpoB,petB,rbcL,rpl16,23SrRNA遺伝子の転写活性は一過的に上昇した後、誘導開始時の数%にまで減少するが、psbA(光化学系II反応中心32kDaタンパク質遺伝子)とpsaA-B(光化学系Ip700アポタンパク質A1/A2遺伝子)の転写活性はアミロプラスト分化過程を通じて比較的高く、誘導開始時の30-50%に保たれることが分かった。さらに、色素体転写産物蓄積量の変化を調べたところ、多くの色素体遺伝子の転写産物量は誘導開始直後に一過的に上昇した後、そのまま一定の値を保つか漸減したのに対し、psbaおよびpsaA-B転写産物量はアミロプラスト形成過程を通じて増加を続けた。転写産物蓄積量の変化とin vitro転写活性の変化は概ね一致する。しかし、多くの色素体遺伝子の転写活性が著しく低下した後においてもそれらの転写産物量は比較的一定のレベルを保っていることから、アミロプラストにおける転写産物の分解速度は極めて遅いことが示唆された。, kaken
- 重点領域研究, 1992 - 1992, 04257203, 単離オルガネラ核の解体と再構成による高等植物の色素体遺伝子発現機構の解析, 河野 重行; 酒井 敦, 日本学術振興会, 科学研究費助成事業, 東京大学, 2900000, 2900000, 細胞分化の柔軟性は植物細胞で特に著しく,オルガネラの分化にも高い柔軟性が要求される.なかでも色素体は著しい可塑性を有しており,細胞機能に応じて様々に発達・分化し得る.色素体の遺伝子発現制御機講を解析するためには,特微的な遺伝子発現パターンを持つ異なる型の色素体核を無傷単離し,それらを解体・再構成し、転写パターンを比較検討できることが望ましい.
タバコ培養細胞BY-2はこのような研究に理思的な系であるが,培養過程でのオルガネラの発達・分化の様式は明らかにされていない.DAPI染色-抗BrdU抗体免疫蛍光顕微鏡法や定量的サザンブロット法を用いて,通常の培養過程や培養条件を変更した際のオルガネラの挙動を解析した。その結果,原色素体やミトコンドリアのDNA合成は細胞増殖が活発になる直前に,最大になり,細胞核の分裂が活発になる時期には逆に減少することを明らかにした.また,培養7日目の定常期にあるBY-2を2,4-Dを除き6-benaylaminopurine(1mg/l)を添加した改変Linsmaier and Skoog培地に植え継ぐと,原色素体は12時間目頃から澱粉を蓄積し,48時間目頃にはほとんどがアミロプラスト化することを明らかにした.
このような培養系を用いて,原色素体核を無傷単離し,単離色素体核の解体・再構成実験を行った.単離原色素体核をNaCl処理し,タンパク質を段階的に外し,原色素体核の構造と転写活性を解析した.NaCl濃度を上げると,0.1Mで31,30kDaの,0.7Mで69,14kDaのタンパク質が外れ,原色素体核の構造が拡散し,転写活性は著しく減少し,転写産生の構成も著しく変化した.解体した原色素体核を含む溶液のNaCl濃度を段階的に下げると,原色素体核が再構成されるが,再構成した色素体核のin vitro転写効率は解体時を上回ることはなかった.このような再構成系では,核構造が見かけ上再成されても,転写機能に関与する要素の再構成が十分でないことがわかる.今後,核構造の構築に関与するDNA結合タンパク質とRNA合成酵素等の転写機能に直接関与する因子を分別単離し,転写機能を保持した色素体核の再構成系の開発を試みる予定である., kaken
■Ⅲ．社会連携活動実績
１．公的団体の委員等（審議会、国家試験委員、他大学評価委員，科研費審査委員等）
