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.     

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.     

MACROMOLECULAR SYNTHESES RELATED TO THE REPRODUCTIVE CYST OF TETRAHYMENA PATULA

Macromolecular syntheses in encysted Tetrahymena patula were studied using Feulgen fluorescence cytophotometry, autoradiography, and inhibitors of RNA and protein synthesis. Cycloheximide significantly depressed protein synthesis and D-actinomycin effectively blocked RNA synthesis. Under these conditions, the cells within the cyst were unable to divide. Both cytophotometric measurements and autoradiographic data with tritiated thymidine show that DNA synthesis does not occur during the encystment divisions. Excysted cells placed in nutrient broth medium showed a prolonged generation time after the first cell growth cycle, and by the third generation the mean DNA content per cell was almost triple that of starved excysted cells. These findings indicate that (a) the encystment divisions require RNA and protein synthesis, which are apparently effected through turnover, (b) the encystment division cycles occur in the absence of DNA synthesis, and (c) excysted cells placed in culture medium may go through more than one DNA replication per cell cycle.     

Synchronous growth and synthesis of macromolecules in a naturally wall-less volvocale,Dunaliella bioculata

Summary Dunaliella bioculata, a naturally wall-less unicellular green alga, can be induced to divide synchronously when subjected to a 12 hours light-12 hours dark cycle. This rhythmic cell division will last for at least 15 days under a subsequent constant illumination. Synchronization can be improved when cells are submitted to 8 hours light-16 hours dark cycles under bright white light (10,000 lux). In these conditions the cell division gives rise to two daughter cells: The chronology of DNA, RNA and proteins synthesis has been studied during such a synchronized cell cycle. DNA synthesis begins 4 hours before the outset of cell division and is completed after two hours in the dark; in difference, illumination seems necessary to the synthesis of RNA and proteins.     

Degradation of cell constituents during starvation of Salmonella enteritidis

Salmonella enteritidis starved in Fernbach flasks used acid-alcohol-soluble material and RNA as endogenous reserves during starvation. Organisms starved in a fermentor system consumed acid-alcohol-soluble material, RNA and protein to maintain viability. Half-life survival times were 132 h and 118 h for the Fernbach and fermentor-starved cells, respectively. The acid-alcohol-soluble fraction of the cell consisted mainly of peptides or protein. This fraction accounted for most of the loss of label from 14C-labelled cells during the first 5 days of starvation and presumably contains the primary endogenous reserve. Although the residue fraction of fermentor-starved cells provided 35% of the total loss of 14C from the cells by the 5th day, a small net increase in 14C activity of the residue fraction of Fernbach-starved cells was observed. Differences observed appeared to be due to the method of starvation.     

Flow cytometric analysis of the cellular DNA content of Salmonella typhimurium and Alteromonas haloplanktis during starvation and recovery in seawater.

Flow cytometry was used to investigate the heterogeneity of the DNA content of Salmonella typhimurium and Alteromonas haloplanktis cells that were starved and allowed to recover in seawater. Hoechst 33342 (bisbenzimide) was used as a DNA-specific dye to discriminate between DNA subpopulations. The DNA contents of both strains were heterogeneous during starvation. S. typhimurium cells contained one or two genomes, and A. haloplanktis cells contained up to six genomes. S. typhimurium genomes were fully replicated at the onset of starvation. Each replication cycle was completed in the early stage of starvation for A. haloplanktis by stopping cells in the partition step of the cell cycle prior to division. Multigenomic marine cells can undergo rapid cell division without DNA synthesis upon recovery, resulting in large fluctuations in the DNA contents of individual cells. In contrast, the heterogeneity of the DNA distribution of S. typhimurium cells was preserved during recovery. The fluctuations in the DNA fluorescence of this strain seem to be due to topological changes in DNA. Flow cytometry may provide a new approach to understanding dynamic and physiological changes in bacteria by detecting cellular heterogeneity in response to different growth conditions.     

Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris

In situ hybridization with a fluorescently labeled 16S rRNA-targeted probe was examined using Rhodopseudomonas palustris as a model organism, which had been grown at different rates and under different conditions of growth and starvation. The specific growth rate did not affect the percentage of hybridized cells in aerobically grown R. palustris cultures. However, significant changes in the percentage of hybridized cells occurred during extended periods of starvation. These changes were observed both in batch cultures grown and starved aerobically in the dark, and in cultures grown phototrophically and starved anaerobically in the dark. Aerobic growth in batch culture and subsequent starvation resulted in a complete lack of detectable hybridization after 20 days of starvation. In contrast, even after 30 days of starvation, 50% of all cells were still detectable in cultures grown aerobically at growth rates <0.06 h(-1) and then starved aerobically in the dark. The same was true for phototrophically grown cells that were starved anaerobically in the light. During starvation there was a clear, though non-linear, positive correlation between the percentage of hybridized cells and the RNA content. In contrast, no direct correlation was observed between the number of hybridized cells in a culture and the viability of this culture. Thus, in habitats with growing, non-growing, and starving bacteria, data on quantitative detection of populations based on 16S rRNA-targeted probing should be used with extreme caution as the detectability of the individual cells is strongly influenced by their physiological history and current physiological state.     

RNA synthesis in myeloma cells synchronized by isoleucine starvation.

Myeloma cells have been synchronized by isoleucine starvation. Changes in RNA synthetic rates as a result of starvation have been studied. The ability of isolated nuclei to synthesize RNA declines on starvation and increases subsequently on refeeding isoleucine. There is a coordinate drop in synthetic rate for all three polymerases both in vivo and in vitro. The chain elongation rate in vitro is the same in starved and normal cells, so the difference is in the number of active polymerases in vitro. However, the nuclei do not exactly parallel the state of the cell from which they were isolated, but the in vitro RNA synthesis increases more slowly than the in vivo RNA synthesis. There is no change in relative amounts of synthesis by the different RNA polymerases. The in vitro RNA product is similar in starved and growing cells.     

The developmental fate of Dictyostelium discoideum cells depends greatly on the cell-cycle position at the onset of starvation

The relationship between the development of Dictyostelium discoideum Ax-2 and the cell cycle at the onset of starvation was analysed with special reference to sorting behaviors during the formation of polarized cell masses (slugs), using a method for inducing good synchrony. Cells starved at different cell-cycle positions showed different developmental features during further culture. For example, cells just before mitosis and dividing cells were sorted out into the anterior prestalk zone of migrating slugs, while cells starved during most of the G2-phase, into the posterior prespore zone. Time courses of cell aggregation and tip formation were also found to vary greatly in a cell-cycle-related manner, and cells starved during the late G2-phase showed the most rapid development. Differential chemotaxis and cohesiveness are generally considered to be important for cell sorting in Dictyostelium development. In fact, remarkable differences in the chemotactic ability to a chemoattractant, cAMP, were detected among cells starved at any particular phase of the cell cycle. EDTA-resistant cohesiveness was also acquired differently depending on the cell cycle, and it was stronger in the cells showing more rapid aggregation. These findings indicate a close relation of the cell cycle to the cell sorting and pattern formation. The possible significance of the cell-cycle-related events presented here is discussed, with special emphasis on the process of cell aggregation.     

The effect of nalidixic acid on growth and reproductive events in nucleocytosolic and chloroplast compartments in the alga<Emphasis Type="Italic">Scenedesmus quadricauda</Emphasis>

The courses of rRNA accumulation, DNA replication, and nuclear division were followed both in the chloroplast and the nucleocytosolic compartments during the cell cycle in synchronized populations of the chlorococcal alga Scenedesmus quadricauda. Control and nalidixic acid-treated cultures were compared. Nalidixic acid (150 mg/L) was added either at the beginning of the cell cycle or consecutively during the cell cycle to subcultures transferred into the dark. If the inhibitor was applied at the beginning of the cell cycle, chloroplast DNA did not replicate and nucleoids did not divide. Chloroplast division, however, was coordinated in a timely fashion with cytokinesis even under conditions of blocked chloroplast DNA replication. While the growth rate was slowed down, the courses of reproductive processes in the nucleocytosolic compartment were not affected and their timing and the number of rounds were coordinated with growth rate as in the control culture. The rate of cytosolic rRNA synthesis was lower but no apparent effect was seen on the amount of rRNA that accumulated during the cell cycle. In contrast, lower levels of chloroplast rRNA were found at the end of the cell cycle compared with the control culture. Experiments in which cells were transferred to the dark during the cell cycle showed that the inhibitor affected none of the reproductive events in the nucleocytosolic compartment. In the chloroplast compartment, DNA replication was inhibited in inhibitor-treated cultures, but was unaffected in controls. The chloroplast nucleoids themselves divided even in the presence of the inhibitor, reducing their DNA content to a level which corresponded to that in freshly formed control daughter cells.     

Addition of basic amino acids prevents G-1 arrest of nitrogen-starved cultures of Saccharomyces cerevisiae.

Arginase-minus mutants of Saccharomyces cerevisiae were arrested in growth and accumulated at the unbudded G-1 stage of the cell cycle when starved for nitrogen. If, however, arginine was added to the culture medium at the time of starvation, growth ceased but the cells did not collect at the unbudded G-1 stage. We suggest that arginine addition prevented the cells from collecting at the G-1 stage by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle.     

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.     

High survival efficiency and ribosomal RNA decaying pattern of Desulfobacter latus, a highly specific acetate-utilizing organism, during starvation

Abstract: Exponentially grown Desulfobacter latus cells were transferred to anaerobically prepared minimum medium without a carbon or energy substrate and incubated under anaerobic conditions. Changes in 16S ribosomal RNA (rRNA) of individual cells and the viable fraction in a population were monitored. The cell preparation was stained with a phylogenetic DNA probe labelled with fluorescent dye and the fluorescence of each cell was determined with confocal scanning laser microscope. Viable cells were defined as those capable of reducing a tetrazolium salt (the INT method [1]). The viability of a Desulfobacter starvation culture decreased to 85% in 48 h, but further decrease was not observed during prolonged starvation. The mean amount of 16S rRNA in individual cells decreased exponentially for 48 h to 30% the mean value obtained for exponentially growing cells, but did not decrease by prolonged starvation. About 30% of the mean content of 16S rRNA in growing cells was found in the starved cell population, suggesting that most individual cells in the starved population were not metabolically active. The difference between gross pixel intensity of cells having <8% of 16S rRNA in growing cells and those with a negative control probe was not significant. Thus, non-viable cells may not show positive signals by phylogenetic staining.     

Phase of the circadian clock is accurately transferred from mother to daughter cells in the dinoflagellate Gonyaulax polyedra.

In the first cycle following transfer from a 12 h light-12 h dark cycle (LD12:12) to constant darkness (DD), the standard deviation in circadian phase among individual clocks in populations of Gonyaulax polyedra is approximately 60 min. When a culture is transferred to constant light conditions (LL) from an LD 12:12 cycle, the standard deviation increases in the first 2-3 d, but then remains unchanged, suggesting a lack of observable desynchronization in LL after the transient period. The synchrony in a cell population is preserved even after several cell divisions. The results indicate that variations in period among cells are small, that the period of an individual clock does not fluctuate randomly from day to day, and that the circadian phase of a mother cell is faithfully passed to the clocks of the daughter cells.     

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.     

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.     

Tumor cytokinetic effects of acute starvation versus polyamine depletion in tumor-bearing mice.

Previous investigations in our laboratory have demonstrated that both acute host starvation and polyamine depletion by means of the irreversible ODC-inhibitor (ODC = ornithine-decarboxylase) fluoro-methylornithine (DFMO) lead to pronounced growth retardation of rapidly proliferating tumors. The aim of this investigation was to elucidate how these different interventions affect cell kinetics and cell cycle phases in vivo. Adult nongrowing mice (C57Bl/J) bearing a poorly differentiated rapidly growing methylcholanthrene induced sarcoma were used. Combined measurements of bromodeoxyuridine incorporation into DNA and flow cytometric techniques were used. Starvation and DFMO treatment resulted in a prolonged cell cycle transit compared to freely fed animals. Tumor cells from DFMO-treated mice demonstrated an increased time for DNA synthesis and a relatively larger accumulation of cells in the G2M phase, whereas tumor cells from starved animals were accumulated in the G0G1 phase. The fractional cell loss of tumor cell during proliferation was calculated to be around 18% higher in DFMO-treated animals compared to starved and freely fed tumor-bearing mice. This study demonstrates that different mechanisms are involved in tumor growth suppression from substrate deficiency (starvation) and from inhibition of polyamine synthesis.