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Results for “DNA Replication Timing”

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PubMed1992

Responsiveness of tumorigenic and non-tumorigenic CHEF18 Chinese hamster cells to 1-beta-D-arabinofuranosylcytosine treatment.

In cultured mammalian cells, sister chromatid exchanges are easily induced by agents that perturb the scheduled timing of DNA replication. In this work a blockage of DNA synthesis induced by 1-beta-D-arabinofuranosylcytosine was applied to non-tumorigenic and tumorigenic CHEF18 Chinese hamster cells, and their responsiveness was compared. The data show that both the induction of sister chromatid exchanges and the reduction of the colony-forming ability were less extensive in non-tumorigenic than in tumorigenic CHEF18 cells. The results suggest that a tight control of the scheduled timing of DNA replication is present in non-tumorigenic CHEF18 cells and perhaps this feature avoids the generation of those chromosomal structures that are responsible for the abnormal induction of sister chromatid exchanges and for the elevated cytotoxicity seen in tumorigenic cells.

Animals↗

PubMed1990

The role of dam methyltransferase in the control of DNA replication in E. coli.

The timing and control of initiation of DNA replication in E. coli was studied under conditions where the cellular level of dam methyltransferase was controlled by a temperature-inducible promoter. Flow cytometry was used to demonstrate that the synchrony of initiation at the several origins within each cell was critically dependent on the level of dam methyltransferase. Initiations were shown to be synchronous only in a narrow temperature range. The data are explained by a model where a newly replicated and therefore hemimethylated oriC is inert for reinitiation. Such a model may be applicable to eukaryotic cells, where classes of origins are initiated in synchrony and only once per cell cycle.

DNA Replication↗

PubMed2026

Replication stress links Geminin depletion to centrosome amplification.

The timing of DNA replication and centrosome duplication is tightly regulated with cell cycle progression to ensure the faithful duplication of the genome during cell division. Both DNA and centrosomes are licensed for replication in late telophase/early G1, replicated in S phase and segregated during mitosis; yet how defects in DNA replication licensing are coupled to centrosome homeostasis remains poorly understood. Here, we show that depletion of the replication licensing inhibitor Geminin in proliferating mouse embryonic fibroblasts induces robust centrosome amplification together with impaired primary cilium assembly. Rather than promoting whole-genome reduplication, knockdown of Geminin triggers a replication stress response, characterized by DNA damage accumulation throughout the cycle, and activation of an ATR-dependent DNA damage response. Mechanistically, Geminin depletion-induced replication stress activates the ATR-Chk1-Wee1 checkpoint axis prolonging G2 and leading to premature centriole disengagement and centrosome amplification. These findings identify replication stress as the signaling module that couples defective DNA replication licensing to centrosome amplification.

DNA damage↗

PubMed1986

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv.

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37 degrees C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13-18 times slower than that of E. coli.

Chloramphenicol↗

PubMed1987

A model for the spatio-temporal organization of DNA replication in mammalian cells.

The spatio-temporal organization of chromosomal DNA replication was analyzed using a model based on a "DNA unit" (or decondensation unit) hypothesis. The model is an extension of the fork movement theory of Huberman & Riggs (1968) and can account for a partially deterministic and partially stochastic order of DNA replication in chromosomes. It presumes that each chromosome is composed of DNA units that are arranged in sequence and that are replicated in parallel. A deterministic wave of chromatin decondensation propagates along the DNA unit continuously and progressively providing a field for the random activation of replication origin. Assignment of replication times to DNA compartments by a Monte Carlo method was programmed based on the model and the program was used to stimulate DNA synthesis rate curves that can be measured by the method of Dolbeare et al. (1983, 1985). The shape of the curve is shown to constrain possible parameter values of the model, which include the rate of fork movement, the fraction of chromatin that is decondensed at the start of S-phase, the initial number of origins activated, the rate at which new origins are activated, etc. The chromosomal organization that controls the molecular level of DNA replication is briefly reviewed and its relevance to the model is also discussed.

Algorithms↗

PubMed1988

Intrinsic resistance to viral infection. Mouse macrophage restriction of herpes simplex virus replication.

Macrophages isolated from mice resistant to acute (lethal) infection with a neurovirulent isolate of HSV-1 express intrinsic resistance to viral infection in vitro. Bone marrow (BM), spleen (S), peritoneal (P), and thioglycolate-stimulated peritoneal (Pthio) macrophages isolated from resistant C57BL/6 Cr (B6) mice consistently restrict HSV-1 macromolecular synthesis earlier in the viral replicative cycle than do macrophages isolated from the same tissue sources from more susceptible DBA/2Cr (D2) mice. B6-BM (BM macrophages from B6 mice) restrict HSV macromolecular synthesis at least at two points in the replicative cycle: 1) before the onset of alpha-protein synthesis and 2) between the onset of gamma 1 protein and DNA synthesis. D2-BM macrophages restrict HSV replication at about the time of DNA synthesis. B6-P macrophages restrict HSV replication shortly after gamma 1 protein synthesis, and D2-P macrophages inhibit the virus slightly later, but before DNA synthesis. B6-S macrophages restrict HSV replication at about the time of DNA synthesis, and D2-S macrophages inhibit replication after the onset of gamma 2 protein synthesis. Pthio macrophages are more permissive to HSV infection than BM, P, or S macrophages: restrictions in viral replication occur at the time of DNA synthesis in B6-Pthio macrophages, and after the onset of gamma 2 protein synthesis in D2-Pthio cells. These studies demonstrate that isolated macrophages from inbred mouse strains express intrinsic resistance to HSV infection that correlates with in vivo resistance to acute (lethal) infection. Intrinsic resistance to HSV-1 infection is due to restriction of viral macromolecular synthesis. HSV replication is inhibited in macrophages at multiple points in the viral growth cycle, depending on the tissue from which the cells are isolated.

Acute Disease↗

PubMed1976

[Subsequent mutagenesis under the action of UV rays in a culture of Actinomadura carminata, a producer of carminomycin].

The succession of some markers on the chromosomes of Actinomadura carminata was shown with the method of successive mutagenesis based on the preferable effect of the mutagen on DNA in the site of replication. The spore germination was synchronized by selection of the spores of the definite size and maintenance at a temperature of 0 degrees. The periods of formation of the highest numbers of morphological and auxotrophic mutants were compared with the time of DNA replication.

Actinomycetales↗

PubMed1990

Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei.

We have used immunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order to determine the timing of initiation and the duration of nuclear and kinetoplast S-phases within the procyclic stage of the Trypanosoma brucei cell cycle. Both nuclear and kinetoplast S-phases were shown to be periodic, occupying 0.18 and 0.12 of the unit cell cycle, respectively. In addition, initiation of both of these S-phases were in approximate synchrony, differing by only 0.03 of the unit cell cycle. We have also used a monoclonal antibody that recognises the basal bodies of T. brucei in order to visualise cells possessing a new pro-basal body and hence determine the time of pro-basal body formation within the cell cycle. Pro-basal body formation occurred within a few minutes of the initiation of nuclear S-phase, at 0.41 of the unit cell cycle. This provides detection of the earliest known cell cycle event in T. brucei at the level of the light microscope. Cell cycle events including initiation of nuclear and kinetoplast DNA replication and pro-basal body formation may be strictly coordinated in T. brucei in order to maintain the precise single-mitochondrion (kinetoplast), singleflagellum status of the interphase cell.

Animals↗

PubMed1992

RecA protein of Escherichia coli and chromosome partitioning.

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.

Cell Cycle↗

PubMed1989

Sequence of centromere separation: kinetochore formation and DNA replication in dicentric chromosomes showing premature centromere separation in rat cerebral cells.

A subpopulation of rat cerebral endothelial cells, designated B1, exhibits an array of multicentric chromosomes. Because of the formation of bridges at anaphase, this cell population produced new types of multicentrics at every cell division. These chromosomes showed kinetochore proteins at every centromeric site and all centromeric regions replicated their DNA at the end of the S phase, more or less simultaneously. A new subpopulation of cells, designated B2, obtained from the original sample frozen at Wayne State University displayed several dicentrics. In contrast to B1 these chromosomes exhibit premature centromere separation as reported for mouse and human cell lines. These B2 dicentrics show only one site of kinetochore protein deposition. The timing of DNA replication around the centric region of prematurely separating centromere is also changed similar to the earlier reported premature DNA synthesis for mouse dicentrics. These observations suggest a universality of relationship between premature centromere separation, a lack of kinetochore formation, and early replication of the centric/pericentric DNA associated with these centromeres. The cause of sudden change from activity to inactivity of these chromosomes, though interesting, is not clear.

Animals↗

PubMed1992

Increase of cytokeratin D during liver regeneration: association with the nuclear matrix.

An increase of a 45 kD protein (p45) in the nuclear matrix has been observed when rat liver cells were proliferatively activated in vivo by a partial hepatectomy. The maximal levels of the association of p45 with the nuclear matrix have been detected 24 hr after hepatectomy just at the time when DNA replication is also maximal. By amino acid sequence analysis, immunoblotting and immunocytochemical methods, it has been demonstrated that p45 is identical to rat cytokeratin D. Immunogold staining of nuclear matrix-intermediate filament preparations from cultured hepatocytes indicated that p45 is associated with cytoskeletal filaments that are strongly interconnected to the lamina, whereas no intranuclear localization of the protein has been detected. With an overlay assay a specific binding of labeled p45 to two nonidentified high-molecular weight proteins and also to lamin B has been observed. Northern blot analysis revealed a biphasic pattern of expression of the messenger RNA for cytokeratin D during liver regeneration. A sharp increase in the messenger RNA levels occurred in the prereplicative phase of liver regeneration a few hours before the accumulation of the protein in the nuclear matrix fraction, and a second peak occurred 48 hr after partial hepatectomy.

Amino Acid Sequence↗

PubMed1976

[Feeding rhythms and the diurnal rhythm of cell proliferation in pharmacologically induced liver growth (author's transl)].

Stimulation of hepatic DNA synthesis can be achieved in the intact rat by alpha-hexachlorocyclohexane (alpha-HCH = alpha-benzene hexachloride). The extent of stimulation is high in the morning and low in the evening. These rhythmic variations in the rate of DNA synthesis are synchronized indirectly by the light-dark rhythm, but directly by the animal's feeding habits: Rats eat preferentially during the night. If the diurnal rhythm of food intake is abolished, the rhythmic fluctuations in the rate of DNA synthesis are no longer detectable; if rats are adapted to daily feeding periods of only 5 h, these fluctuations are pronounced and almost synchronized. Further experiments show that the time of feeding determines the time of DNA replication. It is concluded that food intake provides a "2nd stimulus" or permissive factor, which is required for the induction of DNA synthesis in a certain critical stage of the prereplicative phase. Labelling experiments with orotic acid suggest that foot intake initially induces an increase of RNA synthesis. The results indicate that controlled feeding schedules provide the possibility to synchronize, in the living animal, a proliferating population of hepatocytes. A hypothesis is derived which offers an explanation for the generation of the diurnal rhythm of cell proliferation in the liver.

Animals↗

PubMed1985

Cellular and viral DNA hypomethylation associated with induction of Epstein-Barr virus lytic cycle.

Epstein-Barr virus (EBV) producer and nonproducer cell lines have been treated with a combination of phorbol 12-myristate 13-acetate and n-butyrate (sodium salt). These inducers caused a massive hypomethylation of the EBV producer line P3HR-1 DNA (about 30%) at the time when DNA replication was inhibited. The viral DNA in these cells is heavily methylated as judged by digestion with Hpa II and probing with the Bam HI H fragment of EBV. However, upon induction with phorbol 12-myristate 13-acetate and n-butyrate, total hypomethylation of this viral DNA region was observed within 24 hr. This hypomethylation preceded EBV amplification, which became apparent only 32-36 hr after induction. When induction was carried out in the presence of retinoic acid, hypomethylation of cellular and viral DNA, viral DNA amplification, and production of the viral early antigen and viral capsid antigen were substantially inhibited. EBV DNA in another producer line (Jijoye nude) and in the nonproducer line Raji was hypomethylated and did not undergo further hypomethylation in response to induction. The observed hypomethylation of P3HR-1 and EBV DNA in the absence of DNA replication suggests that it is achieved by an active demethylation mechanism. This changes our perception of the DNA methylation phenomenon, since it has been generally accepted that hypomethylation of DNA takes place by a passive mechanism that involves DNA replication in the absence of methylation.

Butyrates↗

PubMed1979

Partial triplication and deletion of 13q: study of a family presenting with bilateral retinoblastomas.

This report compares the pathogenetic influences of selective deletion and triplicaton of chromosome 13 derived from a familial 12;13 insertional translocation. In the proband a heritable chromosomal basis for his bilateral retinoblastomas is established [46,XY,del (13) (pter leads to q12.5: :q22.1 leads to qter)mat], and in his sister the relatively modest effects of triplication of the mid-portions of 13q are demonstrated [46,XX,ins(12;13) (12pter leads to 12p11.2: :13q22.1 leads to 13q12.5: :12p11.2 leads to 12qter)mat]. Qualitative and quantitative gene marker studies and chromosomal staining techniques to differentiate timing of DNA replication failed to indicate functional gene changes about the breakpoints.

Child, Preschool↗

PubMed1979

Altered patterns of ribonucleic acid synthesis during the cell cycle: a mechanism compensating for variation in gene concentration.

In the fission yeast Schizosaccharomyces pombe, a series of diploid mutants divides at smaller cell sizes than wild type. In these smaller strains, the mean gene concentration (defined by previous authors as the DNA to protein ratio) is higher than in wild type. Such an increase in gene concentration should also increase the concentration of those components such as messenger and ribosomal RNA, whose rate of synthesis is determined by gene dosage. We show that the mean concentrations of these 2 RNA species in the small cells are not increased, but are the same as in wild type. The small mutant cells are thus able to compensate for changes in gene concentration. This compensation is shown to operate through differences in the patterns of synthesis of RNA during the cell cycle. In all the strains of the diploid series, the rates of synthesis of messenger and ribosomal RNA double as steps once in each cell cycle. The timings of the steps in the cell cycle appear to be cell-size related, since the smaller the cell at division, the later are the steps in the cell cycle. In contrast, there is comparatively little variation in the timing of DNA replication in the cycles of cells of different sizes. We propose that after DNA replication, there is a delay before doubling in the rate of transcription. Such a cell mass-related delay is all that is required to compensate for increased gene concentration, and results in the same mean functional DNA concentration in all strains. This mechanism will maintain the same mean messenger and ribosomal RNA concentrations in cells dividing at different sizes. Ways in which the cell size-related control over transcription may operate are discussed.

Adenine↗

PubMed1971

Synthesis of virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus.

By using the technique of deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybridization, virus-specific RNA (cRNA) was detected 6 hr after infection in preparations of total RNA from cells infected with type 2 adenovirus in the presence of 2 mum 5-fluorodeoxyuridine. In the absence of 5-fluorodeoxyuridine, there was a continuous increase in the incorporation of (3)H-uridine into viral cRNA until 20 hr after infection, at which time approximately 40% of the (3)H-uridine entering RNA was found in virus-specific RNA. When RNA was prepared from polyribosome fractions obtained from cytoplasmic extracts of infected cells, virus-directed transcription was detected at 3 hr after infection (i.e., 3 to 4 hr before the initiation of viral DNA synthesis). Viral cRNA species synthesized at different times after infection were compared by the technique of DNA-RNA hybridization-inhibition ("presaturation" hybridization-competition). Three hybridization-inhibition techniques were compared. The techniques differed in the manner in which the DNA-RNA complex was isolated after the first hybridization reaction. Depending on the procedure employed, various degrees of inhibition were measured. The variation could be essentially eliminated if prior to hybridization the inhibitory RNA species were alkali-degraded to a uniform size of about 4S. Undegraded RNA could be used if the DNA-RNA complex was isolated by using a procedure involving rigorous washing (preferably including ribonuclease treatment) before the second hybridization with labeled RNA. When a rigorous hybridization-inhibition procedure was used, three classes of virus-specific RNA species could be distinguished: (i) early RNA class I whose synthesis began prior to viral DNA replication and stopped at some time after the initiation of viral DNA replication-it comprised about 70% of the early RNA species and was apparently degraded by 18 hr after infection; (ii) early RNA class II whose synthesis began prior to viral DNA replication and apparently continued at an enhanced rate late in infection; and (iii) late RNA whose synthesis began after the initiation of viral DNA synthesis.

Adenoviridae↗

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↗

PubMed1992

The effect of 2,2'-dichlorodiethyl sulfide on DNA synthesis of a murine stratified keratinocyte culture system.

A primary stratified keratinocyte culture resembling the epidermis in situ was used as a model for studying the effects of exposure to 2,2'-dichlorodiethyl sulfide, or sulfur mustard (SM), on DNA synthesis. A method that distinguishes between semi-conservative (s.c.) DNA synthesis and repair synthesis was used to determine if the former was inhibited following treatment with SM. In this method the density of the newly synthesized DNA was increased by incorporation of 5-bromo-2-deoxyuridine. Density gradient centrifugation was then used to isolate the heavy DNA for quantification. It was demonstrated that topically applied SM in the dose range of 1-10 nmole/cm2 inhibited s.c. DNA synthesis (replication) in a dose and time related manner. Inhibition of DNA replication by SM would result in inhibition of cell division which must be preceded by s.c. DNA synthesis. This failure to replace damaged germinative cells may lead to the destruction of the basal layer which is observed in vivo and in our epidermal culture following exposure to SM. This may also be related to development of vesication observed in exposed intact human skin.

Animals↗