scholarly journals The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts

1991 ◽  
Vol 100 (4) ◽  
pp. 869-876 ◽  
Author(s):  
I.R. Kill ◽  
J.M. Bridger ◽  
K.H. Campbell ◽  
G. Maldonado-Codina ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Indirect Immunofluorescence ◽  
Laser Scanning ◽  
S Phase ◽  
Nuclear Antigen ◽  
In Vitro Techniques ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts

The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence microscopy and laser scanning confocal microscopy (LSCM). In common with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly, nuclei which had been isolated from quiescent cultures of HDF were innoculated into cell-free extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated at multiple discrete spots some 15 min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.

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 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 Cell population heterogeneity in the inducibility of DNA synthesis in human diploid fibroblasts.

1981 ◽  
Vol 89 (2) ◽  
pp. 194-197 ◽  
Author(s):  
M V Rao
Keyword(s):  
Dna Synthesis ◽  
Cytochalasin B ◽  
Nuclear Dna ◽  
S Phase ◽  
Population Heterogeneity ◽  
Diploid Fibroblasts ◽  
Binucleate Cells ◽  
Human Diploid Fibroblasts ◽  
Significant Difference ◽  
Sister Cells

The initiation of nuclear DNA synthesis has been studied in cytochalasin B (CB)-induced binucleate human diploid fibroblasts (WI-38 cells). Mitotic cells from different passage levels were rendered binucleate by a brief pulse of CB. The cells were then washed free of the drug, and DNA synthesis was studied by [3H]thymidine labeling. The results showed that, in a small percentage of binucleate cells, one nucleus was labeled (S phase) and the other nucleus was unlabeled (G1 phase). There was no significant difference in the percentage of these cells with increasing passage levels. The results of this study suggest that some WI-38 cells retire from the cell cycle at different passage levels, and thereby become refractory to inducers of nuclear DNA synthesis generated by sister cells in S phase.

scholarly journals INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE

1974 ◽  
Vol 60 (1) ◽  
pp. 249-257 ◽  
Author(s):  
Jeffrey E. Froehlich ◽  
Martin Rachmeler
Keyword(s):  
Cell Growth ◽  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Fresh Medium ◽  
G1 Phase ◽  
Cyclic Monophosphate ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G1 phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N6, O2-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.

scholarly journals Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites.

1987 ◽  
Vol 105 (4) ◽  
pp. 1549-1554 ◽  
Author(s):  
R Bravo ◽  
H Macdonald-Bravo
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Quiescent Cells ◽  
Replication Sites ◽  
Nuclear Structures ◽  
Proliferating Cell ◽  
Two Populations

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.

scholarly journals Cell proliferation and gill morphology in anoxic crucian carp

2005 ◽  
Vol 289 (4) ◽  
pp. R1196-R1201 ◽  
Author(s):  
Jørund Sollid ◽  
Aina Kjernsli ◽  
Paula M. De Angelis ◽  
Åsmund K. Røhr ◽  
Göran E. Nilsson
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Crucian Carp ◽  
Fold Increase ◽  
S Phase ◽  
Nuclear Antigen ◽  
Tyrosyl Radical ◽  
Radical Generation ◽  
Proliferating Cell

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp ( Carassius carassius), by examining 5′-bromo-2′-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G0 cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells (∼1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.

Nuclear distribution of centromeres during the cell cycle of human diploid fibroblasts

1991 ◽  
Vol 99 (2) ◽  
pp. 255-263 ◽  
Author(s):  
M.F. Bartholdi
Keyword(s):  
Cell Cycle ◽  
Nuclear Structure ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
S Phase ◽  
Interphase Nuclei ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Chromosome Domains ◽  
Transcription And Replication

The distribution of centromeres in the interphase nuclei of human diploid fibroblasts was analyzed using anti-centromere immunofluorescence and laser scanning confocal microscopoy. The positions of the centromeres were placed within the nuclear chromatin distribution and presented some aspects of the dynamics of nuclear structure during the cell cycle. During the G1 phase of the cell cycle many of the centromeres were located in association with nucleoli or fused in chromocenters. A few centromeres were dispersed singly in the euchromatin. During S phase, the fused centromeres dispersed, often forming distinct patterns of rings or lines. At prophase, the centromere immunofluorescence condensed into distinct double dots upon the formation of the prophase chromosomes. Quantitative analysis by both image and flow cytometry showed that the intensity of immunofluorescence started to duplicate in mid S phase, well before the appearance of the double dots. The coalesence of the centromeres during G1 indicated that regions of the chromosome domains remain compacted and possibly sequestered from transcriptional activity. During S phase the chromatin and the coalesced centromeres dispersed for DNA replication. The dynamics of the centromeres and chromatin during the cell cycle seen here are evidence for a higher-order organization of nuclear structure that accompanies DNA transcription and replication.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

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