Synchronous growth and genome replication in the blue-green alga Anacystis nidulans

Summary Following the induction of synchronous growth of Anacystis nidulans by light and CO₂ deprivation, cell mass and RNA and DNA content during two cell cycles were measured. Both RNA and DNA synthesis were discontinuous and marked variation in survival to ultraviolet light was related to the state of replication. A model is presented which accounts for the proportion of cells (66%) induced into synchronous genome replication which is also related to the state of replication at the onset of pre-synchrony treatment.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.     

Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus

A synchronous cell division system was established using the double phosphate starvation method, based on the observation that one of the limiting factors in the growth of a suspension culture of Catharanthus roseus (L.) G. Don cells in the medium of Murashige and Skoog was phosphate. In the system, an increase in cell number took place in a short period of only 4 h, while the cell number remained almost constant during other periods of the cell cycle. The synchrony of the culture was confirmed by changes in mitotic index, which increased sharply prior to the increase in cell number. The S phase was determined by measuring incorporation of [³H]-thymidine into the DNA fraction during the cell cycle and synchrony of DNA synthesis was verified likewise. Synchronization by phosphate starvation is discussed in relation to the function of phosphate as a nutrient. The synchronous system thus established will be useful in biochemical studies of the cell cycle in higher plants.     

Cyclic appearance of aerobic nitrogenase activity during synchronous growth of unicellular cyanobacteria

The aerobic synchronous growth of marine unicellular cyanobacteriaSynechococcus spp., with molecular nitrogen as the sole nitrogen growth nutrient source, was experimentally demonstrated. Cell division synchrony was induced by withholding aeration and illumination for 20 h. Three distinct cell division cycles of approximately 20 h each were observed. During the first cell division cycle, high degrees of synchrony of 78%–86% were observed in the series of cultures studied. The aerobic nitrogenase activity appeared shortly while the cell division was completed and the cell size was still small, and it disappeared after the cells were enlarging photosynthetically. Thus, three distinct cycles in aerobic nitrogenase activity were also observed having approximately the same 20-h interval as the cell division cycle. The peak aerobic nitrogenase activity was determined to be as high as 840–1220 nmol C₂H₂ reduced per milligram dry weight per hour.     

Shifts in nuclear and cytoplasmic nucleic acid content in temperature-synchronized Tetrahymena pyriformis (HSM)

Nuclei were isolated by exposing temperature synchronized Tetrahymena pyriformis (HSM) to Triton-X-100. Cell division synchrony was induced with a repetitive 12-hour temperature cycle (9.5 hours at 13°, 2.5 hours at 29°). Increase in nucleic acid content was biphasic: primarily during the last two hours of the cold period well in advance of the synchronous burst of division and secondarily in the last hour of the warm period. Nuclear RNA content rises almost two hours ahead of cytoplasmic RNA which shows a maximum 0.5 hour before the onset of the warm period. The DNA content reaches a peak 30 minutes later. On the basis of these shifts there appears to be not net synthesis of nucleic acids during cell division. The changes in RNA/DNA of the isolated macronuclei and micronuclei suggest enhanced RNA turnover, loss to the cytoplasm and enhanced ribonuclease activity prior to cell division. Cytoplasmic RNA also appears to be subject to enzymic degradation.     

Role of silicon in diatom metabolism

Levels of the cyclic nucleotides, cAMP and cGMP, were determined in four species of pennate diatoms; changes in their levels and ratios were monitored in silicon-starved and light-dark synchronized cultures of Cylindrotheca fusiformis. Content of both cAMP and cGMP changed during the cell cycles: when silicate was added to starved cultures, cAMP, cGMP and DNA levels rose rapidly; cAMP and cGMP declined before DNA synthesis was complete and continued to fall during the events leading to cell separation. In unstarved synchronies, net synthesis of DNA continued until cell separation; 1 h before cell separation cAMP levels fell while those of cGMP rose. The results support the proposal that cAMP and cGMP may play a part in the process of cell division in the diatom, possibly involving silicon.     

Chloroplast replication in synchronously dividing Euglena gracilis

Summary Chloroplast replication was studied in Euglena gracilis Klebs, strain Z, synchronized by appropriate light-dark cycles. The chloroplasts divide synchronously, at the time of cytokinesis, but with a tighter synchrony than cell division itself. The chloroplasts within one cell are not noticeably better synchronized than those in the whole population. Chloroplast replication and cell division could not be separated by resetting the time of the light-dark cycle which induces the synchrony. These results are discussed for their implications concerning the mechanisms of integrating cell and plastid division.     

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.     

Measurements and models of synchronous growth of fission yeast induced by temperature oscillations

Pulsing of temperature in a fermentor at intervals coincident with cell generation time was used to induce synchrony in a population of the fission yeast Schizosaccharomyces pombe. Measurements of culture protein, RNA, and DNA during synchronous growth confirm continuous synthesis of protein and RNA and discontinuos synthesis of DNA as previously reported. Flow microfluorometry of populations at different times during the synchrony cycle was used to monitor the changes in single-cell protein. RNA, and DNA frequency functions. These measurements illustrate very clearly the degree of synchrony and patterns of macromolecular synthesis and also confirm previous estimates of the cellular protein contents characteristic of dividing cells. Additional insights into single-cell kinetics and division controls are provided by two-parameter flow microfluorometry measurements and by mathematical modeling of population dynamics. Such data are necessary foundations for robust population balance models of microbial processes.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

Inhibition of an early event in the cell division cycle of Escherichia coli by FL1060, an amidinopenicillanic acid.

Analysis of exponential and synchronous cultures of Escherichia coli B/r after the addition of FL1060 indicates a block point for division by this agent some 15 to 20 min before the end of the preceding cell division cycle, a time corresponding to the beginning of the C period of the cell division cycle. Morphological examination of FL1060-treated synchronous cultures of E. coli /r was consistent with inhibition by FL1060 of a very early event in the cell division cycle. This event appears to be essential for normal cell surface elongation in a rod configuration. Temporary treatment of synchronous cultures of E. coli B/r with FL1060 resulted in division delay, the extent of which was a function of the duration of exposure to FL1060. However, even after relatively long times of FL1060 treatment the delayed divisions were still synchronous. Although FL1060 had no direct effect on deoxyribonucleic acid (DNA) synthesis, the synchronous delayed division occuring after temporary treatment with FL1060 were accompanied by a delay in the attainment of resistance of cell division to inhibitors of DNA, ribonucleic acid, and protein synthesis. These results suggest aht an FL1060-sensitive event initiates at the beginning of the C period of the cell division cycle of E. coli and is responsible for normal cell elongation. This cell elongation pathway procedes independently of DNA synthesis, but there is an interaction between this pathway and termination of a round of DNA replication in which a normal rod configuration is necessary to allow a signal for cell division to be generated upon completion of DNA replication.     

Wall structure of a bitunicate ascus

Summary Ultraviolet light-induced, bleached Euglena clones exhibit synchronous steps of cell division in response to daily cycles of light and dark. The cyclic division activity, in the bleached cells, will persist in constant lighting conditions with a period, independent of temperature, of about 24 h. This persisting rhythm of cell division supports the hypothesis that this phase of the cell cycle may be coupled to the fluctuations of the endogenous circadian clock of the cell.Newly isolated bleached clones are sensitive to light in their growth rates and metabolic characteristics, showing light induced difference in substrate-stimulated respiration, and production of the polyglucan, paramylon. After repeated subculturing of a bleached clone the photosensitivity of the metabolic characteristics and of the growth rate are diminished along with the ability to photo-entrain division synchrony. Division control and the induction of cell synchrony in this organism apparently involve both the temporal influence of the endogenous cell clock and one or more other photosensitive reactions in the metabolism of the cell.A unique culture mixing technique utilizing the bleached Euglena, failed to support the hypothesis of the involvement of intercellular communication in the maintenance of cell synchrony in constant lighting conditions.     

Bildung Zusätzlicher DNS nach Blockierung der Zellteilung von Tetrahymena durch Actinomycin

Summary The effect of 0,4 g of actinomycin per ml medium on DNA synthesis in synchronous cultures of Tetrahymena pyriformis strain HSM was studied. Synchronous cultures were obtained by selecting cells from a stock culture which were all in the same division phase In this concentration actinomycin inhibits cell division but permits the normal doubling of DNA and furthermore another period of macronuclear DNA synthesis. In this additional DNA production phase the rate and the synchrony of DNA synthesis is reduced as revealed by autoradiography. The production of additional DNA was demonstrated by photometric determination of Feulgen stainable material. These findings indicate that the onset of DNA synthesis is independent of a preceding cell division, of a preceding nuclear division, of the average amount of DNA present, and of the main portion of RNA synthesis.

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Herrn Professor Dr. R. Danneel zu seinem 65. Geburtstag.

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Unterstützt durch Sachbeihilfen der Deutschen Forschungsgemeinschaft.     

The action of 5-amino uracil on log growth and division-synchronized Tetrahymena

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.     

Thymidine as a synchronizing agent. I. Nucleic acid and protein formation in synchronous HeLa cultures treated with excess thymidine

Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.     

Synchrony in human,mouse and bacterial cell cultures--a comparison

Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.     

Stepwise effects of cytokinin activity and DNA synthesis upon mitotic cycle events in partially synchronized tobacco cells

Cytokinin addition to tobacco cell suspensions induced synchronous cell division after an 18 h lag period. Although continuous presence of the cytokinin in the culture medium during this lag period was essential to division, cytokinin was not required during mitosis itself. For each cell generation, cytokinin-dependent events are thus completed before mitosis occurs.Two experiments suggested that these cytokinin-dependent events are independent of DNA synthesis:

1. (i) With or without cytokinin, DNA synthesis proceeded normally in the presence of auxin, for at least the time required for one cell generation in complete medium.

2. (ii) In the presence of cytokinin, when DNA synthesis in the lag period was inhibited by FUdR, one normal cell division occurred when cytokinin was withdrawn and DNA synthesis restored by thymidine addition.

In cytokinin-starved cells, metaphase was greatly prolonged although prophase was unaffected.     

Synchrony in Human,Mouse and Bacterial Cell Cultures: A Comparison

Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.     

Synchronous Growth and Sporulation of Bacillus megaterium

Filtration of late log-phase cultures of Bacillus megaterium ATCC 19213 grown on defined sucrose salts medium (SS) or SS plus glutamate medium (SSG) through nine layers of Whatman no. 40 filter paper in a fritted-glass disc Büchner funnel resulted in filtrates containing cells which showed synchronous growth and proceeded to sporulation. SS cells completed one synchronous division after filtration; sporulation ensued after the cessation of growth. SSG cells completed two synchronous divisions and sporulation occurred during the second division. A high degree of synchrony of vegetative growth of SSG cells was evident by the stepwise pattern of growth, by the doubling of cell numbers at each division, the high division index, and by the rapid formation of sporulation cell types and homogeneity of cell types in the filtered cultures when compared with asynchronous cultures. Because the described system gives both good growth and sporulation synchrony, the method should be useful in delineating early events in sporulation and their regulation.     

Synchronization of division in vitamin B12-starved Euglena gracilis.

Vitamin B12 deficiency arrests cell division in Euglena gracilis. B12 starvation for short periods made it possible to induce synchronous growth by addition of the vitamin. Culture conditions were established to optimize replenishment synchrony. The DNA content of E. gracilis in steady state culture and vitamin B12 deficiency culture was measured by flow cytofluorometry and was consistent with colorimetric determinations. The cell volume and DNA distributions of E. gracilis in synchronous culture were analyzed and the sequential changes during the division cycle were computed. Synchronous culture permits more definitive studies of shifts in cell volume and DNA distributions, in which the biochemical events required for cell division are presumably synchronized.