Variations in biochemical parameters of Heterocapsa sp. and Olisthodiscus luteus grown in 12:12 light:dark cycles I. Cell cycle and nucleic acid composition

The division cycle of two phytoplankton species, Olisthodiscus luteus and Heterocapsa sp. was studied in relation to a 12:12 light:dark cycle. Batch cultures in exponential phase were sampled every three hours during 48 hours. Cell number, cellular volume and DNA and RNA concentrations were measured. Microscopic observations of the nuclei of Heterocapsa sp. were also performed. In both species, cell division took place in the dark. In Heterocapsa sp., DNA and RNA showed a similar diel variability pattern, with synthesis starting at the end of the light period, previously to mitosis and cytokinesis. In O. luteus. Major RNA synthesis occurred during darkness, and DNA was produced almost continuously. Both species presented different values and diel rhythmicity on the RNA/DNA ratios.     

Light regulation of the cell cycle in Euglena gracilis bacillaris

We have studied the light regulation of the cell division cycle in the photosynthetic alga Euglena gracilis bacillaris. Euglena grown under phototrophic conditions are easily synchronized to a 12 h light-12 h dark regime. By inoculating stationary phase, nondividing cells into fresh media and exposing the diluted cells to either light or darkness, we have determined that initiation of DNA synthesis for the cell division cycle is light dependent. By varying the length of time in light to which synchronized cells are exposed, we have shown that commitment to the cell cycle requires exposure to more than 6 h of light. We propose that this is to allow the accumulation, through photosynthetic electron transport, of an initiating factor that will enable DNA synthesis to begin. Flow cytometry analysis also shows that once cells are committed to the cell cycle, they complete the cycle in the dark, so mitosis is a light-independent step.     

The S phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G₁ by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

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.     

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.     

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.     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were grown synchronouslyunder a 12 hr light-12 hr dark regime. Time courses of nucleardivision, chloroplast division, "apparent cytokinesis" and zoosporeliberation were followed during the vegetative cell cycle inthe synchronous culture. Liberation of zoospores occurred atabout 23–24 hr after the beginning of the light periodat 25°C. Four zoospores were produced per mother cell underthe conditions used. At lower temperatures, the process of zoosporeliberation as well as length of the cell cycle was markedlyprolonged, but the number of zoospores produced per mother cellwas approximately the same. At different light intensities,lengths of the cell cycle were virtually the same, while thenumber of zoospores liberated was larger at higher rather thanat lower light intensities. During the dark period, nuclear division, chloroplast divisionand apparent cytokinesis took place, in diis order, and proceededless synchronously than did the process of zoospore liberation.When the 12 hr dark period was replaced with a 12 hr light periodduring one cycle, the time of initiation as well as the durationof zoospore liberation was litde affected in most cases, whereasnuclear division, chloroplast division and apparent cytokinesiswere considerably accelerated by extended illumination. Whenalgal cells which had been exposed to light for 24 hr were furtherincubated in the light, zoospore liberation started much earlierand proceeded far less synchronously, compared with that under12 hr light-12 hr dark alternation. (Received October 12, 1970; )     

The s phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G1 by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

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.     

Synchronous Growth and Plastid Replication in the Naturally Wall-less Alga Olisthodiscus luteus

Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B₁₂ to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B₁₂, plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.     

The effect of light on DNA synthesis and related processes in synchronous cultures of Chlorella

Summary In autotrophic cultures of Chlorella synchronised by alternating light and dark periods of 16:8 hours the DNA content duplicated normally 4 times successively during the S _mphase, i. e. between the 10th and 18th hour after the beginning of the light period. This finding together with electron microscopical observations revealed that one duplication of the DNA and of the nuclei per cell proceeds every 110 minutes. All nuclei of a cell seem to undergo successive DNA syntheses and nuclear divisions synchronously. The rate of DNA synthesis was independent from illumination. On appropriate reduction of the light period the last duplication cycle fell out and the average final spore number per cell was accordingly lower.If a culture was transferred to darkness or low light intensity 3 hours before the normal end of the light period the release of spores was promoted by approximately 1 1/2 hours, provided a strong decrease of metabolically accessible carbohydrates was prevented by either an additional short illumination during the dark period or by continuing the weak light.A possible explanation for the shortening of the cell development is that, by passing over one DNA duplication and one protoplast division, the cell can enter sooner the respective subsequent developmental stages.     

Protein Synthesis in Euglena gracilis is Light-and Temperature-Dependent,Oscillating in a Circadian,Temperature-Compensated Manner

Abstract: Incorporation of radiolabelled amino acids into proteins of Euglena gracilis revealed that the amount of labelled protein depends on the conditions of illumination and temperature of cultivation. Protein synthesis was generally lower under dark conditions except at 37 °C. The largest amounts of labelled protein were measured at 21 °C and decreased at higher and lower temperatures. By separating the labelled proteins of the membraneous cell fraction from subcultures under a range of culture conditions, the synthesis of some specific proteins was found to be light- and/or temperature-dependent. On incubating cells taken at different times during a light/dark cycle and under constant conditions, a circadian rhythm of ³⁵S-methionine- as well as ³⁵S-cysteine-incorporation was detected. Thereby the cells incorporated ten-times less cysteine than methionine. Protein synthesis always peaked during the last quarter of the daily light phase, confirming the rhythmic rise in total protein. The length of the rhythm period, approximately 24 h, was nearly independent of the applied temperature in the range of 16 to 27 °C.     

Growth and synthesis of nucleic Acid and protein by excised radish cotyledons

Nutritional and light requirements for growth and synthesis of RNA, DNA, and protein by cotyledons excised from 5-day-old seedlings of Raphanus sativus L. were investigated, and the course of synthesis was followed through the cell cycle. The minimum requirements for a net increase in nucleic acid and protein were sugar, nitrate, and light. The cotyledons used nitrite at low concentration, but not ammonium ion. Light was required for preliminary steps in synthesis of RNA, DNA, and protein, but the actual polymerization reactions occurred in the dark. The cotyledons contained sufficient endogenous growth factors for about half of the cells to complete 1 cycle on a medium of 1% sucrose, 80 mm KNO₃. The increase in DNA was limited to about 50% and was accompanied by a comparable increase in cell number. Fresh weight, RNA, and protein tended to increase in proportion to DNA. Growth of the isolated cotyledons commenced with cell enlargement. RNA began to increase after about 4 hours, DNA after about 12. The major increase in protein also began at about 12 hours. The maximum rate of increase for all 3 occurred between 12 and 16 hours. Cell counts indicated that by 28 hours most of the cells which had replicated DNA had also completed cell division.     

Shifts in nuclear and cytoplasmic nucleic acid content in temperature-synchronized Tetrahymena pyriformis (HSM)

Nuclei were isolated by exposing temperature synchronized Tetrahymena pyriformis (HSM) to Triton-X-100. Cell division synchrony was induced with a repetitive 12-hour temperature cycle (9.5 hours at 13°, 2.5 hours at 29°). Increase in nucleic acid content was biphasic: primarily during the last two hours of the cold period well in advance of the synchronous burst of division and secondarily in the last hour of the warm period. Nuclear RNA content rises almost two hours ahead of cytoplasmic RNA which shows a maximum 0.5 hour before the onset of the warm period. The DNA content reaches a peak 30 minutes later. On the basis of these shifts there appears to be not net synthesis of nucleic acids during cell division. The changes in RNA/DNA of the isolated macronuclei and micronuclei suggest enhanced RNA turnover, loss to the cytoplasm and enhanced ribonuclease activity prior to cell division. Cytoplasmic RNA also appears to be subject to enzymic degradation.     

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.     

金黄滴虫细胞同步分裂的诱导

无菌培养的金黄滴虫在25℃及连续通气的条件下,经12小时光照(2500Lux)和12小时黑暗的相间处理,可连续出现三次同步分裂,同步分裂率最高可达92％,即细胞数在一小时内可陡增92％。 细胞核动态的观察表明,在黑暗处理期中细胞被阻止于细胞分裂的前期。当黑暗处理11小时后,前期细胞突破阻拦,实现了同步分裂。     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary Cell division in Navicula pelliculosa (Bréb.) Hilse, strain 668 was synchronized with an alternating regime of 5 h light and 7 h dark. Cell volume and dry weight increased only during the light period. DNA synthesis, which began during the third h of light, was followed sequentially by mitosis, cytokinesis, silicic acid uptake, cell wall formation, and cell separation. Silicification and a small amount of net synthesis of DNA, RNA and protein occurred during the dark at the expense of carbohydrate reserves accumulated during the light period. Cells kept in continuous light, after synchronization with the light-dark regime, remained synchronized through a second division cycle; the sequence of morphological events was the same as that in the light-dark division cycle, but the biosynthesis of macromolecular components changed from a stepwise to a linear pattern. The silicon-starvation synchrony was improved by depriving light-dark synchronized cells of silicic acid at the beginning of their division cycle, then resupplying silicic acid to cells blocked at wall formation.Abbreviation L light - D dark Portions based on a thesis submitted by W.M.D. to the University of California, San Diego in partial fulfillment of the requirements for the PH.D degree     

The mechanism of regulation of fibroblastic cell replication. I. Properties of the system

Sixty to eighty per cent of the cells in a culture of human diploid fibroblasts may be stimulated from the state of density dependent inhibition of replication to active DNA synthesis and division. The maximum response is effected by 50% serum within the pH range 7.2–8.0. The proportion of cells responding depends on the concentration of serum protein in the medium which may be effectively substituted by crystalling serum albumin. There is a differential sensitivity to the stimulus of cells in the densely packed centers of whorls and in the less dense areas between the whorls. The cell response is parasynchronous and the median durations of the various phases of the cell cycle are: G₁I 6 β ?æ^® ¿ ∞ 8 hours, G₂ = 6 hours and doubling time = 30 hours. The stimulatory effect of fresh medium is lost during contact with dense cultures so that it has only 50% of its initial capacity after 14 hours. It can be restored by dialysis against serum-free medium. The stimulus must be applied for at least ten hours to be effective in inducing DNA synthesis. During the latter half of ten hour induction period subsequent DNA synthesis becomes exquisitely sensitive to actinomycin D. After this time an increasing number of cells become irreversibly committed to replicate. The data are interpreted to indicate that during contact with serum proteins (including albumin) changes in the cell surface, if continued long enough, trigger a mechanism which involves the synthesis of a unique RNA species during the fifth to tenth hours. After this RNA has been synthesized the cells are then committed to DNA synthesis.     

OBSERVATIONS ON CELL DEVELOPMENT IN CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) AT LOW AND HIGH CARBON DIOXIDE TENSION1

Some cell cycle events were compared in Chlamydomonas segnis Ettl during its development in synchronous cultures (12:12 LD) supplied with air and air enriched with 5% CO₂. In cultures bubbled with air, growth resulted in production of 2 relatively small zoospores. In cultures provided with 5% CO₂, 4 large zoospores were formed but not released in darkness unless the cultures were bubbled with CO₂-free air and/or exposed to light. Respiration in zoospores was inhibited by high CO₂ tension. In cultures maintained under continuous illumination for one cell cycle, provision of 5% CO₂ led to enhanced growth, a relatively long S-phase and a 4 h delay of the second cell division. In such cultures, the DNA content of parental cells (12 h L) was insufficient to support two cell divisions. The RNA/DNA ratio of the resulting zoospores was 10 compared to 4 in air cultures. These results provided evidence that the delay of the second cell division was a consequence of the delay in DNA production.