Peak Of DNA Synthesis Research Articles

Ageing factors affecting the ability of adult lens cell nuclei for cleavage and development

Adult frog lens cell nuclei resting in the G 1 phase of the cell cycle were transplanted into recipient eggs with negative results as far as cleavage or development was concerned. If these donor cells were stimulated to undergo mitosis and they were selected at their peak of DNA synthesis, positive results were obtained. Many of the experimental eggs reached the blastula stage of development. Nuclei derived from cells of these experimental blastula eggs were serially transplanted into prepared recipient eggs. An increase in positive results indicated that the “aged” differentiated nucleus can overcome some of its limitations, if it is allowed to replicate for a prolonged period of time in “young” non-differentiated cytoplasm. Unfortunately complete development was not obtained even with this technique. Ideas resulting from these experiments are discussed and ways whereby these results can be improved are suggested.

Circadian variations of DNA synthesis, mitotic activity, and cell size of hepatocyte population in young immature male mouse growing liver.

Circadian variations in DNA synthesis after tritiated thymidine and autoradiography, mitotic activity with the Colcemid method, and cell size, represented inversely by the number of cells per microscopic field, are reported for the hepatocyte population of the young immature growing liver of the male mouse. The peak of DNA synthesis is at 04:00 hours and that of mitotic activity at 12:00 hours. The 8-hour interval between the two peaks is considered, in agreement with other authors, a good estimation of the lag period between DNA synthesis and the initiation of mitotic activity. The minimum cell number per microscopic field, which is considered to correspond to the maximum cell size, coincides with the starting of the circadian mitotic wave. The significance of this coincidence and the relationships of the circadian curves of the parameters controlled with the feeding pattern and food consummatory curve of standardized mice, are discussed.

Biochemical characterization of prostatic nuclei: II. Relationship between DNA synthesis and protein synthesis

1. The maximum rate of DNA synthesis is observed at 72 h in the ventral prostate of a castrated rat that has been stimulated by exogenous testosterone. However, the maximal rate of the synthesis of all classes of nuclear proteins precedes by 24–48 h the peak of DNA synthesis. 2. The rates of synthesis of various fractions of nuclear proteins were determined. The membrane proteins appeared to have the highest rate of synthesis. 3. Studies with inhibitors indicated that the synthesis of prostatic nuclear proteins is not directly coupled to DNA synthesis. 4. DNA synthesis but not RNA synthesis in the prostate is significantly inhibited if total cellular protein synthesis is completely blocked. Inhibition of RNA synthesis by actinomycin D did not alter the rate of DNA synthesis. 5. In comparison to all the nuclear proteins studied, the inhibition of synthesis of the membrane fractions correlated most closely with the rate of decrease DNA synthesis.

Cellular events concerning the developmental potentiality of the transplanted nucleus, with reference to the aging lens cell

An improved method for isolating donor cells from amphibian lens tissue is described. Nuclei from the embryonic and adult frog lens cells were transplanted into prepared, parthogenetically activated, enucleated eggs. The activity and stage of development reached by these experimental eggs decreases as the level of differentiation and age of the donor increases. The area of the cell cycle in which the cell is residing when the donor nucleus is transplanted is of importance. The best results, as far as the developmental potentiality of the nucleus is concerned, are obtained when the donor material's nuclei are at their peak of DNA synthesis. Reasons for this increase and the factors which may be responsible for the behaviour of the aging cell are discussed.

Gene activation in WI-38 fibroblasts stimulated to proliferate: requirement for protein synthesis.

Confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by fresh medium containing 30% fetal-calf serum. Up to 80% of the cells are stimulated to divide, with a peak of DNA synthesis between 15 and 21 hr. 1 hr after the change of medium there is a 70% rise in chromatin template activity. Cycloheximide inhibited the increase in chromatin template activity. A requirement for RNA synthesis was investigated by incubating stimulated and unstimulated cells with 10 mug/ml of actinomycin D. In spite of a 95% inhibition of RNA synthesis in whole cells, purified chromatin from stimulated cells showed the usual increase in template activity. These experiments implicate a requirement for protein synthesis in template activation, and imply that the synthesis of this (or these) protein(s) is independent of RNA synthesis and regulated by a purely translational mechanism.

DNA synthesis and interaction between controlled feeding schedules and partial hepatectomy in rats.

The rate of DNA synthesis has been measured during liver regeneration in rats adapted to a controlled feeding schedule. The results show two different phenomena in the regulation of DNA synthesis. The first is the appearance of a peak of DNA synthesis following the operation itself and independent of the time of the day; the second one is the presence of constant diurnal variations in the rate of DNA synthesis in response to the partial hepatectomy and following the stimulus or stimuli of the controlled feeding schedule.

Changes in alkaline deoxyribonuclease activity in rat liver following partial hepatectomy

The activity of an endonuclease (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5) which preferentially hydrolyses native DNA has been examined during the regeneration of rat liver. The activity of this enzyme increased 2–3-fold prior to the initial post-operative peak of DNA synthesis and remained elevated for at least 70 h. The control levels of activity seen in the sham-operated animals and at 4 h following partial hepatectomy indicate that the observed increase is not due to a shock reaction.

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium.

AbstractWhen resting WI‐38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine‐3H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl‐soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non‐proliferating cells were stable for at least 12 hours. 2‐mercapto‐1‐(β‐4‐pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI‐38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.

Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts.

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40-75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [(3)H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.

The Synthesis of Acidic Nuclear Proteins in the Prereplicative Phase of the Isoproterenol-stimulated Salivary Gland

Abstract A single injection of isoproterenol results, after a lag period of 20 hours, in a marked stimulation of DNA synthesis and cell proliferation in the mouse salivary gland. The incorporation of leucine-3H into three acidic nuclear protein fractions (nonhistone nuclear proteins) increases within 30 min following the administration of isoproterenol and does not return to control levels until 48 hours later. The three fractions consist of (a) a fraction soluble in Tris-EDTA (ribonucleoprotein fraction); (b) one extractable from nuclei with 0.15 n NaCl; and (c) an acidic fraction insoluble in the above solutions and in 0.25 n H2SO4 (residual fraction). The incorporation of leucine-3H into acidic nuclear proteins reaches its peak 12 hours after isoproterenol injection while the peak activity of the histone fraction coincides with the peak of DNA synthesis. Since the specific activity of the leucine pool does not change in stimulated glands, the increased incorporation of leucine-3H into acidic nuclear proteins indicates an increase in the rate of synthesis of these proteins. Actinomycin D given 30 min prior to isoproterenol inhibits isoproterenol-stimulated DNA synthesis but does not affect the increase in the rate of synthesis of nuclear proteins which occurs 2 hours after isoproterenol. However, the increases in the rates of synthesis occurring at 8 and 12 hours are inhibited. Cycloheximide, administered 1 hour after isoproterenol, inhibits isoproterenol-stimulated DNA synthesis as well as the increase in acidic nuclear protein synthesis at 8 and 12 hours. These results indicate a temporal difference in the synthesis of histones and acidic proteins during the transition of G0 cells to the S phase and suggest that acidic nuclear proteins may be involved in the control of cell proliferation in mammalian cells.

Deoxyribonucleotide Pools and Deoxyribonucleic Acid Synthesis in Cultured Mouse Embryo Cells

Abstract Cellular pools of dATP and dTTP were determined during the growth of secondary cultures of mouse embryo cells with a recently developed, highly sensitive enzymatic method. During the most active phase of DNA synthesis, the cells contained approximately 2.5 pmoles of dATP and 3.0 pmoles of dTTP per µg of DNA. Later, during growth, these values decreased to between one-half and one-third. The pools of the two nucleotides were calculated to suffice for about 2 to 4 min of DNA synthesis. After 3 days of incubation in a medium containing limiting (0.5%) serum, the cells attained a low resting level of DNA synthesis, and the pool sizes of both nucleotides dropped to around 0.5 pmole per µg of DNA. A wave of DNA synthesis was triggered after a well defined lag phase, either by raising the serum concentration of the medium to 10% or by infecting the cells with polyoma virus. With both procedures, the pools of dATP and dTTP, which increased in parallel with the rate of DNA synthesis, reached a maximum either simultaneously with or just after the peak of DNA synthesis. In no case was there a demonstrable increase in pool sizes before the onset of DNA synthesis; in fact, in the case of the dTTP pool, a decrease was observed. After serum stimulation, the activities of ribonucleotide reductase and thymidine kinase increased about 7- and 100-fold, respectively. These shifts in enzyme activities, as well as changes in pool size of deoxyribonucleoside triphosphates, demonstrate a striking parallelism between the stimulatory effects of serum and polyoma virus infection on the initiation of DNA synthesis in mouse embryo cellS.

Studies of folate uptake by phytohaemagglutinin-stimulated lymphocytes.

Summary Cultures of lymphocytes with and without phytohaemagglutinin (PHA) were used as an in vitro model system to study the cellular uptake of tritiated folic acid, [3H]PteGlu and of14C labelled 5 methyltetrahydrofolic acid, 5 [methyl‐14C]‐H4PteGlu. Total folate uptake by PHA‐stimulated transformed lymphocytes, measured both by liquid scintillation counting and by autoradiography, was much greater than by non‐stimulated mature lymphocytes. This suggests that growing and dividing cells take up folate more avidly than mature non‐dividing cells. Uptake of both folate compounds, measured over a 4 hr period, was (a) approximately 80% less at 4°C than at 37°C, (b) exhibited saturation kinetics, and (c) was inhibited by methotrexate in concentrations of 10−3 M, 10−4 M, but was not affected by methotrexate at a concentration of 10−6 M.The results suggest that an active transport mechanism may be at least partly involved in cellular folate uptake. The two folate compounds, PteGlu and 5 methyl H4PteGlu, however, do not appear to share the same uptake pathway, since PteGlu did not inhibit the uptake of 5 [methyl‐14C]H4PteGlu by PHA‐stimulated lymphocytes. Further, the uptake of 5 [methyl‐14C]H4PteGlu by PHA‐stimulated lymphocytes from six patients with untreated pernicious anaemia was found to be significantly impaired, whereas the uptake of [3H]PteGlu by those cells was normal. Diphenylhydantoin did not show any consistent effect on folate uptake by lymphocytes. A sequential relation was found between the peak rates of RNA synthesis, folate uptake and DNA synthesis by PHA‐stimulated lymphocytes. The peak rate of folate uptake occurred at 44–48 hr of culture, and preceded peak DNA synthesis by 24 hr and followed peak RNA synthesis by 24 hr. Folate uptake, however, did not appear to be directly coupled to either DNA or RNA synthesis, since actinomycin D inhibited both DNA and RNA synthesis by transformed lymphocytes over a 4 hr period without significantly affecting folate uptake.

'Delayed' peaks of DNA synthesis in phytohemagglutinin-stimulated human leucocyte cultures.

'Delayed' peaks of DNA synthesis in phytohemagglutinin-stimulated human leucocyte cultures.

Mitomycin C may inhibit mitosis by reducing “G 2” RNA synthesis

In recent studies it was found that mitomycin C blocks mitosis in cultured frog tenses even when it is added to the. system after peak DNA synthesis. The results presented tare suggest that the mitotic inhibition produced by the antibiotic may be due to an effect of the synthesis of RNA.

Thymidine kinase, thymidylate kinase and 32P i and [ 14C]thymidine incorporation into DNA during early embryogenesis of the sea urchin

In the early embryogenesis of the sea urchin, the relationship between the activity of thymidine kinase (ATP:thymidine 5′-phosphotransferase, EC 2.7.1.21) and thymidylate kinase (ATP:thymidinemonophosphotransferase, EC 2.7.4.9) and of DNA synthesis was investigated. Thymidine kinase and thymidylate kinase activities were found in the homogenate and in the crude extract of unfertilized sea-urchin eggs and embryos. Maximal activity of the kinases was found in unfertilized eggs and the activity was decreased after fertilization. Thereafter, the activities changed periodically in parallel during the cell cycle and their maxima appeared at a defined stage of the cell cycle. [2- 14C]Thymidine and 32P i incorporation into the DNA fraction was found usually just after each division, with the exception of the first division cycle, which occurred at the stage when pronuclear fusion may take place. DNA synthesis was observed after the appearance of the peak of activity of these two kinases in the division cycles. The DNA synthesis system was not included in one division cycle (the division took place between the time of the appearance of the activity peak of DNA synthesis and that of the two kinases). In the early embryogenesis of the sea urchin, the mechanism of regulation of thymidine kinase activity was investigated and the following results were obtained. Puromycin and ethionine depressed thymidine kinase activity in vivo. Thus, the increase of thymidine kinase activity may be due mainly to enzyme synthesis de novo. Although thymidine kinase was observed to have a low stability, the enzyme had a similar lability in the homogenates of unfertilized eggs and of embryos. Thymidine protected thymidine kinase activity from heat inactivation. dTTP was a feedback inhibitor, but dCDP was not an activator.

The in vitro response of thymic lymphocytes of the pig to phytohemagglutinin.

AbstractThe response of thymic lymphocytes of the pig to phytohemagglutinin was studied with H3 thymidine in cultures, from 0–72 hours. At the beginning of the culture period 6–18% of lymphocytes were in DNA synthesis. during the first 24 hours a sharp decrease in the number of DNA synthesizing cells was observed in both pha and control cultures, although pha cultures consistently showed small but significantly greater numbers of DNA synthesizing cells. this was followed by a definite peak in DNA synthesis and mitotic response of a minority of the cells in pha cultures between 48–54 hours, whereas in control cultures activity ceased. in addition, a small proportion of the progeny of initially DNA synthesizing medium sized lymphocytes was apparently stimulated by pha and found in mitosis by 48 hours.It was concluded that the thymus contains a fraction of lymphocytes, not in the mitotic cycle, which are capable of being transformed by pha to mitotic activity. the data also suggests some stimulation of cells already in the mitotic cycle.