Drosophila nuclei replicate in Xenopus eggs

Development ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 183-194
Author(s):  
Christina Smith ◽  
David Brill ◽  
Mary Bownes ◽  
Christopher Ford
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Replication ◽  
Xenopus Laevis ◽  
Dna Synthesis ◽  
Cell Line ◽  
S Phase ◽  
Caesium Chloride ◽  
Permanent Cell ◽  
Xenopus Egg ◽  
Invertebrate Organism

Nuclei isolated from a permanent cell line derived from Drosophila melanogaster embryos have been injected, along with a radioactive DNA precursor [3H] TTP, into Xenopus laevis eggs. In culture, less than 7 % of the cells were in S phase. After a 90 min incubation, following injection into eggs, 99% of the nuclei were shown by autoradiography to have synthesized DNA. In a similar experiment, a density label BrdUTP was injected into eggs along with the nuclei. Subsequent analysis on caesium chloride gradients showed that this DNA synthesis was semi-conservative replication. Therefore we conclude that signals present in Xenopus egg cytoplasm can initiate and sustain true semi-conservative DNA replication in nuclei from an invertebrate organism.

scholarly journals Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

1995 ◽  
Vol 15 (6) ◽  
pp. 2942-2954 ◽  
Author(s):  
D M Gilbert ◽  
H Miyazawa ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dihydrofolate Reductase ◽  
Nuclear Membrane ◽  
S Phase ◽  
G1 Phase ◽  
Site Specific ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.

scholarly journals Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.

1993 ◽  
Vol 123 (6) ◽  
pp. 1321-1331 ◽  
Author(s):  
Y Kubota ◽  
H Takisawa
Keyword(s):  
Dna Replication ◽  
Specific Activity ◽  
S Phase ◽  
Egg Cell ◽  
Sperm Dna ◽  
Before And After ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.

scholarly journals LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

1966 ◽  
Vol 31 (3) ◽  
pp. 577-583 ◽  
Author(s):  
J. E. Cummins ◽  
H. P. Rusch
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Nuclear Dna ◽  
S Phase ◽  
Early Prophase ◽  
Late Prophase ◽  
Removal Experiments ◽  
Inhibitor Of Protein Synthesis

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.

scholarly journals MAMMALIAN CHROMOSOMES IN VITRO

1964 ◽  
Vol 23 (1) ◽  
pp. 53-62 ◽  
Author(s):  
T. C. Hsu
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Sex Chromosomes ◽  
Chinese Hamster ◽  
Distal Portion ◽  
S Phase ◽  
Chromosome 1 ◽  
Chromosome 6 ◽  
Chromosome 2

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H3-thymidine labeling and the method of 5-fluorodeoxyuridine block with H3-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.

scholarly journals Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

1982 ◽  
Vol 2 (10) ◽  
pp. 1295-1298 ◽  
Author(s):  
B F Cheetham ◽  
D C Shaw ◽  
A J Bellett
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Ornithine Decarboxylase ◽  
Adenovirus Type ◽  
S Phase ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Cellular Dna ◽  
Adenovirus Type 5

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation.

scholarly journals Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

scholarly journals Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

2008 ◽  
Vol 82 (18) ◽  
pp. 9056-9064 ◽  
Author(s):  
Sally Roberts ◽  
Sarah R. Kingsbury ◽  
Kai Stoeber ◽  
Gillian L. Knight ◽  
Phillip H. Gallimore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Cellular Dna ◽  
In Cells

ABSTRACT Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

scholarly journals REGULATION OF DNA REPLICATION IN THE NUCLEI OF THE SLIME MOLD PHYSARUM POLYCEPHALUM

1968 ◽  
Vol 37 (3) ◽  
pp. 761-772 ◽  
Author(s):  
Sophie Guttes ◽  
Edmund Guttes
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Slime Mold ◽  
S Phase ◽  
G2 Phase

Nuclei in G2 phase of the slime mold Physarum polycephalum, when transplanted, by plasmodial coalescence, into an S-phase plasmodium, failed to start another round of DNA synthesis. In the reciprocal combination, S-phase nuclei in a G2-phase host continued DNA synthesis for several hours without appreciable decrease in rate. It is suggested that the beginning of DNA replication is determined by an event, either during or shortly after mitosis, which renders the chromosomes structurally competent for DNA replication.

scholarly journals Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

2021 ◽  
Author(s):  
Cory Haluska ◽  
Fengzhi Jin ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Protein Phosphatase ◽  
Budding Yeast ◽  
Replication Stress ◽  
S Phase ◽  
Viability Loss ◽  
Phosphatase 2A ◽  
Dna Replication Stress

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and Protein Phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We showed that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1 that encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK ( cdc28F19) also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

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