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 sulphur starvation

Daughter cells of the chlorococcal algaScenedesmus quadricauda were incubated under photosynthesizing conditions in a sulphur-free medium. The course of the cell cycle under these conditions was changed in daughter cells which differed in their stage of development. In absence of sulphur, advanced daughter cells with two nuclei and 2 or 4 genomes passed a cycle identical with that of control in sulphur containing medium. Each cell yielded eight binuclear daughter cells. With less advanced daughter cells (one nucleus and 1 or 2 genomes) restriction of RNA synthesis occurred near to the end of the cell cycle and protein synthesis ceased two hours later (practically at the time of the protoplast fission). The last round of DNA replication found in the control culture was not initiated in sulphur-starved culture and uninuclear daughter cells with one genome were released. If the daughter cells coming from the starved populations were kept further in the sulphur-free medium, macromolecular syntheses were dramatically restricted. Only photosynthesis continued to produce starch at a similar rate as in normally grown cells. Thus, a very large amount of starch accumulated. Supported by these reserves, starved cells refed with sulphur passed an entire cell cycle in the dark and divided into eight daughter cells. In sulphur-supplied cells, both in the dark and in light, RNA, protein and DNA synthesis started without any delay in a similar way as in the control culture. Competition for sulphur reserves occurred between the growth and division processes; the former were preferred in the light and the latter in the dark.     

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.     

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.     

Growth and cell division during nitrogen starvation of the yeast Saccharomyces cerevisiae.

During nitrogen starvation, cells of the yeast Saccharomyces cerevisiae increased threefold in number, and little ribonucleic acid (RNA) and protein were accumulated. Both RNA and protein were extensivley degraded during starvation, suggesting that intracellular macromolecules could supply most of the growth requirements. The types and proportions of stable RNA synthesized during nitrogen deprivation were characteristic of exponentially growing cells; however, the complement of proteins synthesized was different. We conclude that, once events in the deoxyribonucleic acid division cycle are initiated, cells can complete division with little dependence on continued net cell growth.     

DNA synthesis byLactobacillus acidophilus during glutamic acid starvation

The synthesis of DNA was investigated inLactobacillus acidophilus R-26 during starvation for glutamic acid. The synthesis of RNA and of proteins was also examined. The content of DNA in the cell increased by 150–250%. The results show that new DNA-replication cycles can be initiated without simultaneous synthesis of proteins and of RNA.     

Synthesis of inducible enzyme in Escherichia coli recovering from prolonged energy starvation.

A marked breakdown of ribosomes and rRNA occurs in Escherichia coli cells during prolonged deprivation of a carbon source (energy starvation). In E. coli recovering from energy starvation: (a) synthesis of RNA started immediately, total protein synthesis showed a delay of 5 to 10 minutes; (b) beta-galactosidase, tryptophanase and serine deaminase could not be induced in the first 50--70 min; (c) a lag of 60 min in the synthesis of beta-galactosidase was observed in a lac constitutive mutant of E. coli; synthesis of the constitutive enzyme malate dehydrogenase did not shown any delay. RNA synthesized in the early stages of recovery contained a higher percentage of low molecular weight molecules than RNA synthesized after 70 min of recovery or during exponential growth. Messenger RNA specific for beta-galactosidase was not synthesized for the first 50--60 min of recovery even when the specific inducer was added to the cultures.     

MACROMOLECULAR EVENTS LEADING TO CELL DIVISION IN TETRAHYMENA PYRIFORMIS AFTER REMOVAL AND REPLACEMENT OF REQUIRED PYRIMIDINES

Tetrahymena pyriformis were brought to a non-growing state by removal of pyrimidines from their growth medium. During pyrimidine deprivation cell number increased 3- to 4 fold, and this increase was accompanied by one or more complete cycles of macronuclear DNA replication. Autoradiographic studies show that endogenous protein and RNA were turning over throughout starvation and that RNA breakdown products were used to support the DNA synthesis that occurred during the early period of starvation. However, after 72 hours of starvation all DNA synthesis and cell division had ceased. Feulgen microspectrophotometry shows the macronuclei of these cells to have been stopped at a point prior to DNA replication (G1 stage). After pyrimidine replacement the incorporation of H³-uridine, H³-adenosine, and H³-leucine was measured by the autoradiographic grain counting method. The results indicate that RNA synthesis began to increase almost immediately, but that there was a lag of almost an hour before an increase in protein synthesis. In agreement with the autoradiographic data, chemical data also show that cellular content of RNA began to increase shortly after pyrimidine replacement but that cellular protein content did not increase until about one hour later. Pulse labeling of the cells with H³-thymidine at intervals after pyrimidine replacement shows that labeled macronuclei first began to appear at 150 minutes; that 98 per cent of the macronuclei were in DNA synthesis at 240 to 270 minutes; and that the percentage then began to decrease from 300 to 390 minutes, at which time only 25 per cent of the macronuclei were labeled. Cellular content of DNA did not increase for at least 135 minutes after pyrimidine replacement; however, just before the first cells divided (360 minutes) the DNA content had doubled. After pyrimidine replacement the cells first began to divide at 360 minutes, and 50 per cent had divided at 420 minutes; however, all cells had not divided until 573 minutes. This technique of chemical synchronization of cells in mass cultures makes feasible detailed biochemical analysis of events leading to nuclear DNA replication and cell division.     

Effects of nitrogen and phosphorus starvation on nucleic acid and protein content of Heterocapsa sp.

Changes in the protein, RNA and DNA content related to nitrogen(N) and phosphorus (P) starvation were studied in the marinedinoflagellate Heterocapsa sp. grown in batch cultures. In bothcases of nutrient starvation, metabolic adaptations affectedprotein and RNA pools, while the DNA content per cell remainedapproximately constant. N starvation led to a parallel decreasein protein and RNA concentration which caused the protein/RNAratios to remain constant. A dramatic decrease in the RNA contentcharacterized the P-starved cultures, although protein synthesiscontinued. The ribosomal RNA content was lower than expectedgiven the continuation of protein synthesis. It is suggestedthat protein/RNA ratios could be used as an indicator of P starvation,while protein/chlorophyll ratios would characterize N starvation ¹Present address: University of Hawaii at Manoa Soest, BiologicalOceanography, 1000 Pope Road, Honolulu, HI 96822, USA     

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.     

Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956.

Carbon starvation induces the development of a starvation- and stress-resistant cell state in marine Vibrio sp. strain S14 (CCUG 15956). The starved cells remain highly responsive to nutrients during prolonged starvation and exhibit instantaneous severalfold increases in the rates of protein synthesis and RNA synthesis when substrate is added. In order to elucidate the physiological basis for the survival of cells that are starved for a long time, as well as the capacity of these cells for rapid and efficient recovery, we analyzed the ribosome content of carbon-starved Vibrio sp. strain S14 cells. By using direct chemical measurements of the amounts of ribosomal particles in carbon-starved cultures, we demonstrated that ribosomes were lost relatively slowly (half life, 79 h) and that they existed in large excess over the apparent demand for protein synthesis. After 24 h of starvation the total rate of protein synthesis was 2.3% of the rate during growth, and after 3 days this rate was 0.7% of the rate during growth; the relative amounts of ribosomal particles at these times were 81 and 52%, respectively. The ribosome population consisted of 90% 70S monoribosomes, and no polyribosomes were detected in the starved cells. The 70S monoribosomes were responsible for the bulk of the protein synthesis during carbon starvation; some activity was also detected in the polyribosome size region on sucrose density gradients. We suggest that nongrowing carbon-starved Vibrio sp. strain S14 cells possess an excess protein synthesis capacity, which may be essential for their ability to immediately initiate an upshift program when substrate is added.     

Changes in nucleic acid and protein synthesis during starvation and spherule formation inPhysarum polycephalum

Summary The synthesis of protein and nucleic acids was studied by isotope incorporation and dilution in the plasmodia ofPhysarum polycephalum during periods of growth and differentiation (spherule formation). The total protein content decreased during starvation, but protein synthesis still occurred, probably at the expense of proteins previously synthesized during growth. Studies on leucine incorporation showed that protein synthesized during growth had a greater turnover than did protein formed by starving cultures, when both types of cultures were transferred to starvation conditions. Protein synthesis after prolonged starvation was rapidly and markedly decreased following the inhibition of RNA synthesis, whereas no such direct dependence on RNA synthesis was observed in growing cultures or during early starvation.The kinetics of RNA synthesis and the types of RNA formed were also shown to differ in growth and starvation. RNA turnover was low in growing cultures but substantial in starving cultures that were returned to growth medium. Qualitative differences in pulse-labeled RNA extracted from growing or starving cultures were revealed by methylated-albumin-kieselguhr column chromatography and sucrose gradient centrifugation. In starving cultures proportionately more labeled RNA was found in the lighter, non-ribosomal region of the gradient, and RNA from this region hybridized with denatured DNA to a greater extent than did other RNA fractions.This work was supported in part by Grant CA-07175 from the National Cancer Institute and by a grant from the Alexander and Margaret Stewart Trust Fund. The authors express their appreciation to Dr. H. Kubinski for helpful suggestions.One of us (H.W.S.) was in part supported by the Deutsche Forschungsgemeinschaft.     

Genes for a nuclease and a protease are involved in the drastic decrease in cellular RNA amount in fission yeast cells during nitrogen starvation

Cellular RNA in Schizosaccharomyces pombe cells drastically decreases in amount during nitrogen starvation. Previously, we found and purified a soluble RNA-degrading enzyme whose activity drastically increased in the cells of S. pombe undergoing nitrogen starvation. The enzyme was a nuclease encoded by pnu1(+). In this study, the increase in the RNA-degrading activity and the decrease in cellular RNA level are examined in a null-mutant of pnu1(+) (pnu1Delta). During nitrogen starvation, wild-type cells show an apparent increase in RNA-degrading activity, whereas the pnu1Delta cells do not. The wild-type cells show a drastic decrease in cellular RNA amount, whereas the pnu1Delta cells show only a slight decrease. These results suggest that Pnu1 nuclease is implicated in the decrease in cellular RNA amount during nitrogen starvation, probably via the RNA-degrading activity. The increase in the RNA-degrading activity is independent of both the Wis1 stress-activated MAP kinase cascade and Tor1 signaling pathway, but it is strongly dependent on isp6(+), a gene for a possible protease, whose expression is induced during nitrogen starvation. A disruption mutant for isp6(+) (isp6Delta) is deficient in both the increase in the RNA-degrading activity and the drastic decrease in the cellular RNA amount during nitrogen starvation, which suggests that isp6(+) is involved in the RNA degradation via regulating the RNA-degrading activity of Pnu1.     

Synchronization of carrot cell culture by starvation and cold treatment

When a suspension culture of carrot cells in the early stationary phase was allowed to stand at 4 °C for 72 h, the cell population was partially synchronized in relation to their division cycle. Judging from a pattern of increase of cell number, two steps in the cell cycle are thought to be sensitive to these treatments. When an additional cold treatment was applied to the culture, degree of synchronization was markedly increased. Protein content in a synchronized culture increased in a stepwise fashion as well as DNA while RNA increased continuously.     

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     

Correlation between cell enlargement and nucleic acid and protein content of HeLa cells in unbalanced growth produced by inhibitors of DNA synthesis

HeLa cells in monolayer cultures were treated with the following inhibitors of DNA synthesis: mitomycin C, nitrogen mustard, fluorodeoxyuridine, hydroxyurea, arabinofuranosylcytosine and high concentrations of thymidine. The concentration of each inhibitor used was, in most cases, just sufficient to arrest cell multiplication and all produced unbalanced growth in the sense that the synthesis of RNA and protein were only partially inhibited while DNA synthesis stopped. This resulted in approximately 100% increases in RNA and protein content per cell in 48 hours and, since cell volume also increased by 100% during this time, the concentration of RNA and protein per unit cell volume remained constant. It was concluded that cell protein content may be used as an accurate index of variation in cell size in HeLa cells treated with inhibitors of DNA synthesis.     

Incorporation of tritiated thymidine by marine bacterial isolates when undergoing a starvation survival response

Bacterial DNA synthesis, as measured by the incorporation of [methyl-³H] thymidine, was examined during conditions of decreasing biomass and non-growth of three heterotrophic marine bacteria. High rates of [³H] thymidine incorporation were recorded during the initial phase of starvation and two strains exhibited a net increase in DNA during the first few hours of starvation. The decreased rate of [³H] thymidine incorporation with the time of starvation, was in agreement with the decrease in the percentage of the total population that showed uptake of labelled thymidine, as seen by a combined autoradiography-epifluorescence technique. It is suggested that new rounds of replications were initiated after cells had been starved for times that well exceeded the time for replication of genomes during growing The initial increase in cell numbers upon transfer of growing cells to a starvation regime was inhibited by nalidixic acid, suggesting that DNA synthesis, rather than an excess of nuclear bodies, allow for the fragmentation process in these strains.     

Recovery from nutrient starvation by a marine Vibrio sp

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.