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Tsuneyoshi Kuroiwa - One of the best experts on this subject based on the ideXlab platform.
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isolation of dividing chloroplasts with intact plastid dividing rings from a Synchronous Culture of the unicellular red alga cyanidioschyzon merolae
Planta, 1999Co-Authors: Shinya Miyagishima, Ryuuichi Itoh, Saburo Aita, Haruko Kuroiwa, Tsuneyoshi KuroiwaAbstract:In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized Culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions.
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aphidicolin uncouples the chloroplast division cycle from the mitotic cycle in the unicellular red alga cyanidioschyzon merolae
European Journal of Cell Biology, 1996Co-Authors: Ryuuichi Itoh, Haruko Kuroiwa, Hideo Takahashi, Kyoko Toda, Tsuneyoshi KuroiwaAbstract:: The unicellular red alga Cyanidioschyzon merolae possesses one chloroplast, one mitochondrion, and one cell nucleus. Since the division of these organelles and cytokinesis occur in a coordinated manner, mitosis and the organelle division cycles must be tightly coupled. We report here that aphidicolin, a specific inhibitor of DNA polymerase alpha, uncouples the chloroplast division cycle from the mitotic cycle. The effects of aphidicolin on C. merolae cells were examined by both epifluorescence and electron microscopy. When cells at the S phase in Synchronous Culture were treated with aphidicolin, mitosis and cytokinesis did not occur, while chloroplast division did. Moreover, both of the chloroplasts in these cells continued to divide and then generated four or more chloroplasts per cell. The inhibition of cell-nuclear DNA synthesis by aphidicolin was confirmed using microfluorometry. In addition, microfluorometry revealed that the total size and the amount of DNA in chloroplasts in aphidicolin-treated cells remained constant during uncoupled chloroplast division. As a result, the size and amount of DNA per chloroplast decreased stepwise during chloroplast division. Electron microscopic examination of aphidicolin-treated cells showed that the second division of chloroplasts uses a chloroplast-dividing ring similar to that in cells undergoing normal chloroplast division. These results suggest that chloroplast division by the dividing ring is free from a checkpoint control that inhibits the progression of mitosis and cytokinesis in the absence of the completion of cell-nuclear DNA synthesis, and also that chloroplasts lack a checkpoint control mechanism that inhibits its division without growth or DNA synthesis of itself.
Ryuuichi Itoh - One of the best experts on this subject based on the ideXlab platform.
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isolation of dividing chloroplasts with intact plastid dividing rings from a Synchronous Culture of the unicellular red alga cyanidioschyzon merolae
Planta, 1999Co-Authors: Shinya Miyagishima, Ryuuichi Itoh, Saburo Aita, Haruko Kuroiwa, Tsuneyoshi KuroiwaAbstract:In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized Culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions.
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aphidicolin uncouples the chloroplast division cycle from the mitotic cycle in the unicellular red alga cyanidioschyzon merolae
European Journal of Cell Biology, 1996Co-Authors: Ryuuichi Itoh, Haruko Kuroiwa, Hideo Takahashi, Kyoko Toda, Tsuneyoshi KuroiwaAbstract:: The unicellular red alga Cyanidioschyzon merolae possesses one chloroplast, one mitochondrion, and one cell nucleus. Since the division of these organelles and cytokinesis occur in a coordinated manner, mitosis and the organelle division cycles must be tightly coupled. We report here that aphidicolin, a specific inhibitor of DNA polymerase alpha, uncouples the chloroplast division cycle from the mitotic cycle. The effects of aphidicolin on C. merolae cells were examined by both epifluorescence and electron microscopy. When cells at the S phase in Synchronous Culture were treated with aphidicolin, mitosis and cytokinesis did not occur, while chloroplast division did. Moreover, both of the chloroplasts in these cells continued to divide and then generated four or more chloroplasts per cell. The inhibition of cell-nuclear DNA synthesis by aphidicolin was confirmed using microfluorometry. In addition, microfluorometry revealed that the total size and the amount of DNA in chloroplasts in aphidicolin-treated cells remained constant during uncoupled chloroplast division. As a result, the size and amount of DNA per chloroplast decreased stepwise during chloroplast division. Electron microscopic examination of aphidicolin-treated cells showed that the second division of chloroplasts uses a chloroplast-dividing ring similar to that in cells undergoing normal chloroplast division. These results suggest that chloroplast division by the dividing ring is free from a checkpoint control that inhibits the progression of mitosis and cytokinesis in the absence of the completion of cell-nuclear DNA synthesis, and also that chloroplasts lack a checkpoint control mechanism that inhibits its division without growth or DNA synthesis of itself.
Haruko Kuroiwa - One of the best experts on this subject based on the ideXlab platform.
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isolation of dividing chloroplasts with intact plastid dividing rings from a Synchronous Culture of the unicellular red alga cyanidioschyzon merolae
Planta, 1999Co-Authors: Shinya Miyagishima, Ryuuichi Itoh, Saburo Aita, Haruko Kuroiwa, Tsuneyoshi KuroiwaAbstract:In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized Culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions.
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aphidicolin uncouples the chloroplast division cycle from the mitotic cycle in the unicellular red alga cyanidioschyzon merolae
European Journal of Cell Biology, 1996Co-Authors: Ryuuichi Itoh, Haruko Kuroiwa, Hideo Takahashi, Kyoko Toda, Tsuneyoshi KuroiwaAbstract:: The unicellular red alga Cyanidioschyzon merolae possesses one chloroplast, one mitochondrion, and one cell nucleus. Since the division of these organelles and cytokinesis occur in a coordinated manner, mitosis and the organelle division cycles must be tightly coupled. We report here that aphidicolin, a specific inhibitor of DNA polymerase alpha, uncouples the chloroplast division cycle from the mitotic cycle. The effects of aphidicolin on C. merolae cells were examined by both epifluorescence and electron microscopy. When cells at the S phase in Synchronous Culture were treated with aphidicolin, mitosis and cytokinesis did not occur, while chloroplast division did. Moreover, both of the chloroplasts in these cells continued to divide and then generated four or more chloroplasts per cell. The inhibition of cell-nuclear DNA synthesis by aphidicolin was confirmed using microfluorometry. In addition, microfluorometry revealed that the total size and the amount of DNA in chloroplasts in aphidicolin-treated cells remained constant during uncoupled chloroplast division. As a result, the size and amount of DNA per chloroplast decreased stepwise during chloroplast division. Electron microscopic examination of aphidicolin-treated cells showed that the second division of chloroplasts uses a chloroplast-dividing ring similar to that in cells undergoing normal chloroplast division. These results suggest that chloroplast division by the dividing ring is free from a checkpoint control that inhibits the progression of mitosis and cytokinesis in the absence of the completion of cell-nuclear DNA synthesis, and also that chloroplasts lack a checkpoint control mechanism that inhibits its division without growth or DNA synthesis of itself.
Shinya Miyagishima - One of the best experts on this subject based on the ideXlab platform.
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Additional file 2 of Evolution of cytokinesis-related protein localization during the emergence of multicellularity in volvocine green algae
2017Co-Authors: Yoko Arakaki, Shinya Miyagishima, Takayuki Fujiwara, Hiroko Kawai-toyooka, Kaoru Kawafune, Jonathan Featherston, Pierre Durand, Hisayoshi NozakiAbstract:Figure S1. Alignment of DRP1A from Arabidopsis thaliana (At) and DRP1 from Chlamydomonas reinhardtii (Cr), Tetrabaena socialis (Ts), Gonium pectorale (Gp) and Volvox carteri (Vc). Black and gray background indicates identical or similar amino acid, respectively. GTPase domain, dynamin middle domain, and GTPase effector domain are indicated by pink, green, and yellow background color, respectively. The region corresponding to the antigen for an anti-TsDRP1 antibody is showed under the alignment (gray bar). Figure S2. Specificity of the affinity-purified anti-TsDRP1 antibody. The specificity of the anti-TsDRP1 antibody was validated in three volvocine algae by western blotting. A single band was detected in each lane (~75 kDa) with the antibody that was incubated with acetone powder of E. coli with the empty vector (left) while no signal was detected with the antibody that was incubated with acetone powder of E. coli expressing TsDRP1 (middle). For details of the methods, see Information S1 (Additional file 2). Figure S3. Western blot analyses of DRP1 proteins of Chlamydomonas reinhardtii (CrDRP1), Tetrabaena socialis (TsDRP1), and Gonium pectorale (GpDRP1) using anti-TsDRP1 antibody. Time-course of Synchronous Culture and western blot (WB) of C. reinhardtii, T. socialis, and G. pectorale are shown in a, b, and c, respectively. Time-course samples were obtained from five points (arrows in each line graph): the greatest number of dividing cells (0), three (−3) and six (−6) hours before 0 point, and three (+3) and six (+6) hours after 0 point. Coomassie brilliant blue (CBB) staining of a duplicate gel shows the equal protein loading in each lane. Information S1. Methods for specificity of the affinity-purified anti-TsDRP1 antibody (Additional file 2: Figure S2). (PDF 1785 kb
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isolation of dividing chloroplasts with intact plastid dividing rings from a Synchronous Culture of the unicellular red alga cyanidioschyzon merolae
Planta, 1999Co-Authors: Shinya Miyagishima, Ryuuichi Itoh, Saburo Aita, Haruko Kuroiwa, Tsuneyoshi KuroiwaAbstract:In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized Culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions.
Joseph L Derisi - One of the best experts on this subject based on the ideXlab platform.
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rna seq analysis of splicing in plasmodium falciparum uncovers new splice junctions alternative splicing and splicing of antisense transcripts
Nucleic Acids Research, 2011Co-Authors: Katherine Sorber, Michelle T Dimon, Joseph L DerisiAbstract:Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly Synchronous Culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5′ GU-AG 3′ and 5 new 5′ GC-AG 3′ junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasite’s transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasite’s transcriptome.
