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.     

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.     

Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine.

Flow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker-256 carcinoma cells. After both in vivo and in vitro pulse labelling with BrdUrd, Walker-256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase. BrdUrd-labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle. To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro. The results indicated exponential growth characteristics for this tumor between days 5 and 8 after implantation. Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar. The mean time for DNA synthesis was estimated from the relative movement of BrdUrd-labelled cells towards G2. The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration. This study demonstrates that BrdUrd labelling of rat Walker-256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor-bearing rat.     

Linear Cell Growth in Escherichia coli

Growth was studied in synchronous cultures of Escherichia coli, using three strains and several rates of cell division. Synchrony was obtained by the Mitchison-Vincent technique. Controls gave no discernible perturbation in growth or rate of cell division. In all cases, mean cell volumes increased linearly (rather than exponentially) during the cycle except possibly for a small period near the end of the cycle. Linear volume growth occurred in synchronous cultures established from cells of different sizes, and also for the first volume doubling of cells prevented from division by a shift up to a more rapid growth rate. As a model for linear kinetics, it is suggested that linear growth represents constant uptake of all major nutrient factors during the cycle, and that constant uptake in turn is established by the presence of a constant number of functional binding or accumulation sites for each growth factor during linear growth of the cell.     

Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry

The cell cycle kinetics of Escherichia coli B/r A and B/r K cells were studied by flow cytometry. Three-dimensional histograms of cell cultures show the number of cells as a function of cellular DNA and protein contents and give detailed pictures of the cell cycle distribution with regard to these parameters. Histograms of slowly growing chemostat cultures showed that cell cycle periods B and C + D increase with a decreasing growth rate and that the B period occupies an increasing fraction of the cycle. The DNA replication patterns of B/r A and K were found to be quite similar. At extremely low growth rates (doubling time [T] = 17 h), B/r A cells had a B period of 0.8 T, a C period of 0.1 T, and a D period of 0.1 T, and B/r K cells (T = 16 h) had a B period of 0.6 T, a C period of 0.15 T, and a D period of 0.25 T. Mass increase, i.e., essentially protein synthesis, was seen in all three periods of the cell cycle. For B/r A cells, the average rate of mass increase was 11 times greater in the D period than in the B period, whereas for B/r K cells the rate of mass increase was twice as great in the D period as in the B period. The DNA and cell size distributions of batch cultures in exponential growth were found to vary with time, indicating that such cultures are not suitable for studies of cell cycle kinetics.     

Cell elongation and division probability during the Escherichia coli growth cycle.

A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions. The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history. This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min. The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division. The average value of the coefficient of variation was 13.5% for all strains and growth rates. These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E. coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential.     

Growth kinetics under adenine-limiting conditions of Bacillus subtilis KYA 741, an adenine-requiring strain

The growth kinetics of Bacillus subtilis KYA 741, an adenine-requiring strain, was investigated under adenine-limiting conditions. The concentration of adenine (the limiting substrate for cell growth) in the culture filtrate remained constant during the stationary phase. In this phase, DNA turnover was active and the DNA content per cell was constant throughout the cultivation period. When cells were transferred to medium without adenine, the cell concentration began to decrease immediately and then reached a constant level due to the supply of adenine from lysing to growing cells. The rates of degradation of cells and DNA were both found to be 0.2 hr^?1. An equation for cell growth in this pseudostationary phase was obtained by combining Contois' equation, in which the apparent saturation constant was a function of the cell concentration, with a term for cell degradation. This equation satisfactorily expressed the feature of cell growth and adenine consumption by B. subtilis KYA 741 under adenine-limiting conditions.     

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the process of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Athough the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation.

F plasmid replication during the Escherichia coli division cycle was investigated by using the membrane-elution technique to produce cells labeled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The F plasmid replicated, like the minichromosome, during a restricted portion of the bacterial division cycle; i.e., F plasmid replication is cell-cycle specific. The F plasmid replicated at a different time during the division cycle than a minichromosome present in the same cell. F plasmid replication coincided with doubling in the rate of enzyme synthesis from a plasmid-encoded gene. When the cell cycle age of replication of the F plasmid was determined over a range of growth rates, the cell size at which the F plasmid replicated followed the same rules as did replication of the bacterial chromosome--initiation occurred when a constant mass per origin was achieved--except that the initiation mass per origin for the F plasmid was different from that for the chromosome origin. In contrast, the high-copy mini-R6K plasmid replicated throughout the division cycle.     

Kinetics of human lymphocyte responses in vitro: the phase of exponential growth

The kinetics of thymidine uptake in human peripheral lymphocytes stimulated by allogenic cells, antigen E (ragweed allergen) and a variety of mitogens can generally be divided into four consecutive phases. First, a lag period with no increase in thymidine uptake, then a short period of rapid change in uptake, followed by a log-linear growth period and finally a decay phase. In this report we examine in detail the characteristics of the third, log-linear growth phase. Since, as discussed in the preceding paper, thymidine uptake is proportional to the number of cells acumulating thymidine, we can calculate from the log-linear growth period an apparent doubling time. We show that for five different stimulating agents the cells reach a log-linear growth phase of varying length and that the doubling times show little variation. This invariance indicates that, despite possible variation in cell death and recruitment rates, the rate of proliferation is in all cases dominated by the generation time of human lymphocytes.     

PERIODIC CHANGES IN RATE OF AMINO ACID UPTAKE DURING YEAST CELL CYCLE

Uptake of amino acids is a complex process but in cells growing with ammonia as sole nitrogen source the initial uptake rate of amino acids is a measure of the transport capacity of the uptake system (permease). In synchronous cultures of Saccharomyces cerevisiae amino acids were transported at all stages of the cell cycle. However, for any one amino acid the initial uptake rate was constant for most of the cycle and doubled during a discrete part of the cycle. Thus, for a variety of amino acids the functioning amino acid transport capacity of the membrane doubles once per cycle at a characteristic stage of the cycle. Arginine, valine, and phenylalanine exhibit periodic doubling of uptake rate at different stages of the cell cycle indicating that the transport of these amino acids is mediated by three different systems. Serine, phenylalanine, and leucine exhibit periodic doubling of the uptake rate at the same stage of the cycle. However, it is unlikely that serine and phenylalanine share the same transport system since the uptake of one is not inhibited by the other amino acid. This phenomenon is analogous to the periodic synthesis of soluble enzymes observed in S. cerevisiae.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Actinomycin D: Effects on Mouse L-Cells

The lethal and inhibitory effects of actinomycin D (Act D) on asynchronous and synchronized populations of mouse L-cells have been studied. It has been shown that the survival curve of populations in the logarithmic phase of growth can be approximated by two exponential survival curves corresponding to a sensitive and resistant moiety. The size and sensitivity of both moieties vary during the growth of the population. As the cell population moves through logarithmic and into stationary phase, the sensitive moiety becomes smaller but more resistant whereas the resistant moiety increases in size and also becomes more resistant. This variation appears to be related to a reduced uptake of Act D and also a reduced rate of DNA and RNA synthesis. Variations in sensitivity to the drug have also been observed during the division cycle of synchronized cells with cells in the S phase showing the greatest uptake of the drug and also the greatest sensitivity. However, no direct correlation between uptake and sensitivity has been established. Actinomycin D has inhibitory effects on both RNA and DNA synthesis. RNA synthesis is inhibited rapidly but does not seem to drop to less than 5% of the control value. The inhibition of DNA synthesis appears to occur over a longer period and may reach values as low as 0.25% of control. In both cases the degree of inhibitions appears to be dependent on both the length of exposure and the concentration of the drug. Certain similarities between the response of cells to Act D and X-rays have been observed and are discussed.     

Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations.

A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.     

KINETICS OF HUMAN LYMPHOCYTE RESPONSES IN VITRO: THE PHASE OF EXPONENTIAL GROWTH

The kinetics of thymidine uptake in human peripheral lymphocytes stimulated by allogenic cells, antigen E (ragweed allergen) and a variety of mitogens can generally be divided into four consecutive phases. First, a lag period with no increase in thymidine uptake, then a short period of rapid change in uptake, followed by a log-linear growth period and finally a decay phase. In this report we examine in detail the characteristics of the third, log-linear growth phase. Since, as discussed in the preceding paper, thymidine uptake is proportional to the number of cells acumulating thymidine, we can calculate from the log-linear growth period an apparent doubling time. We show that for five different stimulating agents the cells reach a log-linear growth phase of varying length and that the doubling times show little variation. This invariance indicates that, despite possible variation in cell death and recruitment rates, the rate of proliferation is in all cases dominated by the generation time of human lymphocytes.     

Rate and topography of cell wall synthesis during the division cycle of Salmonella typhimurium.

The rates of synthesis of peptidoglycan and protein during the division cycle of Salmonella typhimurium have been measured by using the membrane elution technique and differentially labeled diaminopimelic acid and leucine. The cells were labeled during unperturbed exponential growth and then bound to a nitrocellulose membrane by filtration. Newborn cells were eluted from the membrane with fresh medium. The radioactivity in the newborn cells in successive fractions was determined. As the cells are eluted from the membrane as a function of their cell cycle age at the time of labeling, the rate of incorporation of the different radioactive compounds as a function of cell cycle age can be determined. During the first part of the division cycle, the ratio of the rates of protein and peptidoglycan synthesis was constant. During the latter part of the division cycle, there was an increase in the rate of peptidoglycan synthesis relative to the rate of protein synthesis. These results support a simple, bipartite model of cell surface increase in rod-shaped cells. Before the start of constriction, the cell surface increased only by cylindrical extension. After cell constriction started, the cell surface increased by both cylinder and pole growth. The increase in surface area was partitioned between the cylinder and the pole so that the volume of the cell increased exponentially. No variation in cell density occurred because the increase in surface allowed a continuous exponential increase in cell volume that accommodated the exponential increase in cell mass. Protein was synthesized exponentially during the division cycle. The rate of cell surface increase was described by a complex equation which is neither linear nor exponential.     

The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts

Histone variant synthesis patterns from human diploid fibroblast-like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors.

A mouse-mouse hybridoma cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration. The transient kinetics of cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods. The cell-specific kinetic parameters (growth and metabolic rates) as well as cell size were constant throughout the exponential phase. Intracellular protein and RNA content followed a similar trend. Cell growth stopped when the glutamine in the medium was depleted. Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion. Ammonia and lactate production followed closely glutamine and glucose consumption, respectively. Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed. The cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation. The antibody was produced at a constant rate in both the exponential and decline phases of growth. The intracellular antibody content of the cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards.