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At least 487 records · Page 27Linked to original sources
PubMed1985

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state.

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.

Animals↗
PubMed1979

Purification and properties of DNA polymerase gamma from rabbit intestinal epithelial cells.

1. DNA polymerase gamma from the cytoplasmic fraction of rabbit intestinal epithelial cells has been purified 120 000-fold and was free of phosphatase and nuclease activities towards deoxyribonucleoside-5'-triphosphates and polynucleotides. 2. The enzyme exhibited maximal activity for activated DNA and poly(A) . oligo(dT)12--18 at pH 8.5 IN 0.25 AND 0.15 M-KCl, respectively. Km values for dTTP with these two templates were 0.5 and 3.8 microM, respectively. 3. In contrast to DNA polymerases alpha and beta, the enzyme replicated poly(A) . oligo(dT)12--18 10 times faster and poly(dA) . oligo(dT)12--18 5 times slower than activated DNA. 4. DNA polymerase gamma did not replicate poly(C) . oligo(dG)12--18 or poly(Cm) . oligo(dT)12--18. The reaction with poly(I) and poly(U) did not exceed 1% of that observed with poly(A). 5. The enzyme was inhibited in 60% by antiserum against DNA polymerase gamma from human lymphoblasts. 6. The nuclear fraction of rabbit intestinal epithelial cells contained DNA polymerase gamma with the same characteristics.

Animals↗
PubMed1986

DNA replication is required for abundant expression of a plasmid-borne late US11 gene of herpes simplex virus type 1.

During herpes simplex virus type 1 (HSV-1) infection, the appearance of true-late gene products is severely reduced under conditions of DNA synthesis inhibition. This report describes the use of a plasmid-borne promoter of a true-late HSV-1 gene (US11), linked to the rabbit beta-globin gene, to study the requirement of DNA replication for late gene expression. The activity of the plasmid-borne US11 promoter in constructs containing or lacking an HSV-1 origin of replication (ORIS) was analysed by quantitative S1 mapping of correctly initiated hybrid transcripts. Following HSV-1 superinfection of transfected HeLa cells, the US11 promoter in ORI+ plasmids was expressed with similar kinetics to the viral US11 promoter. US11 promoter activity was first detected at the same time as the onset of DNA template replication. Expression of US11 RNA was detectable from non-replicating ORI- plasmids, although transcript accumulation was reduced by greater than 90%. Sequences containing the IE-5 promoter (a 3' co-terminal gene whose transcription starts 5' of US11) also played a positive role in achieving normal US11 gene expression.

Cloning, Molecular↗
PubMed1995

Are there DNA damage checkpoints in E. coli?

The concept of regulatory 'checkpoints' in the eukaryotic cycle has proved to be a fruitful one. Here, its applicability to the bacterial cell cycle is examined. A primitive DNA damage checkpoint operates in E. coli such that, after exposure to ultraviolet light, while excision repair occurs, chromosome replication continues very slowly with the production of discontinuous daughter strands. The slower the rate of excision of photoproducts, the greater the delay before the normal rate of DNA replication is restored, the additional time for repair ensuring that normal survival is maintained. A model is proposed in which replication rate is controlled by the ratio of RecA-coated to uncoated single stranded regions of DNA in the replication fork. There are also two cell division inhibitors SulA (= SfiA) and SfiC under the control of the SOS system and sensitive to DNA damage, but they are irrelevant to the survival of wild-type bacteria under normal conditions. In strains where SulA and SfiC do not operate, inhibition is not influenced by the rate of excision repair and so fails one of the criteria for a DNA damage checkpoint, namely the monitoring of the DNA for the level of residual damage.

Cell Cycle↗
PubMed1999

Effect of alpha-tocopherol and silibin dihemisuccinate on the proliferation of human skin fibroblasts.

Cell proliferation is a complex and important event in atherosclerosis, aging and cancer, and is under the control of signalling pathways. These signalling pathways in turn are effected by the presence of a number of chemicals. For this purpose, we have checked the effect of two chemicals on the proliferation of skin fibroblasts. alpha-Tocopherol and silibin dihemisuccinate (SDH) negatively regulate proliferation of human skin fibroblasts. To check the cell-cycle time intervals, a [3H]thymidine incorporation assay was performed, showing DNA replication at around 24 h; this indicated the time required for the incubation with the chemicals. When alpha-tocopherol was added to the growth medium at a physiological concentration of 50 microM, cell proliferation was inhibited by 40% in 72 h. A similar inhibitory effect of cell proliferation was achieved when 500 microM SDH was used (39% inhibition in 72 h). From the dose-response curves obtained it was concluded that both duration of treatment and the concentration of the chemicals are important parameters. The actual mechanism of the inhibition of cell proliferation may be due to the anti-oxidative potential of these chemicals as well as another mechanism effecting signal transduction pathways.

Antioxidants↗
PubMed2004

Cell cycle checkpoints -- molecular background.

Cell cycle checkpoints are the surveillance mechanisms monitoring both the fidelity and accuracy of DNA replication and the segregation of chromosomes. By delaying progression through the cell cycle, checkpoints provide more time for repair before the critical phases of DNA replication and ensure the proper segregation of chromosomes during mitosis. The paper provides basic information about the molecular mechanisms operating in various cell cycle checkpoints activated by DNA damage or disturbances in mitotic spindle assembly.

Animals↗
PubMed1977

Features of the structure of replicating and non-replicating chromatin in chicken erythroblasts.

The digestion by DNAase I of DNA synthesised by isolated chicken erythroblasts was examined in isolated nuclei. It was found that newly synthesised DNA was susceptible to DNAase I but matured to a relatively resistant form with increasing time after replication as observed in mammalian systems. The presence of trypsin in the digestion exposed all of the DNA to DNAase I action. Examination of the digestion products showed that the newly replicated DNA differed little from the more mature form in the structure of the DNA-protein complex but that the difference in susceptibility was probably a result of a differential rate of access of the DNAase to the new and old DNA.

Animals↗
PubMed1999

Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFNalpha), which results in efficient reduction of the viral load only in 10-20% of treated patients. The mechanisms induced by IFNalpha resulting in reduction of viremia in responding patients are unknown. The aim of this study was to characterize HBV-specific IFNalpha-induced intracellular inhibitory mechanisms and IFNalpha-sensitive HBV targets. METHODS: To determine the antiviral activity, cells transiently transfected with HBV DNA were treated with IFNalpha and thereafter, viral products were quantified at different time points. RESULTS: Time-dependent reduction of RNA, replicative DNA-intermediates, core protein and secreted HBsAg/HBeAg levels was observed in IFNalpha-treated cells. Viral RNA levels were reduced most effectively early post-treatment whereas those of core protein and replicative intermediates decreased later. By expression of subgenomic HBV sequences, an RNA target region mediating IFNalpha-induced RNA degradation was mapped. CONCLUSIONS: These data indicate that HuH7 cells transiently transfected with HBV-DNA represent a system well suited for detailed analysis of IFNa-induced antiviral mechanisms and HBV targets. At least two IFNalpha-induced HBV-specific antiviral activities are active in this system: one reduces the levels of core protein and replicative intermediates, the other leads to posttranscriptional degradation of HBV-RNA. Based on the established in vitro system a detailed characterization of the IFNalpha-sensitive RNA-region and of factors mediating this intracellular antiviral effect is feasible. This may lead to the development of novel strategies for therapy of chronic hepatitis.

Carcinoma, Hepatocellular↗
PubMed1988

Modulation of a human dihydrofolate reductase minigene following release from amino acid deprivation involves both 5' and 3' nucleotide sequences.

A dihydrofolate reductase (DHFR) expression system composed of a DHFR minigene constructed from human DHFR genomic and cDNA sequences stably transfected into DHFR- Chinese hamster ovary cells was used to study the modulation of DHFR levels in response to release from amino acid deprivation. The addition of complete medium to cells grown for 48 hr in medium lacking isoleucine and glutamine caused the transfected cells to undergo a synchronous cycle of DNA replication. When DHFR protein levels assayed at the time of maximum DNA synthesis were compared to that present in the deprived state, levels rose 3.2- to 4.9-fold. By contrast, DHFR levels in cells transfected with a DHFR expression construct made from mouse DHFR cDNA fused to viral promoter, intervening, and polyadenylation sequences were not inducible under the identical conditions. Human DHFR minigene deletion or substitution constructs were used to determine which nucleotide sequences were responsible for amino acid-modulated expression. Although deletion of sequences upstream from 322 base pair 5' to the start of transcription did not affect DHFR expression, removal of sequences between 322 and 113 base pairs reduced DHFR induction by approximately 50%. Deletion of nucleotide sequences within the 3' nontranslated region of the gene also reduced the level of induction by approximately 50%. Reduction in the levels of DHFR RNA relative to total cellular RNA was also found. Thus, both 5' and 3' nucleotide sequences are involved in the modulation of DHFR levels following release from amino acid deprivation.

Amino Acids↗
PubMed2004

The effect of nalidixic acid on growth and reproductive events in nucleocytosolic and chloroplast compartments in the alga Scenedesmus quadricauda.

The courses of rRNA accumulation, DNA replication, and nuclear division were followed both in the chloroplast and the nucleocytosolic compartments during the cell cycle in synchronized populations of the chlorococcal alga Scenedesmus quadricauda. Control and nalidixic acid-treated cultures were compared. Nalidixic acid (150 mg/L) was added either at the beginning of the cell cycle or consecutively during the cell cycle to subcultures transferred into the dark. If the inhibitor was applied at the beginning of the cell cycle, chloroplast DNA did not replicate and nucleoids did not divide. Chloroplast division, however, was coordinated in a timely fashion with cytokinesis even under conditions of blocked chloroplast DNA replication. While the growth rate was slowed down, the courses of reproductive processes in the nucleocytosolic compartment were not affected and their timing and the number of rounds were coordinated with growth rate as in the control culture. The rate of cytosolic rRNA synthesis was lower but no apparent effect was seen on the amount of rRNA that accumulated during the cell cycle. In contrast, lower levels of chloroplast rRNA were found at the end of the cell cycle compared with the control culture. Experiments in which cells were transferred to the dark during the cell cycle showed that the inhibitor affected none of the reproductive events in the nucleocytosolic compartment. In the chloroplast compartment, DNA replication was inhibited in inhibitor-treated cultures, but was unaffected in controls. The chloroplast nucleoids themselves divided even in the presence of the inhibitor, reducing their DNA content to a level which corresponded to that in freshly formed control daughter cells.

Chloroplasts↗
PubMed2002

Response of renal inner medullary epithelial cells to osmotic stress.

As part of the urinary concentrating mechanism, renal inner medullary epithelial (IME) cells are normally exposed to variable and often very high interstitial levels of NaCl and urea, yet they survive and function. We have been studying the mechanisms involved, using an established cell line (mIMCD3). Acute increase of NaCl or urea from 300 to >500 mOsmol/kg causes cell cycle delay and apoptosis. High NaCl, but not high urea, causes DNA double strand breaks. At 500-600 mOsmol/kg inhibition of DNA replication following high NaCl depends on activation of the tumor suppressor protein, p53, and provides time for DNA repair. If p53 expression is suppressed, cells continue to replicate DNA, and many of those cells die. At higher levels of NaCl (>650 mOsmol/kg) the mitochondria rapidly depolarize and most cells die within a few hours despite a high level of p53 protein (which, however, is less phosphorylated than at 500 mOsmol/kg). Since the levels of NaCl and urea that kill mIMCD3 cells are much lower than those that exist in vivo, we investigated the difference, using early passage mouse IME cells under various conditions. Passage 2 IME cells survive higher levels of NaCl and urea than do mIMCD3 cells, but still not levels as high as in vivo. However, when the osmolality is increased linearly over 20 h, as occurs in vivo, rather than as a single step, cell survival increases to levels close to those found in vivo. We conclude that a more gradual increase in osmolality provides time for accumulation of organic osmolytes and activation of heat shock protein, previously known to be important for cell survival.

Animals↗
PubMed1981

On the 'clock' mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis. II. Evidence for association of the clock with the cycle of DNA replication.

Acetylcholinesterase (AChE) is a tissue-specific enzyme of the muscle cells of ascidian embryos and its synthesis begins at the neurula stage. Embryos which had been permanently cleavage-arrested with cytochalasin B could develop AChE activity. The time of first AChE occurrence in embryos which had been arrested in the 32-cell stage with cytochalasin was about the same as in normal embryos. The nucleus in the cell of cytochalasin-arrested embryos divided in good synchrony with that of normal embryos. Embryos which had been continuously arrested with colchicine could also produce AChE activity at nearly the same time as did normal embryos. In the cell of colchicine-arrested embryos normal nuclear divisions did not occur, but the cell showed repeated cycles of nuclear envelope breakdown and nuclear envelope reformation in almost parallel with cell cycles of normal embryos. The cell of colchicine-arrested embryos incorporated [3H]thymidine. Aphidicolin, a specific inhibitor of DNA synthesis, prevented cleavages of ascidian eggs. Embryos which had been permanently arrested with aphidicolin in the cleavage stages up to the 64-cell stage did not develop AChE activity, while embryos which had been treated with it from the 76-cell stage onwards were found to be able to differentiate AChE activity. Based on these findings it was proposed that DNA replication is prerequisite for development of the histospecific protein and that the cycle of DNA replication is closely associated with the clock mechanism which is determining the time of initiation of the enzyme development.

Acetylcholinesterase↗
PubMed2006

Distinctive activities of DNA polymerases during human DNA replication.

The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.

Chromatin↗
PubMed2001

Timely release of both replication forks from oriC requires modulation of origin topology.

Initiation of DNA replication at oriC occurs bidirectionally both in vivo and in vitro. Although the proteins involved in establishing the replication forks are known, little is known about the events that ensure that initiation is bidirectional. We show here that in the absence of DNA gyrase, replication fork progression from oriC on a plasmid template in vitro is unidirectional, although both replication forks have formed at the origin. There was no bias in the release of one fork or the other, ruling out protein blockage of one fork as a possible reason for the asymmetric release. Timely release of both forks required the presence of either DNA gyrase or topoisomerase IV, suggesting that modulation of the topology of the origin region is the governing factor.

Bacterial Proteins↗
PubMed2006

Genome-wide sequence and functional analysis of early replicating DNA in normal human fibroblasts.

BACKGROUND: The replication of mammalian genomic DNA during the S phase is a highly coordinated process that occurs in a programmed manner. Recent studies have begun to elucidate the pattern of replication timing on a genomic scale. Using a combination of experimental and computational techniques, we identified a genome-wide set of the earliest replicating sequences. This was accomplished by first creating a cosmid library containing DNA enriched in sequences that replicate early in the S phase of normal human fibroblasts. Clone ends were then sequenced and aligned to the human genome. RESULTS: By clustering adjacent or overlapping early replicating clones, we identified 1759 "islands" averaging 100 kb in length, allowing us to perform the most detailed analysis to date of DNA characteristics and genes contained within early replicating DNA. Islands are enriched in open chromatin, transcription related elements, and Alu repetitive elements, with an underrepresentation of LINE elements. In addition, we found a paucity of LTR retroposons, DNA transposon sequences, and an enrichment in all classes of tandem repeats, except for dinucleotides. CONCLUSION: An analysis of genes associated with islands revealed that nearly half of all genes in the WNT family, and a number of genes in the base excision repair pathway, including four of ten DNA glycosylases, were associated with island sequences. Also, we found an overrepresentation of members of apoptosis-associated genes in very early replicating sequences from both fibroblast and lymphoblastoid cells. These data suggest that there is a temporal pattern of replication for some functionally related genes.

Cell Proliferation↗
PubMed1999

Site-specific in vivo cleavages by DNA topoisomerase I in the regulatory regions of the 35 S rRNA in Saccharomyces cerevisiae are transcription independent.

Eukaryotic type I DNA topoisomerase controls DNA topology by transiently breaking and resealing one strand of DNA at a time. During transcription and replication its action reduces the torsional stress derived from these activities. The association of DNA topoisomerase I with the nucleolus has been reported and this enzyme was shown to be involved in yeast rDNA metabolism. Here, we have investigated the in vivo presence of DNA topoisomerase I cleavage sites in the non-transcribed spacer of the rDNA cluster. We show a specific profile of highly localized cleavage in relevant areas of this region. The sites are detected in the promoter and in the enhancer regions of the 35 S gene. The analysis of mutants in which transcription is prevented and/or reduced, namely a strain lacking the 43 kDa subunit of RNA polymerase I, a second one that does note transcribe, lacking a subunit of the core factor and another member of the RNA polymerase I transcription factors lacking one of the UAF component which transcribes at very low level, show that DNA topoisomerase I cleavage sites are not related to transcription by RNA polymerase I. These findings point to a role for DNA topoisomerase I that is additional to the commonly recognized function in removing the transcription-induced topological stress.

Camptothecin↗
PubMed1990

Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells.

Simian virus 40 (SV40) DNA molecules chromosomally integrated at different sites in three Chinese hamster lung fibroblast lines replicated during the middle portion of S phase but not precisely at the same time in all three cell lines. The time of replication was unrelated to the presence of T antigen or to its relative activity in promoting SV40 replication. SV40 sequences and chromosomal DNA sequences adjacent to the SV40 insert in one cell line expressing a temperature-sensitive T antigen showed a T-antigen-independent difference in replication timing from the homologous, allelic locus not linked to SV40. Our results indicate that the timing of replication of these integrated SV40 molecules is dependent upon the site of integration and is not determined by the level of T antigen replication-promoting activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1983

The dual role of mevalonate in the cell cycle.

It is well established that either exogenous or endogenous cholesterol is required for both cell growth and proliferation. This laboratory has recently discovered that, in baby hamster kidney-21 cells, independent of its role as a cholesterol precursor, mevalonic acid plays an essential role in S phase DNA replication. It was later shown that isopentenyl adenine, a known product of mevalonate in prokaryotes and lower eukaryotes, is 100 to 200 times more effective than mevalonate in restoring DNA replication in cells in which mevalonic acid synthesis is blocked with the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor, compactin. The present study was designed to determine the relationship in the cell cycle between the known requirement for cholesterol and the newly discovered effect of mevalonic acid and isopentenyl adenine on S phase DNA synthesis. Employing cells arrested by serum depletion, it was shown that the cholesterol requirement is limited to the early and mid-G1 phases, whereas the isopentenyl effect is required at the late G1-S interphase of the cell cycle. The evidence supporting these conclusions involves: first, in serum-arrested cells blocked early in G1 by compactin, only the combination of cholesterol added in early G1 and either mevalonate or isopentenyl adenine in late G1 permitted progression through the G1 and S phase DNA synthesis. Neither isopentenyl adenine added early in G1 nor cholesterol in late G1 was capable of restoring DNA synthesis in this system. Second, in accord with the above formulation, inhibition of cholesterol synthesis with the oxidosqualene cyclase inhibitor, dl-4,4,10 beta-trimethyl-trans-decal-3 beta-ol, affected only the early G1 phase of the cell cycle, but had no late G1 effect on DNA replication.

Acetates↗