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

Daughter cells of the chlorococcal algaScenedesmus quadricauda incubated under photosynthesizing conditions in a phosphate-free medium accomplished one cell cycle but divided into a lower number of daughter cells compared to the control. RNA synthesis was restricted early in the cell cycle while protein synthesis was retarded as compared to the control only at the end of the cycle. The number of DNA replication rounds (and consequently the number of divisions) was reduced in proportion to the lower content of RNA per cell. Daughter cells produced by phosphorus-starved mother cells and grown further in a phosphorus free medium performed no net RNA, DNA and protein synthesis within the period corresponding to the duration of control cell cycle an o were unable to develop. They accumulated, however, about half the amount of starch found in normally developed mother cells. In a complete medium, the phosphorus-starved daughter cells resumed macromolecular syntheses with a lag of about 5 h. Thereafter, their development and reproductive processes were comparable to those in a healthy population. A similar course of recovery was obtained with starved daughter cells exposed to light in phosphorus-free medium for the period corresponding to one cell cycle. Thanks to the large amount of starch accumulated in these cells, they were able to run through an entire cell cycle in the dark after being supplied with phosphorus. The first response to phosphorus withdrawal from the nutrient medium was the restriction of RNA synthesis. This occurred in spite of the fact that phosphorus reserves in the cell were still abundant, which suggests an intimate link between the supply of exogenous phosphorus to the cell and RNA synthesis.     

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.     

Effects of 3,4-benzopyrene on the course of cell cycle events in the chlorococcal alga Scenedesmus quadricauda

Synchronous cultures of the chlorococcal alga Scenedesmus quadricauda were grown under optimal growth conditions. The mean length of their cell cycle was approximately 20 h. The cultures were treated at the start, at the 4th, and 8th hour of the cell cycle with 3,4-benzo(a)pyrene (BP) in the range of 0.1–0.5 g ml^-1 of final concentration. A period about 4 h was found within which no inhibitory effects could be detected even at the highest BP concentrations used. In presence of BP the rates of RNA and protein syntheses gradually decreased until complete inhibition of net syntheses occurred. In a similar way chlorophyll synthesis was inhibited, and this was followed by gradual degradation of the chlorophyll. The higher the concentration of BP the more rapid the decrease of the rates of syntheses and the earlier their complete inhibition. At low BP concentrations while DNA replications were initiated, the number of replications was lowered. At higher concentrations the initiations of DNA replications were delayed or completely suppressed. Syntheses of saccharides were the least inhibited processes in presence of BP. Starch synthesis was slowed down at the end of the cell cycle and fructose synthesis (free and sucrose bound) was even stimulated later in the cell cycle. The release of daughter coenobia, and protoplast fissions were most susceptible to BP treatment, being affected at concentrations which produced no measureble disturbances of macromolecular syntheses. At BP concentrations at which the inhibition of macromolecular syntheses occurred, the delay or suppression of mitoses was observed.Abbreviations BP 3,4-benzo(a)pyrene - PhAR photosynthetically active radiation     

Effect of irradiance on the course of RNA synthesis in the cell cycle ofScenedesmus quadricauda

In synchronous populations ofScenedesmus quadricauda the RNA amount in the cells increases in waves: periods of a high rate of RNA synthesis alternate with periods of a low rate in the course of the cell cycle. Each wave usually leads to the doubling of the RNA amount per cell. In cells growing under normal conditions the waves of RNA synthesis seem to be linked with consecutive rounds of DNA replication. The pattern of RNA synthesis in the course of the cell cycle, however, does not change, if DNA replication is prevented by application of 5-fluoro-deoxyuridine. In darkness the rate of RNA synthesis drops to zero and thereafter the RNA amount per cell decreases. In cells which have been induced to cellular division RNA synthesis may become restored in the dark in newly formed daughter cells. The lowering of RNA amount and its new increase during the dark period become more pronounced with increasing irradiance in the previous light period as well as with its increasing length. In the period of protoplast fissions RNA synthesis is arrested even if the cells divide in the light; whether a similar inhibition occurs during mitoses is not clear.     

Uncoupling of chloroplast reproductive events from cell cycle division processes by 5-fluorodeoxyuridine in the algaScenedesmus quadricauda

Summary FdUrd (5-fluorodeoxyuridine), a specific inhibitor of thymidylate synthase, was used to study the relationship between reproductive processes in chloroplast and nucleocytoplasmic compartments of the chlorococcal algaScenedesmus quadricauda. The courses of DNA replication and nuclear division in both the compartments were followed in populations synchronised by the alternation of light and dark periods. DAPI-staining of DNA-containing structures was used for their visualisation and quantification. In contrast with cellular reproductive events, those in chloroplasts were not substantially affected by the presence of FdUrd (25 g/ml). It was shown that FdUrd specifically blocked nucDNA replication but not ptDNA replication. Thus, cells which had attained commitment to ptDNA replication, fission of pt-nuclei and chloroplast kinesis triggered and terminated these processes while the corresponding cellular processes were blocked. The courses of reproductive processes in chloroplasts were also substantially unaffected in cells grown in the presence of FdUrd for the whole cell cycle. This provided evidence that attainment of commitment to and termination of the entire sequence of reproductive events, including chloroplast fission, were controlled by different mechanisms than the reproductive processes in the nucleocytoplasmic compartment.Abbreviations DAPI 4,6-diamidino-2-phenylindole - ptDNA DNA of chloroplast nuclei - nucDNA DNA in cell nuclei - FdUrd 5-fluorodeoxyuridine     

Variations in chloroplast nucleoid number during the cell cycle in the algaScenedesmus quadricauda grown under different light conditions

Summary Synchronous cultures of the green algaScenedesmus quadricauda were grown at different mean irradiances (ranging from 15 Wm^–2 to 130Wm^–2). At each irradiance, the algae were exposed to illumination regimes which differed in light duration and dark intervals (222 to 240 hours). The cells from these cultures were sampled during their cycles, stained with DAPI and the number of nuclei and chloroplast nucleoids estimated.The nucleoids divided semisynchronously in steps which represented doublings in their number. For each doubling a constant amount of light energy (defined as the product of irradiance and light duration) had to be converted by the cells to become committed to this division. The times to the start of the nucleoid divisions were therefore inversely proportional to the irradiances applied and the final number of nucleoids was proportional to the light duration.Temporal relationships between nuclear and nucleoid divisions were also light dependent. Shortage of light energy caused delay in nucleoid division. The cell division rate was higher than the rate of nucleoid division and consequently, the cells tended to decrease their nucleoid number with decreasing irradiance. With increasing irradiance the start of nucleoid division was gradually shifted toward the beginning of the cell cycle. The rate of nucleoid division exceeded the rate of nuclear and cellular division, thus with increasing irradiance cells with increasing numbers of nucleoids were formed.Abbreviations DAPI 46-diamidino-2-phenylindole - pt-DNA chloroplast DNA     

Regulation of chloroplast and cytoplasmic rRNA accumulation by light energy and its relation to reproductive events during the cell cycle of the alga Scenedesmus quadricauda

Synchronous cultures of the green alga Scenedesmus quadricauda were grown at different mean irradiances (ranging from 20 W/m² to 150 W/m²). At these irradiances, the algae were exposed to illumination regimes which differed in the ratio between light and dark intervals (from 02.22 to 24.00 h). The patterns of accumulation of chloroplast and cytoplasmic ribosomal RNA (chl- and cyt-rRNA) and variations in their ratio were followed under the above mentioned growth conditions. The chl-rRNA accumulated at a high specific rate even in the dark while the synthesis of cyt-rRNA was depressed. Consequently, the ratio of chl-/cyt-rRNA increased during the dark period to the high value of 0.4. After the start of the light period, this ration decreased gradually to the low value of 0.2. In continuously illuminated cells, the chl-rRNA and cyt-rRNA were accumulated at the same specific rate of that their ratio remained constant (0.2) during entire cell cycle. However, the absolute amount of cyt-rRNA accumulated in light was about 10 times higher than that of chl-rRNA. In an experiment with 5-fluorodeoxyuridine treated cells, the evidence was provided that DNA replications, nuclear divisions and chloroplast nucleoid fissions interferred with the course of neither chl-rRNA nor cyt-rRNA during the cell cycle. Chloroplastkinesis and cytokinesis were the only reproductive events that prevented the accumulation of both chl- and cyt-rRNA.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

Divider size and the cell cycle after prolonged starvation ofTetrahymena corlissi

Cell growth and division of the ciliateTetrahymena corlissi were examined upon refeeding after prolonged starvation of up to 12 days. Division did not automatically occur when a certain critical cell size was reached. Rather, it varied both with the nutritional history of the cell and the nutrient conditions in which the cell was growing. Upon refeeding, cells starved for 12 days divided at a smaller size and later than cells starved for 6 days. Cells refed at high density took longer to begin division than cells refed at low density. The results are discussed with respect to the relative starvation and critical constituent models of the cell cycle and in terms of the polymorphic life cycle ofTetrahymena species.     

Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts

The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.     

Effect of inositol starvation on the in vitro syntheses of mannan and N-acetylglucosaminylpyrophosphoryldolichol in Saccharomyces cerevisiae.

An early consequence of starvation for inositol in yeast is inhibition of synthesis of the major cell wall components mannan and glucan. In looking for the mechanism of this inhibition, we found that the activity of the enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryldolichol was diminished in particular membrane preparations from cells starved for inositol. This loss of reactivity was observed under a variety of in vitro assay conditions and could be restored by the addition of phosphatidylinositol but not by other phosphoinositol-containing sphingolipids known to occur in yeast. When assayed in the presence of high concentrations of Triton X-100, enzyme preparations from both control and inositol-starved cells required phosphatidylinositol for maximal activity. Since this enzyme catalyzed an early step in the synthesis of mannan that is N-linked to protein, a reasonable hypothesis is that inhibition of mannan synthesis in inositol-starved cells results from the depletion of the necessary cofactor phosphatidylinositol.     

Accumulation, activity and localization of cell cycle regulatory proteins and the chloroplast division protein FtsZ in the alga Scenedesmus quadricauda under inhibition of nuclear DNA replication

Synchronized cultures of the green alga Scenedesmus quadricauda were grown in the absence (untreated cultures) or in the presence (FdUrd-treated cultures) of 5-fluorodeoxyuridine, the specific inhibitor of nuclear DNA replication. The attainment of commitment points, at which the cells become committed to nuclear DNA replication, mitosis and cellular division, and the course of committed processes themselves were determined for cell cycle characterization. FdUrd-treated cultures showed nearly unaffected growth and attainment of the commitment points, while DNA replication(s), nuclear division(s) and protoplast fission(s) were blocked. Interestingly, the FdUrd-treated cells possessed a very high mitotic histone H1 kinase activity in the absence of any nuclear division(s). Compared with the untreated cultures, the kinase activity as well as mitotic cyclin B accumulation increased continuously to high values without any oscillation. Division of chloroplasts was not blocked but occurred delayed and over a longer time span than in the untreated culture. The FtsZ protein level in the FdUrd-treated culture did not exceed the level in the untreated culture, but rather, in contrast to the untreated culture, remained elevated. FtsZ structures were both localized around pyrenoids and spread inside of the chloroplast in the form of spots and mini-rings. The abundance and localization of the FtsZ protein were comparable in untreated and FdUrd-treated cells until the end of the untreated cell cycle. However, in the inhibitor-treated culture, the signal did not decrease and was localized in intense spots surrounding the chloroplast/cell perimeter; this was in agreement with both the elevated protein level and persisting chloroplast division.     

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.     

Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Periodic events and cell cycle regulation in the plasmodia ofPhysarum polycephalum

The multinucleated plasmodia ofPhysarum polycephalum, a myxomycete, have been extensively used in cell cycle studies. The natural synchrony of mitosis and DNA synthesis, easy culture methods, the ready fusions obtainable between plasmodia, and the amenability to phase specific studies, employing physical and chemical perturbers, are some of the attractive features of this organism. Because of the absence of a Gl phase in the plasmodia, there is a crowding of cell cycle specific marker events at the G2/M boundary, which reflect features of both the G2/M and the Gl/S boundaries of a typical eukaryotic cell. Prominent among these are the synthesis and overall activity of thymidine kinase, the co-triggering of tubulin and histone genes, translation of their mRNA, the organization and duplication of the microtubular organizing centres of the mitotic spindle and the triggering of cdc 2 kinase activity. These above events have not only served as good markers to monitor the progress of the plasmodial cell cycle, but have also been fairly thoroughly analysed by means of specific perturbers such as DNA synthesis inhibitors, antimicrotubular drugs, UV-irradiation, heat-shock etc. Along with fusion studies, these perturbation studies have been helpful in the formulation of various models on regulation of mitosis. These above aspects as well as prospects for future studies employing this organism are discussed This paper is dedicated to the memory of the late Prof. S C K Nair, formerly University Professor of Physics.     

Key events in the cell cycle, their regulation and organization

The review surveys the studies of molecular genetic mechanisms of the cell cycle control on various eukaryotic models. The major cell cycle phenomena are considered: (1) checkpoints and their role in preserving DNA integrity and fidelity of mitosis, (2) the cell oscillator model, and (3) the role of cyclins in timing of cell division and coordination of mitotic events. The main classes of regulatory proteins involved in the cell cycle are discussed in detail.