Period Of DNA Synthesis Research Articles

BrdU-dye characterization of late replication and meiotic recombination in Armenian hamster germ cells.

In vivo BrdU-dye methodology is used to reveal patterns of replication kinetics and meiotic recombination in male germ cells of the Armenian hamster. Analogue substitution over a fraction of the DNA synthesis period results in distinctive 33258 Hoechst fluorescence staining of late replicating chromatin, detectable at spermatogonial or spermatocyte I and II stages. Spermatogonial cells which are extensively substituted with BrdU over the penultimate synthesis period reveal sister chromatid differentiation in all chromosomes of primary or secondary spermatocytes. In chiasmatic regions, exchanges between unlike-stained non-sister chromatids are indicated by isolatelled segments, while those occurring between like-stained non-sister chromatids are not directly detected. In sex chromosomes from young animals, these alternative images occur in a ratio of 1:1, which supports the concept that homologue non-sister chromatid regions are randomly broken and re-united in the process of chiasma formation. Deviations from randomness appeared to occur in older animals. Sex bivalent chiasmata are either coincident with points of visible exchange, or they appear to have variable degrees of terminalization. Secondary spermatocytes display sharp chromatid contrast, and aid in mapping the positions and frequencies of homologue exchanges.

The duration of DNA synthetic (S) period in Zea mays: A genetic control

The nuclear cycle among several diverse genetic stocks of Zea mays root meristem cells was compared and it was found that there were no significant differences among the nuclear cycle durations and its component phases. The durations of various periods of their mitotic cycles were studied by autoradiography of cells pulse-labelled with tritiated thymidine (3H-TdR). The total nuclear cycle was 10 to 11.5 hours and mitosis was 0.81 to 1.34 hours at 25°C. The S period is the longest interval (50% of the total time) of the nuclear cycle; of the rest of the cycle, G2 is longer than G1 or mitosis among all stocks. The constancy of the nuclear cycle among several stocks was adduced as evidence for strict genetic control of the cycle. Furthermore, it is demonstrated the DNA synthesis period is not dependent upon the amount of DNA present.

Bromodeoxyuridine-induced mutations in synchronous Chinese hamster cells: temporal induction of 6-thioguanine and ouabain resistance during DNA replication.

Mutations were induced in synchronous Chinese hamster cells by bromodeoxyuridine (BUdR) incorporated into cells for one-hour periods in the cell cycle. There was a very pronounced temporal dependence during the first half of the DNA synthesis period for the induction of damage leading to 6-thioguanine (6TG) and ouabain resistance. No mutants above background were induced by exposure to BUdR in G1 and G2 cells, and very few mutants were induced in the latter part of the DNA synthesis period. The peak for the induction of 6TG resistance occurs at about two hr in the DNA synthesis period; one hour later there is a peak for the induction of ouabain resistance. Both peaks occur before the time of maximum incorporation of BUdR into DNA. These results suggest that the mutagenesis by BUdR is associated with at least two nuclear genes, which replicate at two hr and three hr in the DNA synthesis period.

Chromosome replication during the division cycle in slowly growing, steady-state cultures of three Escherichia coli B/r strains

The period of DNA synthesis C during the cell cycle was determined over a broad range of generation times in slowly growing, steady-state batch cultures in the exponential phase and in chemostat cultures of three strains of Escherichia coli, strains B/r A, B/r K, and B/r TT, utilizing measurements of average amounts of DNA per cell and cell survival after radioactive decay of 125I incorporated into the DNA of synthesizing cells. At each growth rate, values for cell survival and for C periods were the same within experimental errors for the three strains. The length of the DNA synthesis period increased linearly with generation (doubling) time T of the culture and approached a limiting value of C = 0.36T at very long generation times. In very slowly growing cultures, DNA replication was limited almost entirely to the final third of the cell cycle. D periods, between termination of DNA replication and cell division, were found to be relatively short at all growth rates for each strain. Average amounts of DNA per cell measured in slowly growing cultures of strains B/r A and B/r TT were indistinguishable from results for strain B/r K at the same growth rates. Amounts of DNA per cell calculated from the cell survival values alone are completely consistent with the measured DNA per cell.

Factors affecting the induction of micronuclei at low doses of X-rays, MMS and dimethylnitrosamine in mouse erythroblasts

In erythrocytes from mouse bone marrow the time schedule of micronucleus formation in relation to the last DNA synthesis was investigated by [ 3H]thymidine labelling in the autoradiographic technique. The results suggest that micronuclei can be produced both in the G 2 and S periods by X-irradiation. Furthermore, X-rays had a delaying effect on the cell cycle leading to a pronounced under-estimation of the dose—effect curve at higher dosages. Even when the cells were harvested as late as 30 h after irradiation, the full effect had most likely not yet appeared at dosages over 100 rad. Combined treatment with caffeine did not influence the dose—effect curve of X-rays, indicating no influence of a caffeine-sensitive repair mechanism. The induction of micronuclei by MMS, in contrast with the effect of X-rays, seems to have been restricted, at least predominantly, to the period of DNA synthesis. The dose—effect relation of MMS was characterized by a threshold giving a weaker effect than expected at low doses. Pretreatment with caffeine enhanced the effect of MMS at high but not at low doses, suggesting an error-free repair process operating at low doses and an error-prone and caffeine-sensitive repair at higher doses. The extent of alkylation in the bone-marrow cells was linear with respect to injected dose of MMS both in the presence and absence of caffeine. Pretreatment with phenobarbital reduced the effect of MMS sixfold, which can be explained by a reduction of alkylation found in the bone marrow. This result is in agreement with the enhanced excretion of MMS or its metabolites into urine and bile after pretreatment of the mice with phenobarbital. DMN had no measurable effect on the frequency of micronuclei. However, in the presence of caffeine a significant effect was observed, which was roughly of the same magnitude for the two dosages used. Pretreatment with phenobarbital also indicated a synergistic effect between DMN and phenobarbital. The treatment with DMN, phenobarbital and caffeine together gave a frequency of micronuclei not different from the control level, suggesting some antagonistic action between phenobarbital and caffeine. The indication that DMN is caffeine-sensitive at low dosages, whereas MMS is not, might be related to the difference in the alkylating properties of these chemicals.

Effects of 5-bromodeoxyuridine on development of Mauthner's neuron and neural retina of Xenopus laevis embryos

Bromodeoxyuridine (BrUdR) injected into Xenopus embryos at stages before, during or after the last period of DNA synthesis in Mauthner's neurons had no serious effect on differentiation of Maunthner's neurons. However, the BrUdR caused widespread cell degeneration in the central nervous system. The action of BrUdR on retinal neuronal differentiation depended on dose and stage of injection. At all doses (0.005–0.9 μg per injection), and all stages (22–29), the main effect was retinal cell degeneration. The drug produced complete absence of differentiated retinal neurons only when given at early stages (22–24) and at the highest doses (0.5–0.9 μg) that enable the embryo to survive to stage 44. At stages 22–24 at lower doses (0.005 or 0.05 μg per injection) and at stages 26–29 at all doses, some retinal neurons differentiated, although retinal cell degeneration was the main effect. In these two system BrUdR did not selectively inhibit neuronal differentiation.

Levels of cellular DNA polymerases in synchronized bovine paravovirus-infected cells

Infection of synchronized bovine fetal spleen cells with bovine parvovirus results in changes in the levels and patterns of DNA polymerases alpha and gamma during the cell cycle. The pattern of DNA polymerase alpha activity closely paralled viral DNA synthesis and the production of progeny virus, and levels, of this enzyme were threefold greater than in mock-infected cells during the period of maximal viral DNA synthesis. DNA polymerase gamma activity remained slightly elevated during viral DNA replication. Levels and patterns of DNA polymerase beta were similar in mock- and virus-infected cells.

Role of silicon in diatom metabolism IX. Differential synthesis of DNA polymerases and DNA-binding proteins during silicate starvation and recovery in Cylindrotheca fusiformis

During recovery from silicate-starvation, a period of active DNA synthesis, synchronized cells of Cylindrotheca fusiformis incorporated 3 times more l-[U- 14C]aspartate than did starved cells. Of the diatom's four DNA polymerases, A and D are synthesized during silicate recovery, indicating that they are involved in silicate-dependent DNA replication. Polymerase B, and the chloroplast enzyme, polymerase C, are synthesized during silicate-starvation and their levels are unaffected by the addition of silicate. DEAE-Sephadex analysis of the DNA-binding proteins, labeled with [ 14C]- and [ 3H]aspartate, shows that only three proteins are synthesized in cells recovering from silicate-starvation. Two of these proteins correspond to polymerases A and D, while the function of the third protein is not known. At least 15 other proteins are present in silicate-starved cells and their synthesis is repressed upon the addition of silicate. Models are proposed which describe the modes by which silicate might regulate DNA synthesis in the diatom.

Comparison of Singlet and Doublet Paramecium tetraurelia: DNA Content, Protein Content and the Cell Cycle*

SYNOPSIS Doublet Paramecium tetraurelia contain either a single macronucleus which is substantially larger than that in a singlet cell, or 2 smaller macronuclei. Doublets have approximately twice the DNA content and twice the total protein content of singlets. The cell cycle of doublets is 164% as long as that of singlets, but the relative position of the macronuclear DNA synthesis period within the cell cycle is the same as in singlets. The DNA content of doublets is regulated so that differences in the number of macronuclei do not produce corresponding changes in DNA content; bimacronucleate doublets have only 27% more DNA than unimacronucleate doublets.

Assay of ribonucleotide reduction in nucleotide-permeable hamster cells.

Ribonucleotide reduction was measured in Chinese hamster ovary cells made permeable to nucleotides by treatment with the detergent Tween-80. When compared to the respective ribonucleotide reductase activity in partially purified cell extracts, CDP and GDP reductase activities in permeabilized cells responded in a similar fashion to dithiothreitol, pH, MgCl2, FeCl3, substrate concentration and the presence of positive or negative allosteric effectors. At low protein concentrations both CDP and GDP reduction with whole cells increased linearly with cell number and was greater than the activity in corresponding cell extracts. Permeabilized cells were used to measure the level of CDP and GDP reductase in a hamster cell line resistant to the cytotoxic effects of hydroxyurea. The hydroxyurea-resistant cell line contained four to ten times more CDP and GDP reductase activity compared to parental or revertant cell lines. The permeabilized cell assay was also used to measure CDP and GDP reductase activities in Chinese hamster ovary cells synchronized by isoleucine starvation. CDP reductase activity was low in G1 arrested cells but increased 10-fold by 16 hours after the readdition of isoleucine to the growth medium. GDP reductase, which is present at much higher levels, is similarly induced after isoleucine addition, but only by 2-fold. The maximum activity of both CDP and GDP reductase occurred from 14 to 16 hours after isoleucine addition, which corresponded to the period of maximum DNA synthesis.

Regulation of macronuclear DNA content in Paramecium tetraurelia.

The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.

An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae

Using alkaline sucrose gradients the mechanism of DNA synthesis has been investigated in both log-phase and synchronised cultures of the yeast Saccharomyces cerevisiae. DNA synthesis proceeds via a heterogeneous population of single-stranded intermediates between 7 and 60 x 10(6) daltons in size. The size of these molecules and a comparison of their behaviour in log-phase and synchronised cultures suggests they are nascent or completed replicons. The progressive increase in molecular weight of these intermediates during S in synchronous cultures was used as a measure of the rate of DNA synthesis per single strand. During the first half of the period of DNA synthesis in the culture, the observed rate of elongation was 0.82 x 10(6) daltons/min. Later in S, an apparent increase in rate was detected, but this may have reflected the joining of completed replicons. In our gradients the pattern of DNA synthesis in the cell cycle mutants cdc2 and 6, thought to make incomplete or faulty DNA at the restrictive temperature (Hartwell, 1974), closely resembled that of the wild-type.

Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light

The combination of 8-methoxypsoralen and near UV light is highly effective in inducing sister chromatid exchanges (SCEs) in Chinese hamster ovary cells. Appreciable increases in SCEs can be effected by treatments compatible with cell survival, and effects of a single dose of alkylation persist over multiple generations. Both the frequency and location of SCEs induced at different times within the DNA synthesis period varies in a manner indicating that exchange induction is restricted to regions which replicated during or after DNA damage.

Investigation of the cytostatic action of cyclophosphamide on esophageal tumors and epithelium in mice

The cytostatic action of cyclophosphamide on sarcoma 37 and the esophageal epithelium was investigated in albino mice by determination of the diurnal rhythms of mitotic activity and the number of labeled nuclei. Tumor cells were shown to be most sensitive to the inhibitory action of the compound evidently in the G1 phase at the beginning of the S phase. As the period of DNA synthesis came to an end, the resistance of the cells to the cytostatic increased. Cells in the G1 phase of the mitotic cycle in the esophageal epithelium are sensitive to the inhibitory action of cyclophosphamide.

Effect of Kupffer cell blockade at different times after partial hepatectomy on hepatocyte regeneration

The system of mononuclear phagocytes of the liver was blocked in male Wistar rats weighing 140–160 g by iron carbonyl (brand R-100 F) with a particle size of 1–1.5 μ. The blockade was carried out 2 h before partial hepatectomy and also 3 and 18 h after the operation. Injection of the iron compound before the operation and in the early prereplicative period of regeneration led to substantial delay of the peak of the index of labeled nuclei and mitotic index of the hepatocytes accompanied by a general reduction in proliferative power of the hepatocytes. Blockade of the system of mononuclear phagocytes in the period of intensive DNA synthesis by hepatocytes of the regenerating liver was less effective. The facts are evidence of the important role of the Kupffer cells in regulation of reparative regeneration of the liver.

Midcycle doubling of uptake rates of adenine and serine in Saccharomyces cerevisiae

Rates of uptake of serine and of adenine were measured as a function of cell size, and therefore age, in asynchronous, exponential phase cultures of diploid Saccharomyces cerevisiae strain Y55. In both cases, uptake rates were constant during the initial third of the cell cycle and doubled during the S period in the middle part of the cycle to a constant value during the final third. Cell size and age at mid-step doubling were indistinguishable for serine and adenine uptake, and occurred during the period of DNA synthesis. The results extend an earlier hypothesis of constancy of cell growth rates (mass accumulation rates) and rates of uptake of all or almost all compounds into cells in exponential phase growth to one of piecewise constancy, with an abrupt doubling of growth and uptake rates during DNA synthesis.

Macromolecular syntheses during the cell cycles of yeast and hyphal phases of Candida albicans.

Synchronous cultures of yeast and hyphal phases of Candida albicans showed exponential increases in RNA content and stepwise exponential increases in DNA content. The periods of DNA synthesis in the two phases coincided with one another and with the budding peaks of the yeast phase. Hyphae grown in synchronous cultures also showed an exponential increase in length. The hyphal phase was therefore normal. Hyphal nuclear division occurred after hyphal DNA synthesis. Germination was a unique event for a hypha and unlike yeast bud formation, preceded the first period of DNA synthesis. The exponential increase in RNA and DNA in asynchronous cultures of hyphae in serum paralleled the exponential increase in the numbers of cells in asynchronous cultures of yeasts in defined media. Thus there are no factors in serum which inhibit the normal exponential growth of C. albicans.

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro. MMS given at 6 h produced a dose-dependent delay in the wave of DNA synthesis which normally follows partial hepatectomy. DMN at 6 h caused a dose-dependent inhibition of synthesis, restoration of total liver DNA occurring gradually during the succeeding week. In each case, the induction of DNA polymerase which normally takes place before the increase in DNA synthesis occurs was inhibited. The results suggest that damage to DNA inhibited induction of the enzymes necessary for DNA replication, and therefore extensive DNA synthesis could not occur until after the relevant damage had been repaired. The low level of synthesis on damaged DNA correlates with the fact that the incidence of tumours resulting from DMN administration at 6 h is low. When DMN or MMS were given 24 h after the operation, by which time DNA polymerase has already been induced, no evidence was obtained for a direct inhibition of the enzyme by the methylating agents. Therefore, as there is no need for further induction of essential enzymes, replication of DNA can occur before repair mechanisms have eliminated damage from the template DNA. The high incidence of tumours induced by DMN given at 24 h could be related to the replication of damaged DNA.

The effect of isoprenaline on induction of tumours by methyl nitrosourea in the salivary and mammary glands of female wistar rats

Pretreatment of rats with isoprenaline sulphate (IPR) stimulated DNA synthesis in both salivary and mammary gland tissues. Salivary gland tumours induced by N-methyl-N-nitrosourea (MNU) were observed for the first time in rats, but occurred only in IPR-pretreated animals given MNU during the period of IPR-stimulated DNA synthesis. The cumulative index of MNU-induced mammary tumours and the number of tumours per tumour-bearing rat were increased by IPR-pretreament only if the animals received MNU during the period of IPR-stimulated DNA synthesis.

DNA and RNA Synthesis by Fertilised, Cleavage Arrested Sea Urchin Eggs

Eggs of Lytechinus variegatus which have been fertilised and maintained at 10 degrees C do not undergo segmentation, but do replicate DNA. It has been found that RNA synthesis is initiated in these eggs following a period of DNA synthesis resulting in amounts of DNA/egg comparable to those amounts found in late blastulae (1000 genome equivalents). The RNA synthesised has been studied by autoradiographic and sucrose density gradient centrifugation. RNAse-sensitive grains appear in autoradiographs of cleavage-arrested eggs exposed to tritiated uridine beginning two days after fertilisation at 10 degrees C. Incorporation of labelled uridine into the 18 and 25 s region of sucrose gradients parallels the appearance of acid-insoluble radioactivity in the uridine autoradiographs. Localisation of the RNA in the autoradiographs was shown to be in close association with Feulgen-positive areas of the egg periphery. Since differentiation is generally thought to involve changing patterns of transcription of the zygote genome, transcription patterns in the absence of cleavage and normal morphogenesis is of considerable interest. The study of molecular events occurring in a time span during which there is no cell division may have bearing on the role of cell division in differentiation.