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Centromere identity in Drosophila is not determined in vivo by replication timing.

Sullivan B, Karpen G - J. Cell Biol. (2001)

Bottom Line: Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase.We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase.These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

ABSTRACT
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

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Drosophila centromeres are replicated primarily in mid to late S phase in vitro in tissue culture cells. (A) Replication labeling strategies used for unsynchronized Drosophila cultured cells. Kc and S2 cells were continuously incubated with BrdU to traverse S and G2 phases. Metaphase arrest served as an anchor point to determine where in S phase replication labeling occurred based on length of exposure to BrdU. In double labeling experiments of interphase nuclei, IdU (early S) was administered for 2 h followed by a chase period of 6 h and then CldU (late S) for 2 h. (B) Kc cells incubated with BrdU for 9 h incorporated label at regions replicated during the last 3 h of S phase. Anti-CID antibodies (red) marked centromeres. The 4th chromosome (arrow), including the centromere, and the centromeres of the X and 3rd chromosomes (arrows) were stained with BrdU. The pericentric region of the 2nd chromosome (arrowhead) adjacent to CID staining was labeled at this time, although the centromere was not. (C) Double labeling of Kc and S2 interphase nuclei with IdU (blue) and CldU (green). Most centromeres (red) did not localize with IdU or CldU, suggesting mid S replication. Merged projections of single optical sections are shown (z = 0.1 μm). Bar units are microns.
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fig1: Drosophila centromeres are replicated primarily in mid to late S phase in vitro in tissue culture cells. (A) Replication labeling strategies used for unsynchronized Drosophila cultured cells. Kc and S2 cells were continuously incubated with BrdU to traverse S and G2 phases. Metaphase arrest served as an anchor point to determine where in S phase replication labeling occurred based on length of exposure to BrdU. In double labeling experiments of interphase nuclei, IdU (early S) was administered for 2 h followed by a chase period of 6 h and then CldU (late S) for 2 h. (B) Kc cells incubated with BrdU for 9 h incorporated label at regions replicated during the last 3 h of S phase. Anti-CID antibodies (red) marked centromeres. The 4th chromosome (arrow), including the centromere, and the centromeres of the X and 3rd chromosomes (arrows) were stained with BrdU. The pericentric region of the 2nd chromosome (arrowhead) adjacent to CID staining was labeled at this time, although the centromere was not. (C) Double labeling of Kc and S2 interphase nuclei with IdU (blue) and CldU (green). Most centromeres (red) did not localize with IdU or CldU, suggesting mid S replication. Merged projections of single optical sections are shown (z = 0.1 μm). Bar units are microns.

Mentions: Centromere replication was visualized cytologically by correlating thymidine analogue incorporation with CID antibody staining. For single labeling, described schematically in Fig. 1 A, Drosophila S2 and Kc tissue culture cells were treated with BrdU for increasing intervals to span S phase and then were blocked in metaphase to regressively determine when labeled sites had replicated. All chromosomes stained equally with CID antibodies, suggesting that the inherent aneuploidy of these tissue culture cells was not likely due to defective kinetochores but perhaps to spindle defects such as multipolar spindles (M. Blower, personal communication). Kc cells contained 10–22 chromosomes and 5–11 CID-staining regions in nuclei, since homologues are paired in Drosophila nuclei. After 9 h of labeling, the entire dot-like 4th chromosomes, including the centromeres, were stained with BrdU (Fig. 1 B). CID and BrdU colocalization was also observed on the metacentric third chromosomes and the acrocentric X chromosomes. The chromosome 2 centromere was not replicated in very late S phase, since CID staining at this time did not colocalize with BrdU staining (Fig. 1 B).


Centromere identity in Drosophila is not determined in vivo by replication timing.

Sullivan B, Karpen G - J. Cell Biol. (2001)

Drosophila centromeres are replicated primarily in mid to late S phase in vitro in tissue culture cells. (A) Replication labeling strategies used for unsynchronized Drosophila cultured cells. Kc and S2 cells were continuously incubated with BrdU to traverse S and G2 phases. Metaphase arrest served as an anchor point to determine where in S phase replication labeling occurred based on length of exposure to BrdU. In double labeling experiments of interphase nuclei, IdU (early S) was administered for 2 h followed by a chase period of 6 h and then CldU (late S) for 2 h. (B) Kc cells incubated with BrdU for 9 h incorporated label at regions replicated during the last 3 h of S phase. Anti-CID antibodies (red) marked centromeres. The 4th chromosome (arrow), including the centromere, and the centromeres of the X and 3rd chromosomes (arrows) were stained with BrdU. The pericentric region of the 2nd chromosome (arrowhead) adjacent to CID staining was labeled at this time, although the centromere was not. (C) Double labeling of Kc and S2 interphase nuclei with IdU (blue) and CldU (green). Most centromeres (red) did not localize with IdU or CldU, suggesting mid S replication. Merged projections of single optical sections are shown (z = 0.1 μm). Bar units are microns.
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Related In: Results  -  Collection

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fig1: Drosophila centromeres are replicated primarily in mid to late S phase in vitro in tissue culture cells. (A) Replication labeling strategies used for unsynchronized Drosophila cultured cells. Kc and S2 cells were continuously incubated with BrdU to traverse S and G2 phases. Metaphase arrest served as an anchor point to determine where in S phase replication labeling occurred based on length of exposure to BrdU. In double labeling experiments of interphase nuclei, IdU (early S) was administered for 2 h followed by a chase period of 6 h and then CldU (late S) for 2 h. (B) Kc cells incubated with BrdU for 9 h incorporated label at regions replicated during the last 3 h of S phase. Anti-CID antibodies (red) marked centromeres. The 4th chromosome (arrow), including the centromere, and the centromeres of the X and 3rd chromosomes (arrows) were stained with BrdU. The pericentric region of the 2nd chromosome (arrowhead) adjacent to CID staining was labeled at this time, although the centromere was not. (C) Double labeling of Kc and S2 interphase nuclei with IdU (blue) and CldU (green). Most centromeres (red) did not localize with IdU or CldU, suggesting mid S replication. Merged projections of single optical sections are shown (z = 0.1 μm). Bar units are microns.
Mentions: Centromere replication was visualized cytologically by correlating thymidine analogue incorporation with CID antibody staining. For single labeling, described schematically in Fig. 1 A, Drosophila S2 and Kc tissue culture cells were treated with BrdU for increasing intervals to span S phase and then were blocked in metaphase to regressively determine when labeled sites had replicated. All chromosomes stained equally with CID antibodies, suggesting that the inherent aneuploidy of these tissue culture cells was not likely due to defective kinetochores but perhaps to spindle defects such as multipolar spindles (M. Blower, personal communication). Kc cells contained 10–22 chromosomes and 5–11 CID-staining regions in nuclei, since homologues are paired in Drosophila nuclei. After 9 h of labeling, the entire dot-like 4th chromosomes, including the centromeres, were stained with BrdU (Fig. 1 B). CID and BrdU colocalization was also observed on the metacentric third chromosomes and the acrocentric X chromosomes. The chromosome 2 centromere was not replicated in very late S phase, since CID staining at this time did not colocalize with BrdU staining (Fig. 1 B).

Bottom Line: Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase.We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase.These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

ABSTRACT
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.

Show MeSH
Related in: MedlinePlus