Synchronization of Division in Vitamin B12-Starved Euglena gracilis

SYNOPSIS 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.     

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.     

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.     

Preparation and characterization of Cryptococcus neoformans synchronous culture

We have developed a method for preparation of synchronous culture in Cryptococcus neoformans. The method is based on age fractionation of exponentially growing asynchronous culture through differential sedimentation in 10-20% (w/v) lactose gradient. C. neoformans capsule thickness should be reduced to a minimum to ensure most accurate age fractionation, which is necessary to obtain a higher degree of synchrony. The C. neoformans synchronous culture system has revealed important characteristics with respect to cellular morphology, DNA content and cell volume distribution. The method can be used for further cell cycle studies.     

NUCLEAR SYNCHRONY IN VALONIA MACROPHYSA(CHLOROPHYTA): LIGHT MICROSCOPY AND FLOW CYTOMETRY1

The coenocytic alga Valonia macrophysa Kützing was selected for an investigation of nuclear synchrony in the order Siphonocladales. Light microscopy reveals that nuclear synchrony is evident as patches of nuclei dividing simultaneously. Flow cytometry was utilized for the first time with a macroalga for cell-cycle analysis. Results indicate that nuclei in the entire cell exhibit a high degree of synchrony throughout the cell cycle. Also it appears that cells within a clonal culture are synchronous with each other, in their progression through the cell cycle. The advantages of using flow cytometry for cell-cycle analysis of coenocytic algae include the rapid collection of quantitative data on relative DNA content for a large number of nuclei.     

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.     

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.     

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.     

Changes in cell wall constituents during the cell cycle in a synchronous culture of Catharanthus roseus

Changes in cell wall constituents during the cell cycle were investigated using a synchronous culture of Catharanthus roseus (L.) G. Don which was obtained by the double phosphate starvation method (S. Amino et al. 1983. Physiol. Plant. 59: 393–396). Cell walls isolated from the cells in each phase of the cell cycle were fractionated into EDTA-soluble (pectin), 5 and 24% KOH-soluble (hemicellulose) and 24% KOH-insoluble (cellulose) fractions. Their sugar compositions were investigated by gas chromatography and methylation analysis. The following changes were observed: (1) a significant increase in total cell walls in the G1 phase after cell division, (2) a temporary increase in the relative amount of the EDTA-soluble fraction during cytokinesis, (3) an increase in the relative amount of galactose, probably 4-linked galactose, in the EDTA-soluble fraction prior to cytokinesis, (4) a temporary increase in the relative amount of 3-linked glucose during cytokinesis, (5) little change in the composition of polysaccharides throughout the cell cycle in the 24% KOH-soluble fraction, which consisted mainly of xyloglucan. The changes observed are discussed in relation to the progression and physiological significance of each phase of the cell cycle.     

Induction and stabilization of synchrony in the cell division of Escherichia coli

Escherichia coli strains B5 and B/r/1 were grown under conditions of periodic glucose starvation in a minimal medium. Such conditions of growth give rise to two synchronous populations that are out of phase regarding their time of division, one dividing shortly after a new supply of fresh medium and the other at a later stage of the feeding cycle. Preferential selection of one of the two populations using heat treatment resulted in a homogeneous synchronized culture that exhibited in a non-limiting medium a high degree of synchrony that was long lasting. Synchrony and its persistence could survive preservation of such a synchronized culture by freeze drying. An explanation of the synchrony persistence was put forward and the practical implications of these findings were discussed.     

Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.     

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.     

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.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

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.     

Macromolecular Synthesis in Saccharomyces cerevisiae in Different Growth Media

Synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein was determined in Saccharomyces cerevisiae during amino acid and pyrimidine starvation and during shift-up and shift-down conditions. During amino acid starvation, cell mass, cell number, and RNA continued to increase for varying periods. During amino acid and pyrimidine starvation, cell mass and RNA showed little increase, whereas total DNA increased 11 to 17%. After a shift from broth medium to a minimal defined medium, increase in RNA and protein remained at the preshift rate before assuming a lower rate. DNA increase remained at an intermediate rate during shift-down, and then dropped to a low rate. During shift-up from minimal to broth medium, increase in cell number, protein, and DNA showed varying lag periods before increasing to the new rate characteristic of broth medium; each of these quantities exhibited a step sometime in the first 2 hr after transfer to rich medium, suggesting a partial synchronous division. Immediately after shift-up, RNA synthesis assumed a high rate, and then dropped to a rate characteristic of growth in the rich medium after about 1 hr.     

Macromolecular syntheses and the course of cell cycle events in the chlorococcal algaScenedesmus quadricauda under nutrient starvation: Effect of nitrogen starvation

Daughter cells of the chlorococcal algaScenedesmus quadricauda incubated under photosynthesizing conditions in a nitrogen-free medium did not make any progress in the cell cycle. Photosynthetic starch formation continued for a period corresponding to a half of the cell cycle and then levelled off. Protein synthesis was very slow and it did not surpass double the initial amount. RNA content decayed from the start of treatment and approached about 2 pg/cell. When a synchronous population was deprived of nitrogen or of light in the middle of the cell cycle RNA synthesis stopped immediately or very soon afterwards and, in spite ofabundant intracellular nitrogen reserves, RNA content slowly declined. This degradation was much extensive in nitrogen starved cells where, eventually, the RNA content attained about half the starting value. In both experimental variants, DNA replications started at the same time as in control culture, but the final amount of DNA attained only half the control value. Protein synthesis stopped immediately in the dark. In the nitrogen-starved cells, it continued for several hours and protein content increased about 70 % of the amount present at the start of starvation. The number of daughter cells formed was proportional to the final protein content in the nitrogen-and light-deprived cells (corresponding division numbers were 6 and 4, respectively). Upon refeeding of daughter cells formed under nitrogen starvation, RNA synthesis started immediately, while protein synthesis displayed a lag of about 5 h. DNA replications were triggered at the time when the ratio of RNA to DNA content attained the same value as in the control culture.     

CHANGE OF UBIQUITINATED PROTEINS DURING THE CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYTA)1

Changes in the amount of heat shock-related ubiquitinated proteins in Chlamydomonas were investigated during the cell cycle and gamete induction. In a division-synchronized culture induced by periodic illumination, the amount of the 28-kDa ubiquitinated protein increased during the dark phase. This increase correlated with the increase of total DNA. Such an increase was repressed when nuclear DNA replication was inhibited with aphidicolin. These results suggest that ubiquitination to form the 28-kDa protein is involved in nuclear DNA replication or during the cell cycle. The amount of 31-kDa ubiquitinated protein gradually increased throughout the light phase and decreased in the dark phase. The amount of 28-kDa ubiquitinated protein also increased during gamete induction caused by nitrogen starvation, while that of the 31-kDa did not. These results suggest that the change of ubiquitination of 28-kDa protein mat play a fundamental role in the cell cycle and gamete induction in Chlamydomonas.     

The dynamic distribution and quantification of DNA cruciforms in eukaryotic nuclei

Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies specific for cruciforms have been produced. The antibody binds to structural determinants at the base of the cruciform stem, the "elbow." Labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary. This pattern of fluorescence changes when these cells are released from synchrony. Using fluorescence flow cytometry to quantify the number of DNA cruciform structures in cells throughout the cell cycle, we observed two major populations of nuclei with different numbers of cruciforms; the modal number of cruciforms in these populations was 0.6 x 10(5) and 3 x 10(5) cruciforms per nucleus. Synchronized cells (doubly arrested by serum starvation and aphidicolin) displayed a biphasic distribution of the number of cruciforms over the first 6 h after release from synchrony with maxima at 0 and 4 h after release.