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Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei.

Bass HW, Hoffman GG, Lee TJ, Wear EE, Joseph SR, Allen GC, Hanley-Bowdoin L, Thompson WF - Plant Mol. Biol. (2015)

Bottom Line: We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals.Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei.These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, 319 Stadium Drive, King Life Sciences Building, Tallahassee, FL, 32306-4295, USA. bass@bio.fsu.edu.

ABSTRACT
Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.

No MeSH data available.


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Cytology of DNA replication in endocycling root tip nuclei. Nuclei were prepared as illustrated in Fig. 1 from the 1 to 3 mm section of pulse-labeled roots and subjected to 3D deconvolution microscopy and image display as described for Fig. 2. Two representative examples are shown for each of three sequential sub-stages of endocycling S phase; EARLY endo-S (a–h), MIDDLE endo-S (i–p), and LATE endo-S (q–x). The location of knobs (k) and nucleoli (n) are indicated. Zoomed sections illustrate replication around, but not within, knobs in early endo-S (c/d) and middle endo-S (o/p), overlapping signals of DAPI and A-488 in middle endo-S bulk chromatin (k/l), detection of A-488 within the interior of the nucleolus at early endo-S (g/h), and bright patchy heterochromatin labeling by A-488 at late endo S (s/t, w/x). All scale bars represent 5 µ
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Fig5: Cytology of DNA replication in endocycling root tip nuclei. Nuclei were prepared as illustrated in Fig. 1 from the 1 to 3 mm section of pulse-labeled roots and subjected to 3D deconvolution microscopy and image display as described for Fig. 2. Two representative examples are shown for each of three sequential sub-stages of endocycling S phase; EARLY endo-S (a–h), MIDDLE endo-S (i–p), and LATE endo-S (q–x). The location of knobs (k) and nucleoli (n) are indicated. Zoomed sections illustrate replication around, but not within, knobs in early endo-S (c/d) and middle endo-S (o/p), overlapping signals of DAPI and A-488 in middle endo-S bulk chromatin (k/l), detection of A-488 within the interior of the nucleolus at early endo-S (g/h), and bright patchy heterochromatin labeling by A-488 at late endo S (s/t, w/x). All scale bars represent 5 µ

Mentions: Among the developmental fates of cells in the maize root tip is endoreduplication, as seen by flow cytometric analysis profiling (Fig. 1c; middle panel). We used the developing root tip system to compare the cytological S phase patterns of mitotic versus endocycling cells. Nuclei from the endocycling population were subjected to 3D deconvolution microscopy and image analysis as described above, with representative nuclei shown in Fig. 5. As expected, the volumes of endocycling nuclei were greater than mitotic nuclei and continued to increase (Table 1) during progression through the endocycle S phase. Observed here but not previously reported, the overall spatiotemporal pattern of DNA replication in nuclei from endocycling cells was remarkably similar at the cytological level to that for cells in the mitotic cycle, including the “red + green” pattern of early endo S (Fig. 5c, g) and the “yellow” pattern of middle endo S (Fig. 5k, o).Fig. 5


Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei.

Bass HW, Hoffman GG, Lee TJ, Wear EE, Joseph SR, Allen GC, Hanley-Bowdoin L, Thompson WF - Plant Mol. Biol. (2015)

Cytology of DNA replication in endocycling root tip nuclei. Nuclei were prepared as illustrated in Fig. 1 from the 1 to 3 mm section of pulse-labeled roots and subjected to 3D deconvolution microscopy and image display as described for Fig. 2. Two representative examples are shown for each of three sequential sub-stages of endocycling S phase; EARLY endo-S (a–h), MIDDLE endo-S (i–p), and LATE endo-S (q–x). The location of knobs (k) and nucleoli (n) are indicated. Zoomed sections illustrate replication around, but not within, knobs in early endo-S (c/d) and middle endo-S (o/p), overlapping signals of DAPI and A-488 in middle endo-S bulk chromatin (k/l), detection of A-488 within the interior of the nucleolus at early endo-S (g/h), and bright patchy heterochromatin labeling by A-488 at late endo S (s/t, w/x). All scale bars represent 5 µ
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4631726&req=5

Fig5: Cytology of DNA replication in endocycling root tip nuclei. Nuclei were prepared as illustrated in Fig. 1 from the 1 to 3 mm section of pulse-labeled roots and subjected to 3D deconvolution microscopy and image display as described for Fig. 2. Two representative examples are shown for each of three sequential sub-stages of endocycling S phase; EARLY endo-S (a–h), MIDDLE endo-S (i–p), and LATE endo-S (q–x). The location of knobs (k) and nucleoli (n) are indicated. Zoomed sections illustrate replication around, but not within, knobs in early endo-S (c/d) and middle endo-S (o/p), overlapping signals of DAPI and A-488 in middle endo-S bulk chromatin (k/l), detection of A-488 within the interior of the nucleolus at early endo-S (g/h), and bright patchy heterochromatin labeling by A-488 at late endo S (s/t, w/x). All scale bars represent 5 µ
Mentions: Among the developmental fates of cells in the maize root tip is endoreduplication, as seen by flow cytometric analysis profiling (Fig. 1c; middle panel). We used the developing root tip system to compare the cytological S phase patterns of mitotic versus endocycling cells. Nuclei from the endocycling population were subjected to 3D deconvolution microscopy and image analysis as described above, with representative nuclei shown in Fig. 5. As expected, the volumes of endocycling nuclei were greater than mitotic nuclei and continued to increase (Table 1) during progression through the endocycle S phase. Observed here but not previously reported, the overall spatiotemporal pattern of DNA replication in nuclei from endocycling cells was remarkably similar at the cytological level to that for cells in the mitotic cycle, including the “red + green” pattern of early endo S (Fig. 5c, g) and the “yellow” pattern of middle endo S (Fig. 5k, o).Fig. 5

Bottom Line: We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals.Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei.These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, 319 Stadium Drive, King Life Sciences Building, Tallahassee, FL, 32306-4295, USA. bass@bio.fsu.edu.

ABSTRACT
Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.

No MeSH data available.


Related in: MedlinePlus