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.     

Induction of synchronous growth in the yeast phase of Wangiella dermatitidis.

Synchronous yeast-phase cultures of Wangiella dermatitidis were induced by starvation, heat shock, and inhibition of deoxyribonucleic acid synthesis by hydroxyurea. Hydroxyurea-induced synchrony resulted in some distortion of the yeast-phase cell cycle. However, induction of synchrony by hydroxyurea is a rapid and simple technique which generates a marked degree of synchronous growth.     

Potassium Uptake in Synchronous and Synchronized Cultures of Escherichia coli

Criteria are presented for distinguishing between synchronous and synchronized cultures (natural vs. forced synchrony) on the basis of characteristics of growth and division during a single generation. These criteria were applied in an examination of the uptake of potassium during the cell growth and division cycle in synchronous cultures and in a synchronized culture of Escherichia coli. In the synchronous cultures the uptake of ⁴²K doubled synchronously with cell number, corresponding to a constant rate of uptake per cell throughout the cell cycle. In the synchronized culture, uptake rates also remained constant during most of the cycle, but rates doubled abruptly well within the cycle. This constancy of ⁴²K uptake per cell supports an earlier interpretation for steady-state cultures that uptake is limited in each cell by a constant number of functional sites for binding, transport, or accumulation of compounds from the growth medium, and that the average number of such sites doubles late in each cell cycle. The abrupt doubling of the rate of uptake of potassium per cell in the synchronized culture appears because of partial uncoupling of cell division from activation or synthesis of these uptake sites.     

Drifts in DNA level in the cyanobacterium Anacystis nidulans during synchrony induction and in synchronous culture

Cultures of the cyanobacterium Anacystis nidulans were synchronized by using alternating light-dark cycles. The DNA level in the cells was determined, at intervals, during pre-synchrony treatment and subsequent synchronous growth. The DNA content/cell gradually increased during synchrony induction and reached a maximum value after about 9–10 dark-light cycles, coinciding with the minimum length of pre-synchrony treatment necessary for obtaining good synchrony of cell division in our system. DNA synthesis was found to be discontinuous in the synchronous cultures. The results suggest two gaps in DNA synthesis, one occurring before and one after cell division. The results are compared with the relevant data published on the life cycle of other prokaryotic microorganisms.     

Production of minimally disturbed synchronous cultures of hematopoietic cells

A method is describedforproducing sizable quantities of synchronously dividing, minimally disturbed mammalian cells. Cultures were grown immobilized on surfaces such that cell division within the population resulted in the continuous release of synchronous newborn cells. As judged by the quality and duration of synchronous growth, cell size distributions, and DNA compositions, newborn mouse L1210 cells grew with a very high level of synchrony without overt evidence of growth disturbances. The technology should be applicable to a variety of hematopoietic cells, as evidenced by similar results with human MOLT-4 and U937 cell lines.     

A model of cell cycle control: effects of thymidine on synchronous cell cultures.

Further evidence is presented in support of a model for growth control in which commitment for cell division is determined by an event in the preceding cell cycle. A study was made of conditions affecting synchronous growth following treatment of murine mastocytoma cells with excess thymidine at different phases of the cell cycle. Cells were synchronized by a physical procedure involving velocity sedimentation in a zonal rotor. Pulse treatment of such cultures with thymidine at times corresponding to the S, G2, and M periods had no effect on further growth. However, addition at G1, although having no immediate effect, arrested cell growth in the next cell cycle. This temporal effect may account for the decay of synchrony observed during double thymidine blockade or thymidine-FUdR blockade. When the time interval between two such blocks was 7 hr or less, P815Y cells were arrested after one synchronous division. At this critical time a majority of cells were at, or near, G1. It is suggested that thymidine exerts a hitherto unrecognized effect at the G1 interval.     

Use of zonal centrifugation for preparing synchronous cultures from Ehrlich ascites cells grown in vivo

A method is described which permits the selection of Ehrlich ascites tumour cells belonging to different stages of the cell cycle. The cells are grown in vivo and separated by zonal centrifugation immediately after being withdrawn from the intraperitoneal cavity of mice. Further in vitro growth of cells derived from different fractions obtained by zonal centrifugation demonstrates that this method permits the preparation of synchronous cultures which begin to grow at different points of the cell cycle and which maintain synchrony for at least two further cycles. The selection procedure seems to cause the subsequent appearance of a shortened cell cycle which develops more gradually in asynchronous primary cultures. Advantages of this method are, the large capacity and easy availability of the starting material.     

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.     

Effects of hydroxyurea on Crithidia fasciculata.

Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45-50 h; if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Effects of Hydroxyurea on Crithidia fasciculata

SYNOPSIS Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45–50 h: if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Bacillus volatiles antagonize cyanobacteria

Abstract Vegetative cells of Dictyostelium discoideum were synchronized by a size selection method which gave good synchrony without indication of any respiratory perturbation or abnormal cellular appearance. During synchronous growth, fluctuations in respiratory activity and in total cellular protein content were consistently observed (14 experiments). Control cultures in which the entire exponential population was selected for under identical centrifugation conditions employed to produce the synchronous cultures did not show this pattern; neither did cells subjected to anaerobiosis, cold shock or centrifugation, which were performed to induce perturbation. It appears that extensive turnover of cellular proteins accompanied by respiratory fluctuations occur in the absence of perturbation during vegetative growth of Dictyostelium discoideum . This picture may represent the changes occurring in single cells which would not be evident in time-averaged observations of exponential cultures.     

Characterization of a CV-1 cell cycle. V. An assessment of velocity sedimentation for cell separation and synchrony.

Velocity sedimentation at unit gravity has been used to enrich populations of logarithmically growing cells in different cell cycle phases. In order to evaluate the degree of synchrony obtained by this method of cell separation, synchronous populations of CV-1 cells, initially obtained by the selective detachment of mitotic cells from roller cultures, were separated by velocity sedimentation. It was found that although the mean cell volume increased linearly, the cells remained heterogeneous with respect to size during all phases of the cell cycle. Since the velocity sedimentation technique depends upon discrimination of cell size, the size heterogeneity of cells throughout the cycle limits the degree of synchrony which can be obtained by this method.     

Partial synchrony in soybean cell suspension cultures induced by ethylene

Partial synchrony of cell division in continuous cultures of soybean cell suspensions was obtained by flushing the cultures with ethylene at intervals of 36 h. The most pronounced synchrony resulted from flushing the suspensions with 3% ethylene for 3 h, followed immediately by 3% CO₂ for 3 h and 30 h aeration prior to the next ethylene treatment. Soybean cells responded to this regime of gassing also with a significant enhancement of growth.     

The basis of synchronization by repetitive dilution of a growing culture

Cultures of Escherichia coli have been synchronized by periodic dilution with fresh growth medium in the laboratory of Francois Kepes. When diluted by a large factor into complete test medium, the treated cultures undergo up to 12 synchronous divisions. This long term synchrony must result from an adjustment process during the periodic dilution procedure so that all cells have nearly identical biochemical properties. Robert Pritchard (University of Leicester, personal communication) suggested that this phasing would happen if the uptake of a critical nutrient was limited by the surface area of the cell during a portion of the dilution cycle. If his suggestion is valid, a general method for synchronization of almost any organism that grows exponentially and divides by binary fission into equal sized daughters should be achievable. A computer program was devised to simulate the growth of an initially asynchronous culture under periodic dilution with medium containing a single limiting nutrient. Various models of cell shape and growth were tested along with various models for the growth-limiting substrate uptake.     

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.     

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.