1887

Abstract

The unicellular rhodophyte , having a single plastid and a single mitochondrion, is suitable for the analysis of the cell cycle involving the division of organelles. In conventional methods of synchronous culture of algae, light/dark cycles have been used as signals for synchronization, and the gene expression promoted by light is not separated from the gene expression related to cell cycle progression. We previously devised a novel synchronous culture system with controlled photosynthesis, which is triggered by 6 h-light/18 h-dark cycles combined with different levels of CO. The cells do not enter S-phase and consequently do not divide after the minimum light period without CO supplementation, but do divide after a light period with 1 % CO. In this way, we can compare a dividing cycle and a non-dividing cycle. We examined changes in the expression of 74 genes throughout the cell cycle by quantitative RT-PCR. The expression of genes for two cyclins (cyclin C and H) and two CDKs (CDKA and CDKD) as well as metabolic enzymes was promoted by light, whereas the expression of genes for G1/S or G2/M cyclins and CDKs as well as DNA replication enzymes and proteins related to organellar division was promoted only in the dividing cycles. These results suggested that has a checkpoint for G1/S progression, which is regulated by nutrients within the 6 h light period.

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2010-06-01
2019-10-20
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References

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List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

PDF

List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

PDF

List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

PDF

List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

PDF

List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

PDF

List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

PDF

List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

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List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

PDF

List of primers [ PDF] (26 kb) Summary of cell-cycle-related genes in [ PDF] (16 kb) Change in cell size during the synchronous culture. Cell size was estimated by forward scatter (FSC) by flow cytometer in the first and the third cycles. The median FSC at each time point is plotted. [ PDF] (258 kb) Determination of S-phase of nucleus and organelles. DNA quantity was determined by qPCR between the start of the light phase and 7 h after the start of the dark phase in the three cycles. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene (a), or second primer sets of DNA polymerase delta gene, the gene and the gene (b) were used. The primer pairs are listed in Supplementary Table S1. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate (first cycle) or four replicates of DNA preparation (second and third cycles). [ PDF] (296 kb) Standard curve of quantitative RT-PCR. The graphs were obtained using the primer sets for 18S rRNA. (a) A series of 1:10 dilutions in template DNA with three runs each of qPCR. The horizontal line indicates a threshold used in graph (b). Delta Rn: the magnitude of the signal. (b) Number of cycles (Ct) to obtain the threshold level with various dilutions. Copy number is shown in arbitrary units. [ PDF] (245 kb) SOM-based classification of gene expression profiles into nine classes. Sixty-six genes were classified into nine classes. Eight genes with small changes in gene expression throughout the cell cycle are shown at the bottom of the figure. [ PDF] (327 kb) Analysis of gene expression by hierarchical clustering. The expression datasets of Cluster 1 (a) and 2 (b) were respectively clustered in a tree structure by hierarchical clustering using Cluster version 3.0 software. [ PDF] (275 kb) Effect of circadian rhythm on the timing of cell division. Protocols of synchronous culture by light-dark cycles (a), and mitotic index in the two regimes (b). Grey and white regions in the panel indicate dark and light periods, respectively. [ PDF] (246 kb) Light or tetrapyrrole does not initiate DNA replication. These are reproducibility test for the experiments of Kobayashi (2009). (a) Cells were cultured in 2x Allen's medium with bubbling with ordinary air at 42 °C, and then subjected to synchronization according to the protocol of Kobayashi et al. (2009), namely, subcultured cells were grown to a density of OD 10, and diluted in fresh medium to a density of OD 0.4. They were then subjected to two 18 h-dark:6 h-light cycles. In the light period, the cells were irradiated by light provided by three 20 W krypton bulbs (150 µE m s ). Experiments were initiated at the end of the second dark period. DNA was purified as described in Methods, but not by the method described in Kobayashi (2009), and DNA quantity was determined by qPCR. For quantitative determination of nuclear DNA (Nuc), plastid DNA (Pt) and mitochondrial DNA (Mt), primer sets of the 18S rDNA, the gene and the gene were used. Grey and white regions in the panel indicate dark and light periods, respectively. Each value is a mean±SD of triplicate of DNA preparations. (b) The effect of protoporphyrin IX as a proposed signal for nuclear genome replication. Protoporphyrin IX was added to the culture from the beginning of the first cycle or the third cycle in the dark. DNA content was determined as described above. [ PDF] (256 kb) Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells. , 803-807.

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