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PubMed1970

Synchronous reinitiation of chromosome replication in E. coli B-r after nalidixic acid treatment.

Nalidixic acid has been used to interrupt DNA synthesis in exponentially growing cultures of E. coli B/r. Within three minutes after removal of the nalidixic acid block, premature rounds of chromosome replication are initiated at the normal origin of vegetative DNA synthesis. The new growing forks traverse the chromosome in about 40 minutes, the normal replication time. These rounds continue synchronously into the second generation after removal of the nalidixic acid. These phenomena are similar in some respect to the premature initiation of chromosomal replication observed after thymine starvation or after release of thermo-sensitive DNA synthesis mutants from the restrictive temperature that prevents DNA synthesis.

Chromosome Mapping↗
PubMed1990

Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.

Previous reports have shown that retrovirus infection is inhibited in nonreplicating (stationary-phase [hereafter called stationary]) cells. Infection of stationary cells was shown to occur when the cells were allowed to replicate at times up to a week after infection, suggesting that an unintegrated retrovirus could persist in a form that was competent to integrate after release of the block to replication. However, those studies were complicated by the use of replication-competent virus, which can spread in the infected cells. We have used a replication-defective retrovirus vector to compare the efficiency of gene transfer in stationary and replicating rat embryo fibroblasts. In agreement with previous results, gene transfer was inhibited 100-fold in stationary versus replicating cells. In contrast to previously reported results, the block to infection could not be relieved by stimulating stationary cells to divide at times from 6 h to 10 days after infection. Thus, for successful retroviral infection, the infected cells must be replicating at the time of infection. These results have important implications for the use of retroviral vectors for gene transfer.

Animals↗
PubMed1992

Molecular analysis of the aberrant replication banding pattern on chromosome 15 in murine T-cell lymphomas.

Cytogenetic techniques revealed an altered early replication banding pattern on the distal part of chromosome 15 in some murine T-cell lymphomas. This pattern reverted back to normal replication in somatic cell hybrids that had become non-tumorigenic after fusion of leukemic cells with normal fibroblasts. The altered banding pattern was correlated with malignancy. To investigate the molecular basis of the aberrant pattern in more detail, centrifugal elutriation of cells containing bromodeoxyuridine labeled DNA was used to prepare newly replicated DNA from selected intervals of the S-phase from tumor cells, as well as from hybrid cells with the revertant phenotype. These different DNA fractions were probed for DNA sequences distributed over the distal half of chromosome 15. Only two out of ten chromosome 15 specific genes tested showed a clear change in replication timing between the two different cell lines tested. These two genes were the lymphocyte antigen-6, Ly-6, and the neighboring thyroglobulin gene, Tgn, which replicated at the beginning of S in the tumor cells and later in S in the non-tumorigenic hybrid cells.

Animals↗
PubMed1984

SCE and DNA methylation.

The interrelationship between sister chromatid exchange (SCE) formation and DNA methylation was studied in Chinese hamster V79 and Indian muntjac cells. A DNA methylation inhibitor, 5-azacytidine (5AzaC), induced SCEs only when it had been present in cells for at least 2 rounds of DNA replication. This result suggests that SCEs are formed during replication of hemimethylated or demethylated DNA possessing 5AzaC, and that hypomethylated sites may become fully methylated after they pass 1 cell division. It also appears that hypomethylated DNA is not more sensitive to ultraviolet light (UV) or 3-aminobenzamide (3AMB) than normal chromosomes, but sensitized to mitomycin C (MMC) for the induction of SCEs. An analysis of sites of SCEs induced by 5AzaC within Indian muntjac chromosomes showed that the SCE frequency was enhanced at the 5 methylcytosine-rich regions where spontaneous SCEs were intensively suppressed. The SCE mechanism at the junction between contiguous replicons with different replication timing was discussed.

5-Methylcytosine↗
PubMed1974

The relative numbers of different genes in exponential microbial cultures.

It is shown that the results of the marker frequency analysis of Sueoka and Yoshikawa (1965) can be derived as very good approximations from a model where the rigid assumptions of their analysis are relaxed to take into account statistical variations in the timing of cell events. It is further shown that the expression for the amount of DNA per cell can be approximated by an elementary exponential function of the growth rate, and this result facilitates genetic mapping by DNA hybridization techniques. An analysis of recent data on gene frequencies in Escherichia coli corroborates a model of symmetric, bidirectional chromosome replication with a replication time of approximately thirty minutes.

Cell Division↗
PubMed1968

Chromosome mapping in Staphylococcus aureus.

The genome of Staphylococcus aureus was mapped by enumerating mutants induced by nitrosoguanidine during synchronous chromosomal replication following release from phenethanol inhibition. Both chromosomal replication time and cell division time were 120 min for this strain of S. aureus. Duplication of genes occurred within a 10-min period of the 120 min required for chromosomal replication. A high-resolution method was devised to determine the gene order of four genes that duplicated in the same 10-min interval of replication of the chromosome. A genomic map locating the positions of 10 genes was derived.

Cell Division↗
PubMed1971

Velocity of chromosome replication in thymine-requiring and independent strains of Bacillus subtilis.

Chromosomes in spores of a thymineless mutant of Bacillus subtilis strain 168 were shown to have a replication fork, unlike chromosomes in spores of the thy(+) strain which are in a complete form. As a consequence the number of replication forks in germinating cultures is higher in the thy(-) strain than in the thy(+) one. Chromosome replication time (C) in the thy(+) strain was found to be about 53 min for growth rates from 20 to 60 min. In the thy(-) strain, C was about 75 min at high growth rates and increased with decreasing growth rate when the thymine concentration was not limiting. With limiting thymine concentrations in the medium replication velocity decreased independently of growth rate.

Adenine↗
PubMed1977

Fluorescent probes of chromosome structure and replication.

Procedures employing fluorescent dyes or Giemsa stain have been utilized to differentiate methaphase chromosomes into longitudinal segments termed bands. In spite of the immense practical utility of chromosome banding, the chemical basis of banding patterns remains incompletely understood. Physical chemical studies have elucidated the modes and specificities of the interaction of fluorescent dyes such as quinacrine, 33258 Hoechst, daunomycin, chromomycin A3 and 7-aminoactinomycin D with DNA and chromatin. However, it is not clear that all aspects of chromosome staining are explainable in terms of the optical properties of soluble dye-DNA complexes. BrdU-dye techniques in which chromosome staining depends on the schedule of BrdU incorporation by cells, have been used for cytological studies of chromosome structure and replication. These procedures have revealed a close association between quinacrine or Giemsa bands and late replicating chromosomal regions. Biochemical studies on chromatin differentially labelled according to replication timing may thus prove useful for investigating the molecular basis of chromosome banding.

Animals↗
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↗
PubMed1990

The probability of G1 cells to enter into S increases with their size while S length decreases with cell enlargement in Allium cepa.

The distribution of cell surface area projection (cell size) has been measured at birth and at initiation of DNA synthesis in steady-state populations of Allium cepa root meristems. The conditional probability, P(I/G1), that initiation occurs given that the event of being in G1 also occurs has been estimated from these data. P(I/G1) was found to increase when cells became larger. The distribution of G1 duration has been constructed from indicated cell size distributions. The absolute frequencies of G1 times showed a maximum in the zone of cells with short G1 periods; about 14% of cells appear to enter into S with G1 congruent to 1 h. These results suggest that the increase of P(I/G1) was due to cell enlargement and not to cell aging. By comparing the cell size distribution at initiation of S and at the end of this period, a drastic reduction of cell size variability during DNA replication was observed and both curves were seen as rather similar in shape although they obviously had different modal points. These observations support that there is a negative correlation between the initiation size and the duration of genome duplication, and that cells which initiate DNA synthesis with the same size have a similar replication time. From this hypothesis, a plot of S duration versus cell size at initiation of this period was constructed by comparing the distributions of cell size at start and end of replication; this plot was also consistent with the existence of a negative correlation between cell initiation size and S length.

Allium↗
PubMed1986

Replication and segregation of eukaryotic chromosomal DNA.

Transmission of the genetic information from the parental to the two daughter cells resulting from each cell division requires this genetic information to be duplicated prior to cell division. This ensures that each daughter cell will inherit an exact and complete copy of the information contained in the parental cell. The duplication of the genetic information, which is codified in deoxyribonucleic acid (DNA), is called "DNA replication". During the last 33 years, since the structure of DNA was discovered by Watson and Crick (1953), DNA replication has been investigated using genetical, cytological, and molecular techniques. The present review is an attempt to summarize most of the knowledge that has been accumulated throughout these years on eukaryotic DNA replication, with special emphasis on the methodologies that have been employed. Besides, some of the current "hot spots" in this subject, such as the controversy concerning whether or not there are specific DNA sequences which are recognized as replication origins, the existence of a close association between replication timing and gene expression, and the mechanisms operating at the time of nascent replicon maturation and segregation of sibling DNA molecules, are discussed with special deference.

Animals↗
PubMed1992

Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe.

In the budding yeast, S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (+/- 300 bp). In contrast, in mammalian cells and Drosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just like S. cerevisiae origins. Because the fission yeast, Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than does S. cerevisiae, we thought that S. pombe replication origins might prove to resemble--and thus be helpful models for--animal cell origins. An attempt to test this possibility using 2D gel techniques resulted in identification of a replication origin near the ura4 gene on chromosome III of S. pombe. The 2D gel patterns produced by this S. pombe origin indeed resemble the patterns produced by animal cell origins and show that the S. pombe origin cannot be precisely located. The data suggest an initiation zone of 3-5 kbp. Some aspects of the 2D gel patterns detected at the S. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.

Chromosomes, Fungal↗
PubMed1981

The initiation of chromosome replication in a dnaAts46 and a dnaA+ strain at various temperatures.

The regulation of chromosome replication initiation was studied at various temperatures with an E. coli dnaA46 strain and its dnaA+ parent. We find that, in both strains, the "initiation mass" varies depending upon growth temperature while the replication time remains constant relative to the cell doubling time. In the permissive temperature range, the initiation mass of the dnaA46 mutant strain is larger by a constant factor than that for a dnaA+ strain. We conclude that, even at temperatures permissive for growth of the dnaA46 strain, the activity of the dnaA46 product is lower than that of the wild-type protein. The dnaA gene product, therefore, plays an important role in regulating initiation.

DNA Replication↗
PubMed1989

Increased exercise tolerance and reduced electrocardiographic ischaemia 3 and 12 hours after oral felodipine in effort angina.

The antianginal properties and the duration of action of two doses of felodipine, a dihydropyridine calcium antagonist with a vascular:myocardial potency ratio approximating 100:1, were investigated in 15 patients suffering from disabling effort angina pectoris with reproducible exercise tolerance. Felodipine (5 mg, 10 mg) and placebo were administered once in the morning on three different days, with a 24 h interval between them, according to a double-blind 3 x 3 latin square design, 5 times replicated. Symptom-limited cycloergometric exercise tests were performed 3 and 12 h after administration. Duration of exercise to ST segment depression of 1 mm and to peak exercise was increased (all P less than 0.01) by both doses of felodipine in comparison with placebo. Twelve hours after administration, the 10-mg dose induced a significant improvement in the exercise time and a smaller ST segment depression (all P less than 0.01) in comparison with the 5-mg dose. The relationship between ST segment depression and the pressure-rate product during exercise was favourably influenced by the 10-mg dose at 3 and 12 h after intake, and by the 5-mg dose only at 3 h after intake. These findings suggest an increase in coronary blood flow induced by felodipine. Apart from mild headache there were no other unwanted effects. In conclusion, felodipine improves exercise tolerance and reduces electrocardiographic ischaemia for up to 12 h after single oral administration in patients with effort angina. Increasing the dose from 5 mg to 10 mg produces a more prolonged effect, with increased exercise tolerance 12 h after intake.

Administration, Oral↗
PubMed1981

Evidence of diurnal variation of human epidermal cell proliferation. II. Duration of epidermal DNA synthesis.

In the epidermis of six healthy male volunteers the DNA synthesis time was measured by in vitro double labeling technique in a longitudinal study (48 h) with taking skin punch biopsies every 6 h. The external conditions of the experiments and one group of volunteers were identical to those of a previous study on LI of the epidermal keratinocytes. 14C-thymidine and 3H-thymidine were used for double labeling the DNA-synthesizing nuclei of keratinocytes. The duration of DNA replication time proved to be nearly constant, with minor fluctuation, during the entire time span of experiments (mean 6.19 +/- 0.3 h). Thus, the present results are in accordance with the conception that a periodically changing entrance rate of G1-cells into the S-phase of the generation cycle of keratinocytes is predominantly responsible for circadian variations in the number of DNA-synthesizing epidermal keratinocytes.

Adult↗
PubMed1981

Vitamin B12-dependent replication of L1210 mouse leukemia cells. A model system for cobalamin-folate inter-relationships.

L1210 mouse leukemia cells were made cobalamin-deficient by propagation in a medium from which cyanocobalamin was omitted and fetal bovine serum (containing protein-bound cobalamins) was replaced by bovine serum albumin. These cobalamin-deficient cells exhibited a normal replication time of 12 h, provided that the medium contained excess folate or 5-formyltetrahydrofolate. The cells responded poorly, however, to 5-methyltetrahydrofolate unless exogenous cobalamin was added. A cobalamin dependency was also observed when low levels of folate or 5-formyltetrahydrofolate were used. With 5-methyltetrahydrofolate, optimal stimulation of growth was observed with free and transcobalamin-II-bound cobalamin at 4,000 pM and 2 pM, respectively. Under cobalamin-replete conditions, cells contained 2,000 to 4,000 molecules of cobalamin/cell, and in the deficient state, this value declined to less than 10 molecules/cell; optimal replication on 5-methyltetrahydrofolate required approximately 180 molecules/cell. Cobalamin-deficient cells cultured in the absence of folate reached an arrested state from which limited replication could be induced by the addition of aquacobalamin; normal replication was induced by aquacobalamin plus 5-methyltetrahydrofolate. Results of this investigation are interpreted in terms of the requirement for tetrahydrofolate in cell replication and the production of this compound from folate and 5-formyltetrahydrofolate (via cobalamin-independent pathways) and from 5-methyltetrahydrofolate (via the cobalamin-dependent methionine synthetase).

Animals↗
PubMedDate not supplied

C-myc transcript is induced in rat liver at a very early stage of regeneration or by cycloheximide treatment.

In rats, partial hepatectomy induces reasonably synchronized DNA replication in the remaining liver after approximately 20 h. Events occurring during the earlier stages of liver regeneration are of interest because they may tell us how cells in vivo respond when they move from a differentiated resting state (G0 phase) to a proliferative state. We report here that the expression of the c-myc oncogene is increased up to 10-15-fold of the normal level within 1-3 h after partial hepatectomy. This expression begins to decrease rapidly after 4 h and has returned to less than double the normal level after 8 h, at which time replicative DNA synthesis has still not begun. A still larger increase in c-myc transcription (approximately 600-fold) is observed in the liver when protein synthesis is inhibited by an injection of cycloheximide. These findings suggest the existence of a short-lived protein that is synthesized soon after partial hepatectomy, and which suppresses the expression of c-myc.

Animals↗
PubMed1990

DNA methylation and late replication probably aid cell memory, and type I DNA reeling could aid chromosome folding and enhancer function.

DNA methylation in mammals is reviewed, and it is concluded that one role of methylation is to aid cell memory, which is defined as the ability of mitotically derived progeny cells to remember and re-establish their proper cellular identity. Methylation of X-linked CpG-rich islands probably stabilizes X-chromosome inactivation, but other mechanisms appear to be involved. Late replication is discussed as a key ancestral mechanism for X inactivation, and it is emphasized that early and late replication domains may each be self perpetuating. Therefore, early-late replication timing becomes another strong candidate mechanism for cell memory. A chromosome-loop folding enigma is discussed, and it is concluded that special mechanisms are needed to explain the formation and maintenance of specific looped domains. DNA reeling, such as done by type I restriction-modification enzymes, is proposed to provide this special mechanism for folding. DNA reeling mechanisms can help to explain the cis-spreading of X-chromosome inactivation as well as long-range action by enhancers.

Animals↗