scholarly journals Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition

2017 ◽  
Author(s):  
Charles A Seller ◽  
Patrick H O’Farrell
Keyword(s):  
Drosophila Melanogaster ◽  
Replication Timing ◽  
Embryonic Cell ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Cell Cycles ◽  
Satellite Sequences

In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the Mid-Blastula Transition, MBT. In Drosophila melanogaster embryos, downregulation of Cdk1 activity initiates this slowing by delaying replication of satellite sequences and extending S phase. We found that Cdk1 inhibited the chromatin association of Rif1, a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 downregulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase, and rescued embryonic cell cycles disrupted by depletion of the S phase-promoting kinase, Cdc7. Thus, Drosophila Rif1 mediates the MBT extension of S phase and functionally interacts with S phase promoting kinases to introduce a replication-timing program.

Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 165-171 ◽  
Author(s):  
G. Hime ◽  
R. Saint
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Regulation ◽  
Single Cells ◽  
Embryonic Cell ◽  
Cell Cycles ◽  
Drosophila Embryogenesis ◽  
Zygotic Transcription ◽  
Homozygous Mutant ◽  
Mitotic Figures

Mutations at the pebble locus of Drosophila melanogaster result in embryonic lethality. Examination of homozygous mutant embryos at the end of embryogenesis revealed the presence of fewer and larger cells which contained enlarged nuclei. Characterization of the embryonic cell cycles using DAPI, propidium iodide, anti-tubulin and anti-spectrin staining showed that the first thirteen rapid syncytial nuclear divisions proceeded normally in pebble mutant embryos. Following cellularization, the postblastoderm nuclear divisions occurred (mitoses 14, 15 and 16), but cytokinesis was never observed. Multinucleate cells and duplicate mitotic figures were seen within single cells at the time of the cycle 15 mitoses. We conclude that zygotic expression of the pebble gene is required for cytokinesis following cellularization during Drosophila embryogenesis. We postulate that developmental regulation of zygotic transcription of the pebble gene is a consequence of the transition from syncytial to cellular mitoses during cycle 14 of embryogenesis.

scholarly journals Replication timing of DNA sequences associated with human centromeres and telomeres.

1990 ◽  
Vol 10 (12) ◽  
pp. 6348-6355 ◽  
Author(s):  
K G Ten Hagen ◽  
D M Gilbert ◽  
H F Willard ◽  
S N Cohen
Keyword(s):  
Dna Sequences ◽  
Cell Sorting ◽  
Replication Timing ◽  
S Phase ◽  
Alpha Satellite ◽  
Chromosomal Dna ◽  
Telomeric Sequences ◽  
Alpha Satellite Dna ◽  
Satellite Sequences ◽  
Satellite Repeats

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.

scholarly journals Replication timing of DNA sequences associated with human centromeres and telomeres

1990 ◽  
Vol 10 (12) ◽  
pp. 6348-6355
Author(s):  
K G Ten Hagen ◽  
D M Gilbert ◽  
H F Willard ◽  
S N Cohen
Keyword(s):  
Dna Sequences ◽  
Cell Sorting ◽  
Replication Timing ◽  
S Phase ◽  
Alpha Satellite ◽  
Chromosomal Dna ◽  
Telomeric Sequences ◽  
Alpha Satellite Dna ◽  
Satellite Sequences ◽  
Satellite Repeats

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.

scholarly journals Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle.

1992 ◽  
Vol 3 (6) ◽  
pp. 687-698 ◽  
Author(s):  
D H Walker ◽  
A A DePaoli-Roach ◽  
J L Maller
Keyword(s):  
Dna Synthesis ◽  
Protein Phosphatase ◽  
Specific Inhibitor ◽  
Embryonic Cell ◽  
S Phase ◽  
Multiple Roles ◽  
Protein Phosphatase 1 ◽  
Cell Cycles ◽  
M Phase ◽  
Cytostatic Factor

Using cytostatic factor metaphase II-arrested extracts as a model system, we show that protein phosphatase 1 is regulated during early embryonic cell cycles in Xenopus. Phosphatase 1 activity peaks during interphase and decreases shortly before the onset of mitosis. A second peak of activity appears in mitosis at about the same time that cdc2 becomes active. If extracts are inhibited in S-phase with aphidicolin, then phosphatase 1 activity remains high. The activity of phosphatase 1 appears to determine the timing of exit from S-phase and entry into M-phase; inhibition of phosphatase 1 by the specific inhibitor, inhibitor 2 (Inh-2), causes premature entry into mitosis, whereas exogenously added phosphatase 1 lengthens the interphase period. Analysis of DNA synthesis in extracts treated with Inh-2, but lacking the A- and B-type cyclins, shows that phosphatase 1 is also required for the process of DNA replication. These data indicate that phosphatase 1 is a component of the signaling pathway that ensures that M-phase is not initiated until DNA synthesis is complete.

scholarly journals A Screen for Modifiers of Cyclin E Function in Drosophila melanogaster Identifies Cdk2 Mutations, Revealing the Insignificance of Putative Phosphorylation Sites in Cdk2

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 233-244 ◽  
Author(s):  
Mary Ellen Lane ◽  
Marion Elend ◽  
Doris Heidmann ◽  
Anabel Herr ◽  
Sandra Marzodko ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cyclin E ◽  
S Phase ◽  
Regulatory Subunit ◽  
Phosphorylation Sites ◽  
Induced Mutations ◽  
Complementation Groups ◽  
Higher Eukaryotes ◽  
Eye Imaginal Disc ◽  
Regulatory Significance

Abstract In higher eukaryotes, cyclin E is thought to control the progression from G1 into S phase of the cell cycle by associating as a regulatory subunit with cdk2. To identify genes interacting with cyclin E, we have screened in Drosophila melanogaster for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that we have identified fall into four complementation groups corresponding to the genes split ends, dacapo, dE2F1, and Cdk2(Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed us to address the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19). The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, our results demonstrate that the equivalent sites in Cdk2 play no essential role.

scholarly journals Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

2012 ◽  
Vol 198 (6) ◽  
pp. 973-980 ◽  
Author(s):  
Kai Yuan ◽  
Jeffrey A. Farrell ◽  
Patrick H. O’Farrell
Keyword(s):  
Cell Cycle ◽  
Embryonic Cell ◽  
S Phase ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Mitotic Entry ◽  
G2 Phase ◽  
Embryonic Morphogenesis ◽  
Do So ◽  
S Phase Checkpoint

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila melanogaster embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. We probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. We conclude that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly.

scholarly journals Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 inDrosophila

2018 ◽  
Author(s):  
Charles A. Seller ◽  
Chun-Yi Cho ◽  
Patrick H. O’Farrell
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Repetitive Dna ◽  
Constitutive Heterochromatin ◽  
Experimental Manipulation ◽  
Embryonic Cell ◽  
Chromatin Modifications ◽  
Cell Cycles ◽  
Heterochromatin Formation ◽  
Cycle Times

Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naïve genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/tri methylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the earlyDrosophila melanogasterembryo the heterochromatin lacks these modifications which only appear later when rapid embryonic cell cycles slow down at the Mid-Blastula Transition or MBT. Here we focus on the initial steps restoring heterochromatic modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided proteins in embryos. Using the JabbaTrap we reveal a major requirement for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute minimally. Live-imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase, but then dissociates in mitosis and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. We propose that developmental slowing of the cell cycle times embryonic heterochromatin formation.

scholarly journals PRESENCE OF A BASE LINE LEVEL OF SISTER CHROMATID EXCHANGES IN SOMATIC CELLS OF DROSOPHILA MELANOGASTER IN VIVO AND IN VITRO

Genetics ◽  
1982 ◽  
Vol 100 (2) ◽  
pp. 259-278
Author(s):  
Hideo Tsuji
Keyword(s):  
Drosophila Melanogaster ◽  
Sister Chromatid ◽  
Ganglion Cells ◽  
Sister Chromatid Exchanges ◽  
Base Line ◽  
Cell Cycles ◽  
Chromatid Exchanges ◽  
Third Instar Larvae

ABSTRACT Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2′-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 vg/ml and 0.25-2.5 vg/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979).

scholarly journals Long-Distance Control of Origin Choice and Replication Timing in the Human β-Globin Locus Are Independent of the Locus Control Region

2000 ◽  
Vol 20 (15) ◽  
pp. 5581-5591 ◽  
Author(s):  
Daniel M. Cimbora ◽  
Dirk Schübeler ◽  
Andreas Reik ◽  
Joan Hamilton ◽  
Claire Francastel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Replication Timing ◽  
S Phase ◽  
Chromatin Conformation ◽  
Erythroid Cells ◽  
Long Distance ◽  
Targeted Deletion ◽  
Globin Gene Expression

ABSTRACT DNA replication in the human β-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the β-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5′HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5′HS2-5 in the normal chromosomal context of the human β-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5′HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5′HS2-5 in the classically defined LCR do not control replication in the human β-globin locus. Recent studies also show that targeted deletion of 5′HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

scholarly journals A Maternal Smad Protein Regulates Early Embryonic Apoptosis in Xenopus laevis

2002 ◽  
Vol 22 (5) ◽  
pp. 1317-1328 ◽  
Author(s):  
Yuko Miyanaga ◽  
Ingrid Torregroza ◽  
Todd Evans
Keyword(s):  
Functional Analysis ◽  
Cell Survival ◽  
Caspase 3 ◽  
Embryonic Cell ◽  
Early Embryogenesis ◽  
Cell Cycles ◽  
Smad Proteins ◽  
Smad Protein ◽  
Smad Signaling Pathway ◽  
A Cell

ABSTRACT We identified cDNAs encoding the Xenopus Smad proteins most closely related to mammalian Smad8, and we present a functional analysis of this activity (also referred to recently as xSmad11). Misexpression experiments indicate that xSmad8(11) regulates pathways distinct from those regulated by the closely related xSmad1. Embryos that develop from eggs depleted of xSmad8(11) mRNA fail to gastrulate; instead, at the time of gastrulation, they initiate a widespread program of apoptosis, via a CPP32/caspase 3 pathway. Embryos that avoid this fate display gastrulation defects. Activation of apoptosis is rescued by expression of xSmad8(11) but not xSmad1. Our results demonstrate an embryonic requirement for Smad8(11) activity and show that a maternally derived Smad signaling pathway is required for gastrulation and for mediating a cell survival program during early embryogenesis. We suggest that xSmad8(11) functions as part of a maternally derived mechanism shown previously by others to monitor Xenopus early embryonic cell cycles.

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