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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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Labeling of Drosophila larval neuroblast chromosomes for early S replication with IdU (red) and late S replication with CldU (green) shows that deletion of centric heterochromatin shifts centromere replication to mid S. (A) Dpγ238 (1.3 Mb) was completely labeled with CldU. No IdU staining was ever observed on this minichromosome. (B) DpJ21A contains a partially deleted centromere that is replicated primarily in mid S phase. In most cells, DpJ21A was unlabeled for either IdU or CldU (B), suggesting that this minichromosome, including the centromere, replicates primarily in mid S phase. (C) In 20% of metaphases, DpJ21A stained with anti-CldU (late S), suggesting it occasionally replicated at the mid to late S transition. Gray-scale images are shown of each DAPI-stained minichromosome. Bar units are in μm.
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fig4: Labeling of Drosophila larval neuroblast chromosomes for early S replication with IdU (red) and late S replication with CldU (green) shows that deletion of centric heterochromatin shifts centromere replication to mid S. (A) Dpγ238 (1.3 Mb) was completely labeled with CldU. No IdU staining was ever observed on this minichromosome. (B) DpJ21A contains a partially deleted centromere that is replicated primarily in mid S phase. In most cells, DpJ21A was unlabeled for either IdU or CldU (B), suggesting that this minichromosome, including the centromere, replicates primarily in mid S phase. (C) In 20% of metaphases, DpJ21A stained with anti-CldU (late S), suggesting it occasionally replicated at the mid to late S transition. Gray-scale images are shown of each DAPI-stained minichromosome. Bar units are in μm.

Mentions: To address whether minichromosomes were replicated throughout S phase or only in a portion of S, neuroblasts were double labeled with IdU and CldU (diagrammed in Fig. 2 A). In these experiments, Dp8-23, Dpγ238, Dpγ1230, and Dp10B were entirely late replicating. For example, Dpγ238 was completely and exclusively labeled by CldU, the late S label (Fig. 4 A). Therefore, these experiments corroborated that centromeres of Dp minichromosomes, even in the absence of flanking heterochromatin, are replicated late along with the endogenous X centromere and the other endogenous centromeres. Double labeling experiments ruled out the possibility that centromeres initiated replication in early S and continued throughout S phase.


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

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

Labeling of Drosophila larval neuroblast chromosomes for early S replication with IdU (red) and late S replication with CldU (green) shows that deletion of centric heterochromatin shifts centromere replication to mid S. (A) Dpγ238 (1.3 Mb) was completely labeled with CldU. No IdU staining was ever observed on this minichromosome. (B) DpJ21A contains a partially deleted centromere that is replicated primarily in mid S phase. In most cells, DpJ21A was unlabeled for either IdU or CldU (B), suggesting that this minichromosome, including the centromere, replicates primarily in mid S phase. (C) In 20% of metaphases, DpJ21A stained with anti-CldU (late S), suggesting it occasionally replicated at the mid to late S transition. Gray-scale images are shown of each DAPI-stained minichromosome. Bar units are in μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196454&req=5

fig4: Labeling of Drosophila larval neuroblast chromosomes for early S replication with IdU (red) and late S replication with CldU (green) shows that deletion of centric heterochromatin shifts centromere replication to mid S. (A) Dpγ238 (1.3 Mb) was completely labeled with CldU. No IdU staining was ever observed on this minichromosome. (B) DpJ21A contains a partially deleted centromere that is replicated primarily in mid S phase. In most cells, DpJ21A was unlabeled for either IdU or CldU (B), suggesting that this minichromosome, including the centromere, replicates primarily in mid S phase. (C) In 20% of metaphases, DpJ21A stained with anti-CldU (late S), suggesting it occasionally replicated at the mid to late S transition. Gray-scale images are shown of each DAPI-stained minichromosome. Bar units are in μm.
Mentions: To address whether minichromosomes were replicated throughout S phase or only in a portion of S, neuroblasts were double labeled with IdU and CldU (diagrammed in Fig. 2 A). In these experiments, Dp8-23, Dpγ238, Dpγ1230, and Dp10B were entirely late replicating. For example, Dpγ238 was completely and exclusively labeled by CldU, the late S label (Fig. 4 A). Therefore, these experiments corroborated that centromeres of Dp minichromosomes, even in the absence of flanking heterochromatin, are replicated late along with the endogenous X centromere and the other endogenous centromeres. Double labeling experiments ruled out the possibility that centromeres initiated replication in early S and continued throughout S phase.

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