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Results for “Replication Timing”

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PubMed1986

5-Azacytidine-induced conversion to cadmium resistance correlates with early S phase replication of inactive metallothionein genes in synchronized CHO cells.

Previous studies have shown both hypermethylation and late replication of DNA sequences to be associated with gene inactivity. To determine whether there is a causal relationship between patterns of DNA methylation and replication timing during S phase, we have examined the timing of replication of the inactive, hypermethylated metallothionein (MT) I and II genes in synchronized, cadmium-sensitive (Cds) CHO cells. The time of S-phase replication of the MT genes was ascertained by determining the period of S phase wherein cadmium-resistant (Cdr) cells could be induced with highest frequency by pulse treatment of synchronized Cds cells with the hypomethylating drug 5-azacytidine (5-aza-CR), and by analyzing Southern blots of density fractionated DNAs isolated from synchronized cells pulse-labeled with BrdU during different intervals after release from hydroxyurea blockade. Southern filter hybridization analyses demonstrated replication of both MTI and II gene sequences within the first half of S phase. Consistent with this result, phenotypic conversion of Cds to Cdr was maximal immediately after hydroxyurea release and decreased abruptly within three hours. The replication of inactive hypermethylated MT genes in early S phase argues that transcriptional inactivity and gene-specific hypermethylation are not sufficient conditions for late DNA replication.

Animals↗
PubMed1975

Retardation time measurementson replicating bacillus subtilis chromosomes: effect of EDTA concentration.

We have found that high concentrations of EDTA (greater than 0.024 M) are necessary to produce large, constant numbers of intact replicating Bacillus subtilis chromosomes in lysates of log phase cells. The retardation time of replicating chromosomes in log phase cell lysates is about double that for chromosomes in stationary phase cell lysates, thus making measurement of retardation time a sensitive way to detect and study replicating chromosomes. A theory is developed to predict retardation times for many possible models of DNA replication. The retardation time data on log phase cells is sufficient to eliminate many replication models, but many possibilities remain.

Bacillus subtilis↗
PubMed1985

DNA hypomethylation causes an increase in DNase-I sensitivity and an advance in the time of replication of the entire inactive X chromosome.

We have examined the effect of 5-azacytidine (5-aza-C) induced hypomethylation of DNA on the time of replication and DNase I sensitivity of the X chromosomes of female Gerbillus gerbillus (rodent) lung fibroblast cells. Using in situ nick translation to visualise the potential state of activity of large regions of metaphase chromosomes we show that 5-aza-C causes a dramatic increase in the DNase-I sensitivity of the entire inactive X chromosome of female G. gerbillus cells and this increase in nuclease sensitivity correlates with a large shift in the time of replication of the inactive X chromosome from late S phase to early S phase. These effects of 5-aza-C on the inactive X chromosome are associated with a 15% decrease in DNA methylation. Our results indicate that DNA methylation concomitantly affects both the time of replication and the chromatin conformation of the inactive X chromosome.

Animals↗
PubMed1992

A position effect on the time of replication origin activation in yeast.

The chromosomes of eukaryotes are characterized by the mosaic nature of their replication--large regions of DNA that replicate early in S phase are interspersed with regions that replicate late. This pattern of early and late synthesis appears to be the consequence of a temporal program that activates replication origins at different times. The basis of this temporal regulation in the yeast S. cerevisiae has been investigated by changing the chromosomal locations of two origins, one activated early in the S phase (ARS1) and one activated late (ARS501). We show that the cis-acting information controlling time of activation can be separated from the element that determines origin function. For the ARS501 origin, late activation appears to be a consequence of its proximity to the telomere.

Chromosomes, Fungal↗
PubMed1984

Changes in gene position are accompanied by a change in time of replication.

The globin and immunoglobulin multigene families have been used to study the effect of chromosomal organization on the time of gene replication. Some of the genes are late-replicating, providing the first identification of late-replicating sequences that are not highly repetitive. One is a member of the mouse alpha-globin gene family, which consists of genes mapping to three different chromosomes. The other genes in this family replicate early during S. Our studies demonstrate that immunoglobulin gene rearrangements and rearrangements between these genes and the c-myc oncogene are accompanied by dramatic differences in their temporal order of replication. We conclude that a gene's position in the chromosome, rather than its sequence, determines the time of replication. We suggest that the differences in association with gene rearrangement result from changes in the proximity of the affected gene to sites that control the temporal order of replication during S.

Animals↗
PubMedDate not supplied

Decreased cultured endothelial cell proliferation in high glucose medium is reversed by antioxidants: new insights on the pathophysiological mechanisms of diabetic vascular complications.

Exposure to hyperglycemia slows the rate of proliferation of cultured human endothelial cells. Recently, it has been reported that glucose may autoxidize generating free radicals which have been hypothesized to delay cell replication time. To test whether oxidative stress has an effect on delaying cell replication time in hyperglycemic conditions, human endothelial cells cultured from umbilical veins were incubated in 5 or 20 mM glucose, either alone or in the presence of one of three different antioxidants: superoxide dismutase (SOD), catalase and glutathione (GSH). Cells grown in medium with 5 mM glucose, with or without antioxidants, yielded similar population doubling times and cell cycle phase distributions. Significantly lower growth parameters were observed in cells grown in medium with 20 mM glucose, without antioxidants. The presence of the antioxidant reverted them to almost normal growth. These data show that high glucose levels may delay endothelial cells replication time through the generation of free radicals, suggesting a possible pathophysiological linkage between the high levels of glucose and the development of microvascular complications of diabetes, possibly suggesting a new therapeutic approach to prevent such complications.

Antioxidants↗
PubMed1976

Sequence of DNA replication in 277 R- and Q-bands of human chromosomes using a BrdU treatment.

Replication times for all important chromosome bands, of both types R and Q (277 structures) are analysed. The R-bands form a group of structures whose DNA replicates during the early S-phase, while the DNA situated in the Q-bands replicates during the late S-phase. There may not exist overlapping between replication times of these two types of structures. The widest R-bands are those which are the earliest to replicate; in general, the most intense Q-bands are those which are the latest to replicate. Especially among these last ones, a certain asynchronism exists between the replication times. Finally the heterochromatin of chromosomes 1, 16 and Y and of the short arms of the acrocentrics could contain two types of DNA which replicate at different times.

Bromodeoxyuridine↗
PubMed1983

High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells.

A new method, utilizing selective photodegradation of 5-bromo-deoxyuridine (BUdR)-substituted DNA and flow cytometry, has been developed for analyzing the timing of replication of specific DNA sequences. Chemically synchronized Chinese hamster ovary cells were given a pulse of the deoxythymidine analogue, BUdR, at different times during S phase, and flow sorted according to DNA content, before DNA isolation. Newly-replicated, unifilarly BUdR-substituted DNA was selectively degraded by treatment with 33258 Hoechst plus near UV light followed by S1 nuclease digestion; the resistant DNA was analyzed for its content of 18s and 28s rDNA or dihydrofolate reductase (DHFR) sequences via Southern blot analysis. Both the rDNA and DHFR sequences were found to replicate almost entirely during the first quarter of S phase. The approach described should have general utility for analyzing replication kinetics of specific DNA sequences in mammalian cells.

Animals↗
PubMed1992

Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy.

The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.

Animals↗
PubMed1990

From gene to chromosome: organization levels defined by the interplay of transcription and replication in vertebrates.

In higher eukaryotes, gene activation is accompanied by an increased sensitivity to DNaseI over a domain that extends beyond the limits of the gene itself, or of the gene cluster to which it belongs. This increased sensitivity probably reflects both the partial decondensation of chromatin and an increased communication with the outside of the nucleus. In addition, gene activation usually causes a coreplication domain that extends much beyond the decondensation domain to switch to an early replication time in S phase. This switch is produced, at least in some cases, by an early firing of origins of replication situated in flanking condensed chromatin. Some of the recently identified DNA domains that tether chromosomal loops to the nuclear matrix do represent the borders of decondensation domains. They may also constitute pausing sites for replication forks. The different replication times of successive 200- to 400-kb regions along the genome may have been the basis for the observed long-term differentiation of very large genomes in domains of different overall sequence composition (G:C content and distribution of short repeated motifs). Chromosomal bands represent a low resolution picture of this pattern. Just like gene methylation, differential replication timing and the consequent compositional differentiation of the genome have probably contributed to making the management of very large genomes workable.

Animals↗
PubMed1991

Replication asynchrony between homologs 15q11.2: cytogenetic evidence for genomic imprinting.

Replication kinetics of the Prader-Willi syndrome critical region (15q11.2) was investigated in seven normal healthy adult females using RBG replication bands. Replication asynchrony between homologs 15q11.2 was identified consistently in about 40% of cells in all individuals. It was limited to the stages in which Xp22, Xp11, Xq13 and Xq24/26 were visible in the late-replicating X chromosome. This asynchrony suggested that replication timing overlapped between 15q11.2 and the early replicating R-bands of the late X chromosome in some cells, and that the difference in replication timing between homologs was probably related to genomic imprinting; the latter has been suggested as a pathogenetic basis of Prader-Willi syndrome. As a result of an analysis of the proportions of asynchronous and synchronous cells in each replication stage, two types of cells were deduced providing 1:1 methylation mosaicism of genomic imprinting was assumed. The first type was composed of cells with normal replication in one homolog and delayed replication in the other. The second type was composed of cells with normal replication in both homologs. Our results provide cytogenetic evidence of methylation mosaicism for mammalian genomic imprinting.

Cell Cycle↗
PubMed1990

Sequence of DNA replication in Macaca fuscata chromosomes: an outgroup for phylogenetic comparison between man and apes.

The relative replication times of every band in the standardized 300 band G-band idiogram of the chromosomes of the Japanese macaque are presented, and compared to the human sequence. Many chromosomes thought to be homologous between Macaca fuscata and man on the basis of standard chromosome banding and gene mapping show a conservation of the replication sequence. Other supposed chromosomal homologies between these two species show no good correspondence, and the replication sequence data suggest that these chromosomes have been subject to complex rearrangements. The replication sequence data also point to possible additional chromosomal homologies between man and M. fuscata. Asynchrony in replication time between homologues from the same cell may also be evolutionarily conserved, because these species share a number of asynchronous homologous bands. Replication band sequence data can provide significant information for comparative cytogenetics. However, usually only the full replication R- or G-band pattern has been used for interspecific comparisons. The dynamic sequence data presented here determine the replication time of every band in the karyotype, and provide a quantitatively and qualitatively more sensitive tool to characterize chromosomes. Such data could provide valuable new information on which to make phylogenetic reconstructions, and shed light on the relationship between chromosome change and evolutionary process. Finally, the M. fuscata replication sequence presented here will provide a necessary foundation for future comparisons between apes and man.

Animals↗
PubMed1989

How does inactivation change timing of replication in the human X chromosome?

The kinetics of replication of the inactive (late replicating) X chromosome (LRX) were studied in karyotypically normal lymphocytes and human amniotic fluid cells. Both cell types were successively pulse labeled with 1-h or 1/2-h thymidine pulses in an otherwise BrdU-substituted S phase after partial synchronization of the cultures at G1/S. For the first time with this technique, the entire sequence of replication was analyzed for the LRX from the beginning to the end of the S phase, with special reference to mid S (R-band to G-band transition replication). The inactive X is the last chromosome of the metaphase to start replication, with a delay of 1 or 2 h, after which time a thymidine pulse results in R-type patterns. In mid S, the inactive X is the first chromosome to switch to G-type replication (without overlapping of both types and without any detectable replication pause). Until the end of S, a thymidine pulse results in G-type patterns. To rule out artifacts that might arise by the synchronization of cultures in these experiments, controls were carried out with BrdU pulses and the BrdU antibody technique without synchronization. In the course of replication, no fundamental difference was seen between the two different cell types examined. In contrast to studies using continuous labeling, this study did not reveal an interindividual difference of replication kinetics in the LRXs of the seven individuals studied; thus it is concluded that the inactive X chromosome shows only one characteristic course of replication.

Amniotic Fluid↗
PubMed1972

Relationship of Flac replication and chromosome replication.

The time of replication of a bacterial plasmid, Flac, during the division cycle of Escherichia coli has been estimated in exponentially growing cultures and at various times after a shift from minimal medium to a richer medium (a shift-up). There is a variation in the cell age at which the capacity to synthesize beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) doubles (assumed to be a measure of the time at which the Flac plasmid replicates) when this capacity is measured at various times during the shift-up, and with increasing steady-state exponential growth rate. Cells growing at slow and moderate growth rates exhibit Flac replication in the middle of the division cycle. With increasing time after a shift-up or with increasing growth rate the plasmid replicates at earlier times, eventually at cell division, and finally in the older cells. This variation in the cell age at which the plasmid replicates is similar to the variation in cell age at which chromosome initiation occurs during a shift-up, although plasmid replication occurs slightly before initiation of chromosome replication.

Amino Acids↗
PubMed1988

Analysis of the chromosomal DNA replication pattern using the bromodeoxyuridine labeling method.

The DNA replication of human lymphocytes was studied using the labeling pattern on chromosomes after pulse labeling with bromodeoxyuridine and immunostaining with the monoclonal antibody to bromodeoxyuridine. The sites of bromodeoxyuridine incorporation were defined by comparison with the same karyotype treated G-banding, and a more precise analysis was performed using the image analyzer, Maziscan III. Early phase DNA synthesis roughly coincided with R-bands and late phase DNA synthesis with G-bands. One case of acute myeloid leukemia which developed from myelodysplastic syndrome, had a 46,XY, t(4;11) (p12;p15) karyotype, and revealed an altered DNA replication time at the translocated site, 4p15, from the late to the early phase. The present method was useful in detecting an alteration of DNA replication timing according to chromosomal translocation.

Bromodeoxyuridine↗