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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↗
PubMed1975

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. II. Alkylation of DNA and inhibition of DNA replication.

Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.

Alkylation↗
PubMed2003

Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli.

Recombinogenic engineering methodology, also known as recombineering, utilizes homologous recombination to create targeted changes in cellular DNA with great specificity and flexibility. In Escherichia coli, the Red recombination system from bacteriophage lambda has been used successfully to modify both plasmid and chromosomal DNA in a highly efficient manner, using either a linear double-stranded DNA fragment or a synthetic single-stranded oligonucleotide (SSO). The current model for Red/SSO-mediated recombination involves the SSO first annealing to a transient, single-stranded region of DNA before being incorporated into the chromosome or plasmid target. It has been observed previously, in both eukaryotes and prokaryotes, that mutations in the two strands of the DNA double helix are 'corrected' by complementary SSOs with differing efficiencies. Here we investigate further the factors that influence the strand bias as well as the overall efficiency of Red/SSO-mediated recombination in E.coli. We show that the direction of DNA replication and the nature of the SSO-encoded mismatch are the main factors dictating the recombinational strand bias. However, the influence that the SSO-encoded mismatch exerts upon the recombinational strand bias is abolished in E.coli strains that are defective in mismatch repair (MMR). This reflects the fact that different base-base mispairs are corrected by the mutS/H/L-dependent MMR pathway with differing efficiencies. Furthermore, our data indicate that transcription has negligible influence on the strand bias. These results demonstrate for the first time that the interplay between DNA replication and MMR has a major effect on the efficiency and strand bias of Red/SSO-mediated recombination in E.coli.

Adenosine Triphosphatases↗
PubMed1993

Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.

Adenosine Deaminase↗
PubMed1981

Quiescent human diploid cells can inhibit entry into S phase in replicative nuclei in heterodikaryons.

Serum-deprived quiescent human diploid cells (HDC) were fused to replicative HDC, and DNA synthesis was monitored in the resulting heterodikaryons. Quiescent HDC had an inhibitory effect on DNA synthesis in replicative HDC nuclei in heterodikaryons. The timing of the inhibitory effect suggests that entry into S phase was inhibited but ongoing DNA synthesis was not inhibited in the replicative HDC nuclei. When quiescent HDC were fused to T98G human glioblastoma cells or SUSM-1 chemically transformed human cells, entry into S phase was similarly inhibited. However, when quiescent HDC were fused to simian virus 40-transformed human cells, adenovirus 5-transformed human cells, or HeLa cells, DNA synthesis was induced in the quiescent HDC nuclei. A simple hypothesis to explain these results is that quiescent HDC contain an inhibitor of entry into S phase. Transformed cells with a dominant replicative phenotype may have gained a factor that overrides the putative inhibitor, perhaps through viral transformation, whereas recessive transformed cells may ahve lost the normal inhibitory mechanism, perhaps through mutation. Senescent HDC behave like quiescent HDC in heterodikaryons formed with the same types of replicative cells, which suggest that senescent HDC and quiescent HDC share elements of a common mechanism for cessation of proliferation.

Cell Fusion↗
PubMed2002

Kinetic model of DNA replication in eukaryotic organisms.

We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition. The model describes well a large amount of different data within a simple theoretical framework. This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis. In particular, we have determined the frequency of origin activation in time and space during the cell cycle. Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast.

Animals↗
PubMed1990

A herpes simplex virus type 1 recombinant with both copies of the Vmw175 coding sequences replaced by the homologous varicella-zoster virus open reading frame.

Varicella-zoster virus (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (VZV 140K) that shares considerable amino acid homology with the immediate early (IE) regulatory protein Vmw175 of herpes simplex virus type 1 (HSV-1) and is believed to be its functional equivalent. We have tested this hypothesis by insertion of VZV gene 62 (expressed from the HSV-1 IE3 promoter) into both IE3 gene loci in the short region repeats of the HSV-1 genome. The parent virus used for this manipulation was D30EBA, which is a variant of HSV-1 from which the majority of the Vmw175 coding sequences have been deleted. Like other HSV-1 viruses lacking Vmw175 functions, D30EBA is able to grow only in cell lines which express Vmw175 constitutively. The resulting recombinant virus. HSV-140, is able to propagate (but unable to form obvious plaques) on normal cell lines. The properties of HSV-140 were studied by monitoring the time course of polypeptide expression and DNA replication during normal infection. We found that at high multiplicity HSV-140 synthesized apparently normal amounts of many viral polypeptides but that the expression of certain late genes was reduced; this slight defect may be related to less efficient DNA replication by HSV-140. At low multiplicity HSV-140 expressed viral proteins inefficiently. Surprisingly, VZV 140K was produced in large amounts at later times of a normal infection, indicating that the polypeptide fails to autoregulate the IE3 promoter. The results strongly suggest that VZV 140K is able to perform most of the functions of Vmw175 during growth of HSV-1, but that differences in detail lead to less efficient virus growth.

Animals↗
PubMed1976

Cell cycle analysis and X-chromosome inactivation in the developing mouse.

The duration of different phases of the cell cycle (G1, S, G2, and M), as well as the exact time of initiation of asynchronous DNA replication pattern in one of the X-chromosomes in the female mouse during the early stages of development, have been investigated. It was found that early developmental stages in the mouse are devoid of G1 period, and from the late blastocyst stage the embryonic cells first acquire all the characteristic stages of the cell cycle (G1, S, G2, and M). Additionally, asynchronous DNA replication pattern in one of the X-chromosomes in the female embryos was found to be initiated with the appearance of the G1 period. I have established a correlation between the onset of the G1 period and the initiation of an asynchronous DNA replication pattern in one of the X-chromosomes of the female mammal during embryogenesis.

Animals↗
PubMedDate not supplied

DNA synthesis in the gastroduodenal mucosa during acute and chronic stress in the rat.

We investigated the effects of acute and chronic stress on the DNA synthesis of the gastroduodenal mucosa of the rat using two different methods of physical stress at various time intervals. Acute stress was produced in the rats being briefly plunged or swimming for two hours (water temperature 37 degrees C). "Sham - transported" rats were used as controls. The results indicate that in the stomach the DNA synthesis was substantially reduced during acute stress in both groups tested (when compared to controls). The DNA synthesis was also reduced in experimental rats after one and two weeks of stress (as compared to day one). By four and eight weeks, the rate of DNA synthesis in the gastric mucosa had significantly increased in the stressed animals. Controls demonstrated significantly lower DNA values following two to eight weeks of stress (as compared to day one). From the outset, the DNA replication values were 2.5 to 3 times higher in the duodenal mucosa than in the gastric mucosa. Following two weeks of stress, the duodenal mucosa of both test groups showed significantly lower DNA values than controls, but significantly higher values after four weeks of stress. By eight weeks, the duodenal mucosa in all rats had reached the same values as that of day one. This was considered a sign of "adaptation to stress" in the duodenal mucosa. The above results suggest that the fluctuations of DNA replication may be connected to compensatory mechanisms aimed at adjusting the gastroduodenal mucosa to protracted stress situations.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1982

Timing mechanisms in early embryonic development.

Embryological development takes place in four dimensions and requires the existence of time measuring processes within the embryo. Evidence is accumulating that suggests that the emergence of many events during early embryonic development is controlled by timing mechanisms or developmental clocks. The purpose of this work is to review recent studies on developmental timing with speculations about underlying possible mechanisms. It is an attractive idea that the development of an embryo is timed by a single clock set in motion at fertilization, but this seems not feasible. The clock mechanism which determines the time of initiation of cellular differentiation may be independent of that for the timing of morphogenesis. The clock mechanism for cellular differentiation may be closely associated with the cycles of DNA replication, while the clock which counts the time to onset of early morphogenetic events is found in the cytoplasm. These ideas can provide a framework which may help to organize existing observations and to stimulate new experimental approaches to the problem.

Acetylcholinesterase↗
PubMed1979

[Replicon size and rate of DNA replication in the macronucleus of Tetrahymena pyriformis].

The size of replication units (or replicons) measured in Tetrahymena pyriformis GL macronuclear DNA reaches 20--30 microns, according to the two independent methods: DNA fiber autoradiography, and alkaline isokinetic sucrose gradient centrifugation. The synthesis of new DNA fragments--replicons and their subsequent assembly are separated by time intervals (30 min). The rate of DNA synthesis for one fork averaged 0.6--0.7 microns/min. These data were obtained for cells of cultures being both in the expotential phase of growth, and those synchronized by starvation-refeeding. The generation time of T. pyriformis cells, calculated by the increase of the part of labeled nuclei, is almost 2 hours; the synthesis lasts 1 hour. Total amount of replication units in polyploid (polygenomic) Tetrahymena macronucleus is about 3000. Their initiation during S-period is presumably asynchronous.

Animals↗
PubMed1997

Cloning of the cDNA encoding rat homologue of the mismatch repair gene MSH2 and its expression during spermatogenesis.

A rat cDNA clone encoding the mismatch repair protein MSH2 has been isolated and characterized. The cDNA has an open reading frame of 2802 nucleotides in length coding for a protein of 933 amino acids (100 kDa). It shows significant homology to human and mouse MSH2. Northern blot analysis of rat MSH2 in the testes of rats of different ages showed maximum expression at 20 days, at which time the germ cells are undergoing premeiotic DNA replication. We observed down-regulation in the expression of rat MSH2 beyond 25 days by which time the germ cells have entered meiotic prophase.

Amino Acid Sequence↗
PubMed2000

A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells.

The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells.

Antibodies↗
PubMed2004

SV40 DNA replication inhibition by the monofunctional DNA alkylator Et743.

Ecteinascidin 743 (Et743) is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC-rich sequences resulting in an unusual bending toward the major groove. The ability of Et743 to block DNA replication was studied using the well-established simian virus (SV40) model for mammalian DNA replication in cells and cell-free extracts. Intracellular SV40 DNA isolated from Et743-treated BSC-1 cells was analyzed by native, two-dimensional agarose gel electrophoresis. A low frequency of Et743 adducts detected at 30-100 nM drug concentrations inhibited SV40 origin activity and induced formation of unusual DNA replication intermediates. Under cell-free conditions, only a high Et743 adduct frequency reduced SV40 DNA synthesis. Comparative studies involving related DNA alkylators, tomamycin and saframycin A, revealed inhibition of SV40 DNA replication in cells at concentrations approximately 10 times higher than Et743. Under cell-free conditions tomamycin- or saframycin-A-adducted DNA templates inhibited DNA synthesis similarly to Et743. Et743 appears to be unusual among other alkylators, because its adducts strongly inhibit intracellular SV40 DNA replication but are relatively weak as cis inhibitors as measured under cell-free conditions.

Animals↗