Analysis of the schedule of DNA replication in heat-synchronized Tetrahymena

The temporal schedule of DNA replication in heat-synchronized Tetrahymena was studied by autoradiographic and cytofluorometric methods. It was shown that some cells, which were synchronized by selection of individual dividing cells or by temporary thymidine starvation, incorporated [³H]thymidine into macronuclei in a periodic fashion during the heat-shock treatment. It was concluded that supernumerary S periods occurred while cell division was blocked by high temperature. The proportion of cells which initiated supernumerary S periods was found to be dependent on the duration of the heat-shock treatment and on the cell cycle stage when the first heat shock was applied. Cytofluorometric measurements of Feulgen-stained macronuclei during the heat-shock treatment indicated that the DNA complement of these cells was substantially increased and probably duplicated during the course of each S period. Estimates of DNA content also suggested that the rate of DNA synthesis progressively declined during long heat-shock treatments. These results indicate that the mechanism which brings about heat-induced division synchrony is not an interruption of the process of DNA replication. Further experiments were concerned with the regulation of DNA synthesis during the first synchronized division cycle. It was shown that participation in DNA synthesis at this time increased as more cells were able to conclude the terminal S period during the preceding heat-shock treatment. It is suggested that a discrete period of time is necessary after the completion of DNA synthesis before another round of DNA synthesis can be initiated.     

THE RELATIONSHIP BETWEEN DEOXYRIBONUCLEIC ACID REPLICATION AND CELL DIVISION IN HEAT-SYNCHRONIZED TETRAHYMENA

The effect of supraoptimal temperature on macronuclear DNA synthesis in Tetrahymena was studied by radioautography during prolonged heat and heat-shock synchronization treatments. Prolonged heat treatments (34°C) delayed the initiation of S, but did not appreciably delay DNA synthesis in progress. Return to optimal temperature (28°C) 50 or 100 min later resulted in initiation of S, in delayed cells, at a rate greater than in controls. During the synchronization treatment, most cells were unable to enter S during a heat shock, but initiated S with a slight delay during the following intershock period. These cells were not appreciably delayed in completion of S by subsequent heat shocks. Supraoptimal temperature appears to affect the DNA synthetic cycle near the G₁ to S transition. Cells subjected to the heat-shock treatment in early G₁ all participated in one S period, and many underwent a succession of two S periods. DNA synthesis occurred in about 50% of the cells between EST and the first synchronous division, with the likelihood of DNA synthesis becoming greater the longer the interval between these two events. In some cells no detectable DNA synthesis occurred between EST and the second synchronous division. It was concluded that a precise temporal alternation of DNA replication and cell division is not obligatory in Tetrahymena.     

Erythropoietic progenitors capable of colony formation in culture: response of normal and genetically anemic W-W-V mice to manipulations of the erythron

The macromolecular reguirements for the initiation and maintenance of macronuclear DNA replication were studied in heat synchronized Tetrahymena pyriformis GL-C. Previous work had established that macronuclear S periods could occur in a consecutive fashion without intervening cell divisions during a multiple heat shock treatment, as well as immediately following the synchronized cell divisions. Cycloheximide treatment prior to or during the S period which follows the first synchronized cell division resulted in abolition of the initiation of DNA synthesis or an almost immediate cessation of DNA synthesis in progress. Temporary inhibition of DNA synthesis occurred when cycloheximide was added late in the S period. Treatment with actinomycin D was found to block the initiation of DNA synthesis but did not appreciably affect the continuation of the S period. It was concluded that RNA synthesis was required for the initiation but not the maintenance of DNA replication, whereas protein synthesis was necessary for both processes. The dependency of the initiation of an S period on prior RNA and protein synthesis was also shown to exist when a second consecutive S period was initiated without a preceding cell division. Treatment with actinomycin or cycloheximide prior to a supernumerary S period during a multiple heat shock treatment completely abolished the initiation of DNA synthesis. In T. pyriformis the synthesis of RNA and protein related to the initiation of the S period is tightly coupled to each cycle of DNA replication.     

Protein synthesis requirements at specific points in the interphase of meristematic cells

A 6 h treatment with anisomycin at a concentration of 1 μg/ml enables us to modify the steady-state kinetics of a meristematic cell population of Allium cepa, and this points to a difference of sensitivity to inhibition of protein synthesis between the several periods of the cell division cycle (G1, S, G2, M). The results show that the cells are incapable of entering the S period in the presence of the inhibitor, but that, where DNA synthesis has already been initiated, the synthesis continues in the cells in question. It was found, moreover, that there is a point in the early G2 period, which has a duration of approx. 3 % of the total duration of the cycle, at which the synthesis of specific proteins appears to determine the progression of cells to mitosis.     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.     

The effect of light on DNA synthesis and related processes in synchronous cultures of Chlorella

Summary In autotrophic cultures of Chlorella synchronised by alternating light and dark periods of 16:8 hours the DNA content duplicated normally 4 times successively during the S _mphase, i. e. between the 10th and 18th hour after the beginning of the light period. This finding together with electron microscopical observations revealed that one duplication of the DNA and of the nuclei per cell proceeds every 110 minutes. All nuclei of a cell seem to undergo successive DNA syntheses and nuclear divisions synchronously. The rate of DNA synthesis was independent from illumination. On appropriate reduction of the light period the last duplication cycle fell out and the average final spore number per cell was accordingly lower.If a culture was transferred to darkness or low light intensity 3 hours before the normal end of the light period the release of spores was promoted by approximately 1 1/2 hours, provided a strong decrease of metabolically accessible carbohydrates was prevented by either an additional short illumination during the dark period or by continuing the weak light.A possible explanation for the shortening of the cell development is that, by passing over one DNA duplication and one protoplast division, the cell can enter sooner the respective subsequent developmental stages.     

THE MECHANISM OF 5-AMINOURACIL-INDUCED SYNCHRONY OF CELL DIVISION IN VI CIA FABA ROOT MERISTEMS

Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the thymine analogue, 5-aminouracil. Recovery of mitotic activity began 8 hours after removal from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By use of single and double labeling techniques, it was shown that synchrony of cell divisions resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G₂ period. It appeared to have no effect on the duration of the G₁ period. When roots were removed from 5-aminouracil, DNA synthesis resumed in all cells in the S phase. Although thymidine antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine.     

Variation in periodic replication of the chromosome in Escherichia coli B/rTT.

A method using 5-bromouracil photolysis induction with 313 nm radiation was employed to estimate the variation in the period between successive rounds of DNA replication in rapidly growing cultures of Escherichia coli$\text{B}\text{rTT}$ The coefficient of variation of this period was 9.3%, which is significantly less than the corresponding value of about 20% reported for variation in the cell interdivision period. Thus chromosome replication is much more tightly controlled than is cell division. The reduced variability of the DNA replication cycle indicates that the period (D) between termination of a round of DNA replication and cell division and the following period ending in initiation of the next round of DNA replication (B) are riot independent of each other but tend to have compensatory variations. The results suggest that other events in the cell cycle are related more closely to DNA replication rather than to the much less regular event of cell division.     

Regulation of Cell Division in Escherichia coli

The rate of cell division was measured in cultures of Escherichia coli B/r strain after periods of partial or complete inhibition of deoxyribonucleic acid (DNA) synthesis. The rate of DNA synthesis was temporarily decreased by removing thymidine from the growth medium or replacing it with 5-bromouracil. After restoration of DNA synthesis, a temporary period of accelerated cell division was observed. The results were consistent with the idea that chromosome replication begins when an initiator complement of fixed size accumulated in the cell. The increase in the potential for the initiation of new replication points during inhibition of DNA synthesis results in an increase in the rate of cell division after an interval which encompasses the time for the arrival of these replication points to the termini of the chromosomes and the time from this event to division.     

CHANGES IN THE DNA SYNTHESIS PATTERN OF PARAMECIUM WITH INCREASED CLONAL AGE AND INTERFISSION TIME

The clonal age in paramecia refers to the total number of vegetative divisions a clone has undergone since its origin at autogamy (self-fertilization). As clonal age increases, the interfission time usually increases. The DNA synthesis pattern of cells of different ages was compared by autoradiographic analysis of the DNA synthesis of synchronized cells at various time intervals during the cell cycle (from one division to the next). The study showed that the G₁ period (the lag in DNA synthesis post division) was constant, irrespective of interfission time or clonal age; but the duration of the DNA synthesis period increased with increased interfission time or clonal age. Therefore, we have shown for the first time that the G₁ period is fixed, and the S period is increased in a eukaryotic unicellular organism as a function of interfission time and clonal age.     

MACROMOLECULAR SYNTHESES RELATED TO THE REPRODUCTIVE CYST OF TETRAHYMENA PATULA

Macromolecular syntheses in encysted Tetrahymena patula were studied using Feulgen fluorescence cytophotometry, autoradiography, and inhibitors of RNA and protein synthesis. Cycloheximide significantly depressed protein synthesis and D-actinomycin effectively blocked RNA synthesis. Under these conditions, the cells within the cyst were unable to divide. Both cytophotometric measurements and autoradiographic data with tritiated thymidine show that DNA synthesis does not occur during the encystment divisions. Excysted cells placed in nutrient broth medium showed a prolonged generation time after the first cell growth cycle, and by the third generation the mean DNA content per cell was almost triple that of starved excysted cells. These findings indicate that (a) the encystment divisions require RNA and protein synthesis, which are apparently effected through turnover, (b) the encystment division cycles occur in the absence of DNA synthesis, and (c) excysted cells placed in culture medium may go through more than one DNA replication per cell cycle.     

Methionine biosynthesis in Saccharomyces cerevisiae: mutations at the regulatory locus ETH2. I. Genetic data

Summary Evidence has been obtained that sodium azide is an inhibitor of cell division in wild-type and aziA strains of Salmonella typhimurium. The bacteria grown in media containing sodium azide and glucose formed long filaments. It has been found that sodium azide had a stronger inhibitory effect on DNA synthesis than on cell mass increase. When filaments produced by azide action were transferred to azide-free medium very rapid increase in DNA content was observed during the first 45 min. After this time, when relative DNA content was increased the rate of DNA synthesis was reduced and cell divisions reappeared.Inhibitory effect of azide on DNA biosynthesis in vitro was observed with toluenized cells of S. typhimurium. Only ATP-dependent radioactive dTMP incorporation into DNA was affected by sodium azide. It had no effect on the incorporation in the absence of ATP.Mutant aziC was isolated in S. typhimurium by scoring for clones with normal cell division in the presence of sodium azide. Azide had much less effect on DNA biosynthesis in vivo and in vitro in aziC cells as compared with isogenic controls.This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1.     

Requirements for DNA replication preceding cell division in Tetrahymena pyriformis

Populations of Tetrahymena pyriformis were synchronized by 30 min heat shocks at 34 °C separated by 160 min intervals at the normal growth temperature. The cells initiate DNA synthesis immediately after the cellular division, and the S period of the population lasts about 80 min. It was found that DNA replication is a prerequisite for the following synchronous division. Inhibition of the DNA synthesis in early S by starvation of the cells for thymidine prevents the forthcoming division. However, inhibition in the latter half of S does not prevent the subsequent division. Thus the cells have synthesized enough DNA to permit cell division before the end of a normal S period. These results are discussed in relation to the organization of the genome replication in the highly polyploid macronucleus.     

On the mode of action of 5-diazouracil on bacterial cell division

Cell division by strains ofEscherichia coli andSalmonella typhimurium is inhibited by 5-diazouracil (5-DU). Division recovers in the presence of the inhibitor after a period which is temperature-dependent. Recovery is probably due to breakdown of 5-DU and the rate of this breakdown is apparently increased at alkaline pH. Growth with 5-DU caused only a slight reduction in the rate of murein synthesis and no alteration in the properties or composition of membranes ofS. typhimurium. The agent caused chaining inStreptococcus fecalis and inhibition of the penicillin-induced lysis ofS. typhimurium. These effects may have been due to direct inhibition of lysin activity but an indirect effect seems more likely. The most marked effect of 5-DU onS. typhimurium was to cause a transient inhibition of DNA synthesis. Since 5-DU did not stop uncoupled cell division (i.e. division occurring independently of DNA replication) and sincelon^? strains were more sensitive to 5-DU thanlon⁺ strains, it was concluded that 5-DU acts on cell division via an inhibitory effect on DNA replication.     

Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents

Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.     

Coordination of division events in theChlamydomonas cell cycle

Summary At concentrations that did not affect growth, hydroxyurea and 2¹-deoxyadenosine inhibited DNA synthesis inChlamydomonas. Evidence that initiation of mitosis is dependent upon completion of DNA replication was provided by the arrest of inhibited cells with undivided nuclei containing undispersed nucleoli. Initiation of cytokinesis is not dependent upon progress of nuclear division since, in arrested cells, cleavage microtubules became deployed in a phycoplast and a cleavage furrow developed fully, until obstructed by the undivided nucleus. Chloroplast constriction and division also continued independently of nuclear division. It is concluded that nuclear division, cytoplasmic cleavage and chloroplast division are in separate sequences of dependent events. This is supported by flexibility of their relative timing in successive divisions, since after the first commitment to divide nuclear division is followed by initiation of cleavage and then chloroplast division, whereas following subsequent commitments these events occur in reverse time order. This flexibility of order indicates changing rates of progress through separate sequences of events.Deposition of wall material was dependent upon the completion of cytokinesis, but this inhibition of wall deposition by incomplete cytokinesis did not extend to other daughters within the same mother cell.These observations are correlated with our earlier data concerning the rate-limiting control points for division and a model for the coordination of division events is presented. The relationships between different plant cell cycles is discussed in view of the findings presented.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : II. Recovery from Unbalanced Growth Induced by Vitamin B12 Deficiency

When vitamin B₁₂ is added to B₁₂-deficient cultures of Euglena gracilis, the cells undergo two relatively synchronous cell divisions within a shorter than usual period of time, apparently as a result of a transitory shortening of the cell cycle. The first cell division pulse, occurring 4.5 h after addition of B₁₂, is preceded by the completion of DNA duplication, but appears to involve no net synthesis of RNA or protein. Before the second round of cell division at about 11 h, a significant amount of DNA synthesis is observed. This time it is accompanied by a minor increase in the RNA and protein content of the culture. The cellular contents of RNA and protein were observed to decrease steadily after the resumption of cell division in B₁₂-depleted cultures receiving the vitamin. Ultimately all three macromolecules returned to their nondeficient, plateau stage levels; by this time, cell division had ceased.     

CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS

The question of amino acid requirements for DNA synthesis and cell division has been studied in Tetrahymena pyriformis by depriving cells of histidine and tryptophan at defined stages in the interdivision interval. Deprivation any time before DNA synthesis does not prevent the initiation of such synthesis but completely inhibits the following division and limits the increase in DNA, as measured microspectrophotometrically, to 20 per cent. H³-thymidine added to the medium is not incorporated during the 20 per cent increase. Deprivation after DNA synthesis is initiated does not prevent the continuation (to completion) of DNA synthesis, and cell division ensues. H³-thymidine added to the medium under these conditions is incorporated into macronuclear DNA. The data indicate that some amino acid-dependent event occurs, about the time of the beginning of the DNA synthesis period, which is not essential for initiation of DNA synthesis but which is essential for the maintenance of synthesis once it has begun. These results are further discussed in terms of enzymes required to convert thymidine (and possibly the other three deoxyribonucleosides) to the immediate precursor of DNA synthesis.     

Bacterial filaments recover by successive and accelerated asymmetric divisions that allow rapid post-stress cell proliferation

Filamentation is a reversible morphological change triggered in response to various stresses that bacteria might encounter in the environment, during host infection or antibiotic treatments. Here we re-visit the dynamics of filament formation and recovery using a consistent framework based on live-cells microscopy. We compare the fate of filamentous Escherichia coli induced by cephalexin that inhibits cell division or by UV-induced DNA-damage that additionally perturbs chromosome segregation. We show that both filament types recover by successive and accelerated rounds of divisions that preferentially occur at the filaments' tip, thus resulting in the rapid production of multiple daughter cells with tightly regulated size. The DNA content, viability and further division of the daughter cells essentially depends on the coordination between chromosome segregation and division within the mother filament. Septum positioning at the filaments' tip depends on the Min system, while the nucleoid occlusion protein SlmA regulates the timing of division to prevent septum closure on unsegregated chromosomes. Our results not only recapitulate earlier conclusions but provide a higher level of detail regarding filaments division and the fate of the daughter cells. Together with previous reports, this work uncovers how filamentation recovery allows for a rapid cell proliferation after stress treatment.     

Arrest of cell division blocks the utilization of polyamines in synchronized cultures of photoautotrophically grown Euglena

A trimodal change in the cellular levels of three major polyamines: spermidine, N,N′-bis(3-aminopropyl)-1, 3-propanediamine (BAP) and 3,3′-diaminodipropylamine (DAD) was observed during two successive cell cycles in synchronously dividing cultures of the algal flagellate, Euglena gracilis Z photoautotrophically grown in a 24-h light-dark cycle. The intracellular levels of these three polyamines decreased as cells divided and then were enhanced as cells exited the G₁ phase and proceeded through the S and G₂ phases. Spermidine, BAP and DAD concentrations increased about 2.5-fold during the S phase. Putrescine and 1,3-diaminopropane levels did not vary significantly. One peak of polyamine synthesis occurred in the G₁ phase prior to DNA synthesis, followed by a second more important peak during the S-G₂ phases before cell division; both peaks were observed during the light period. A third minor peak was observed during the pre-G₁ (or G₀) phase in the dark period after mitosis had been completed. In contrast, when the cells attained the “stationary” phase of growth, there was no significant increase in the content of polyamines during the light period although spermidine and BAP increased slightly twice during the dark period (putrescine and 1,3-diaminopropane and DAD levels remained almost constant). To ascertain whether the synthesis of polyamines was merely a direct effect of the photoperiod, parallel experiments with synchronous cultures were carried out in the presence and absence of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, a photosynthetic inhibitor. Although a slight decrease in the concentration of polyamines was observed, the three maxima of polyamines synthesis were observed as in normal cultures. These results clearly suggest that polyamine biosynthesis is closely related to DNA replication and cell division in Euglena cells.