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; )     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture IV. Effects of 6-methyl purine on the revolution of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were synchronouslygrown under a 12 hr light-12 hr dark regime. The algal cellcycle under these conditions starts with a light-induced reaction(s)at the beginning of the light period and ends, after a definiteperiod of time (23–24 hr at 25°C), in zoospore liberation.When cells were exposed to 6-methyl purine for short periods(0.5–2.5 hr) at different times during the early and intermediatephases of the cell cycle, it exerted, as an analogue of adenine,two different effects on the revolution of the cell cycle: onea "lengthening" effect seen at its low concentrations in whichthe length of the cell cycle was somewhat prolonged, the othera "return to start" effect at higher concentrations. In thelatter a short exposure of cells to 6-methyl purine broughtthem to the starting point of the cell cycle concurrent withthe abortion of the cycle in process. When 6-methyl purine wasapplied during the later phase of about 1/4 the length of thecell cycle, it casued no effect. Control of the revolution ofthe algal cell cycle by an "adenine-involving reaction(s)" disturbedby this adenine analogue is discussed. (Received September 1, 1975; )     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture III. Some notes on the process of zoospore liberation

When Chlamydomonas reinhardi cells liberate zoospores, theyexcrete into the medium a factor(s) which induces zoospore liberationof other cells that are not yet ready to liberate zoosporesby themselves. The "factor" is contained within cells at laterstages of the cell cycle, but its action is suppressed untilthe regular time of zoospore liberation in the cell cycle. (Received November 18, 1974; )     

Occurrence of wall-less cells during synchronous gametogenesis in Chlamydomonas reinhardtii

Synchronous gametogenesis in Chlamydomonas reinhardtii is accompaniedby a round of cell division. Some of the unmated gametes becomenaked at daughter cell liberation. A sporangial wall-lytic enzyme,which is excreted into the medium at zoospore liberation, actson the wall of the gametic cell. (Received August 28, 1980; )     

Occurrence of wall-less cells during synchronous gametogenesis in Chlamydomonas reinhardtii

Synchronous gametogenesis in Chlamydomonas reinhardtii is accompaniedby a round of cell division. Some of the unmated gametes becomenaked at daughter cell liberation. A sporangial wall-lytic enzyme,which is excreted into the medium at zoospore liberation, actson the wall of the gametic cell. (Received August 28, 1980; )     

Inhibitor effects during the cell cycle in Chlamydomonas reinhardtii. Determination of transition points in asynchronous cultures

A wide variety of inhibitors (drugs, antibiotics, and antimetabolites) will block cell division within an ongoing cell cycle in autotrophic cultures of Chlamydomonas reinhardtii. To determine when during the cell cycle a given inhibitor is effective in preventing cell division, a technique is described which does not rely on the use of synchronous cultures. The technique permits the measurement of transition points, the cell cycle stage at which the subsequent cell division becomes insensitive to the effects of an inhibitor. A map of transition points in the cell cycle reveals that they are grouped into two broad periods, the second and fourth quarters. In general, inhibitors which block organellar DNA, RNA, and protein synthesis have second-quarter transition points, while those which inhibit nuclear cytoplasmic macromolecular synthesis have fourth-quarter transition points. The specific grouping of these transition points into two periods suggests that the synthesis of organellar components is completed midway through the cell cycle and that the synthesis of nonorganellar components required for cell division is not completed until late in the cell cycle.     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture II. Effects of chloramphenicol and cycloheximide on the length of cell cycle

Cells of Chlamydomonas reinhardi Dangeard were synchronouslygrown under a 12 hr light— 12 hr dark regime. When thesecells were brought into contact with chloramphenicol for a shortperiod at early stages in the cell cycle, zoospore liberationwas delayed for a period which was nearly the same as that ofthe duration of contact with the antibiotic. When given at laterstages, the antibiotic caused no such effect. Cycloheximide,on the other hand, caused—when provided at some intermediatestage of the cell cycle— two different prolonging effectson the length of the cell cycle: one doubled the normal length(observed when the drug was administered at certain stages)and the other caused a delay similar to that caused by chloramphenicol.Interestingly, no prolonging effect was observed when cycloheximidewas given either at early stages or at later stages, such asduring the last 1/4 period of the cell cycle preceding zoosporeliberation. Based on these results, three phases were distinguishedin the algal cell cycle: "chloramphenicolsensitive", "cycloheximide-sensitive"and "insensitive" phases. Considering the known facts aboutthe modes of action of the two antibiotics inhibiting proteinsynthesis, discussions were made on the significance of proteinsynthesis in chloroplasts and in cytoplasm in determining thelength of the cell cycle. (Received October 12, 1970; )     

Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro

Erosions and ulcerations of the intestinal epithelium are hallmarks of inflammatory bowel diseases (IBD). Intestinal epithelial cell migration (restitution) and proliferation are pivotal mechanisms for healing of epithelial defects after mucosal injury. In addition, the rate of apoptosis of epithelial cells may modulate intestinal wound healing. The purine antagonists azathioprine (AZA) and 6-mercaptopurine (6-MP) are widely used drugs in the treatment of IBD. In the present study, the hitherto unknown effects of AZA as well as its metabolites 6-MP and 6-thioguanine (6-TG) on repair mechanisms and apoptosis of intestinal epithelia were analysed. Intestinal epithelial cell lines (human Caco-2, T-84 and HT-29 cells, rat IEC-6 cells) were incubated with AZA, 6-MP or 6-TG for 24 h (final concentrations 0.1-10 microM). Migration of Caco-2 and IEC-6 cells was analysed by in vitro restitution assays. Caco-2 and IEC-6 cell proliferation was evaluated by measurement of [3H]thymidine incorporation into DNA. Apoptosis of Caco-2, T-84, HT-29 and IEC-6 cells was assessed by histone ELISA, 4'6'diamidino-2'phenylindole-dihydrochloride staining as well as flow cytometric analysis of Annexin V/propidium iodide (PI)-stained cells. Cell cycle progression was evaluated by PI staining and flow cytometry. Epithelial restitution was not significantly affected by any of the substances tested. However, proliferation of intestinal epithelial cells was inhibited in a dose-dependent manner (maximal effect 92%) by AZA, 6-MP as well as 6-TG. In HT-29 cells, purine antagonist-effected inhibition of cell proliferation was explained by a cell cycle arrest in the G2 phase. In contrast, AZA, 6-MP and 6-TG induced no cell cycle arrest in Caco-2, T-84 and IEC-6 cells. AZA, 6-MP as well as 6-TG induced apoptosis in the non-transformed IEC-6 cell line but not in human Caco-2, T-84 and HT-29 cells. In summary, AZA and its metabolites exert no significant effect on intestinal epithelial restitution. However, they profoundly inhibit intestinal epithelial cell growth via various mechanisms: they cause a G2 cell cycle arrest in HT-29 cells, induce apoptosis in IEC-6 cells and dose-dependently inhibit intestinal epithelial proliferation.     

A short note on the effects of 6-methyl purine on the revolution of the cell cycle of Chlorella pyrenoidosa in synchronous culture

Short exposure of Chlorella pyrenoidosa cells to a high concentrationof 6-methyl purine atan early stage of the cell cycle causedan apparent "return-to-start" effectin which the cellsseemedto return to the starting point of a new cell cycle, with concurrentabortion of the cell cycle in process. (Received November 25, 1975; )     

The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle.

Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously. In this paper, one of them, dbf2, is characterized in detail. The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined. This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids). The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases. DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene. Its first function was completed very near to initiation of DNA synthesis. However, DNA synthesis in the mutant was only delayed at 37 degrees C, and the cells blocked in nuclear division. Consistent with this finding, the execution point occurred about 1 h after DNA synthesis, and the nuclear morphology of the mutant at the restrictive temperature was that of cells blocked in late nuclear division. DBF2 is therefore likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division.     

Die Teilung und Verformung des gegliederten Chromatophors vonStigeoclonium stagnatile (Chlorophyceae)

Division and plastic remodelling of the highly differentiated chromatophore inStigeoclonium stagnatile (Hazen)Collins is followed in living cells during their life cycle. In contradistinction to the unicellular algae both processes are separated: During the cell division, when the cell is growing, the highly differentiated chromatophore is simply divided without plastic remodelling and its division is finished before the nuclear division starts. The mode of the chloroplast division is identical with that of other algae. In contrast, during zoospore formation plastic remodelling of the chromatophore takes place: The lobed gutter-shaped chromatophore is transformed into a cup-like one which is adapted to the shape of the zoospore. After the zoospore has changed into a germling the cupshaped chromatophore is turned again into the original lobed gutter-like form of the vegetative cells. The precursory chromatophore division with regard to mitosis as well as the uniform mode of chromatophore division in various algae is stressed.

     

Infection of the diatom Asterionella by a chytrid. I. Effects of light on reproduction and infectivity of the parasite

The influence of hight limitation of the diatom Asterionellaformosa Hass, on the growth-determining parameters of its fungalparasiteRhizophydium planktonicum Canter emend, was measured,using laboratory cultures of both organisms. The experimentswere earned out at 6°C under a 15:9 h light-dark cycle.At saturating light conditions, the mean zoospore productionof the parasite was 23.4 zoospores sporangium^–1, and themean development time of the sporangia was 7 9 days. Light limitationof the host caused a substantial decrease of the zoospore production,while the development time was only slightly reduced. The improvedzoospore production at high light intensities was mainly theresult of incorporation of photosynthetic products generatedby the host after infection. Under limiting light conditions,Asterionella cells were less susceptible to infection withfungal zoospores. No infection at all occurred below 2 µEm^–2 s^–1, a light intensity that still supportedsome algal growth The maximum infection rate indicated thatchemotactic attraction of the parasite's zoospores by extracellularproducts of the host is involved. The infective lifetime ofthe zoospores of the parasite did not depend on light conditions,and was estimated at 8 days. The measured zoospore productionrates, both under limiting and saturating light conditions,enable the parasite to exceed the specific growth rate of thehost, and thus become epidemic, at sufficiently high host densities.     

Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney

We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus. immunohistochemistry; cell cycle; proliferation; renal stem cells; proximal tubule; renal epithelial cells     

Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110^Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34^cdc2 synthesis, and cyclin A synthesis. In control cultures, p110^Rb phosphorylation was detected within 24 hr of PHA stimulation; p34^cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110^Rb phosphorylation and other key regulatory events signaling G₁/S transition. © 1993 Wiley-Liss, Inc.     

The cell cycle of<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: the role of the commitment point

Chlamydomonas reinhardtii cells can double their size several times during the light period before they enter the division phase. To explain the role of the commitment point (defined as the moment in the cell cycle after which cells can complete the cell cycle independently of light) and the moment of initiation of cell division we investigated whether the timing of commitment to cell division and cell division itself are dependent upon cell size or if they are under control of a timer mechanism that measures a period of constant duration. The time point at which cells attain commitment to cell division was dependent on the growth rate and coincided with the moment at which cells have approximately doubled in size. The timing of cell division was temperature-dependent and took place after a period of constant duration from the onset of the light period, irrespective of the light intensity and timing of the commitment point. We concluded that at the commitment point all the prerequisites are checked, which is required for progression through the cell cycle; the commitment point is not the moment at which cell division is initiated but it functions as a checkpoint, which ensures that cells have passed the minimum cell size required for the cell division.     

Yeast-phase cell cycle of the polymorphic fungus Wangiella dermatitidis.

The yeast-phase cell cycle of Wangiella dermatitidis was studied using flow microfluorimetry and the deoxyribonucleic acid (DNA) synthesis inhibitor hydroxyurea (HU). Exposure of exponential-phase yeastlike cells to 0.1 M HU for 3 to 6 h resulted in the arrest of the cells in DNA synthesis and produced a nearly homogeneous population of unbudded cells. Treatment of the yeast-phase cells with HU for 9 h or longer resulted in the accumulation of the cells predominantly as budded forms having either a single nucleus in the mother cell or a single nucleus arrested in the isthmus between the mother cell and the daughter bud. Exposure of unbudded stationary-phase cells to 0.1 M HU resulted in the accumulation of the cells in the same phenotypes. Analysis by flow microfluorimetry and cell counts of HU-inhibited mithramycin-stained cells indicated that the eventual progress of HU-inhibited cells from unbudded to the two budded forms was due to the limited continuation of the growth sequence of the cell cycle even in the absence of DNA synthesis, nuclear division, and in some cases nuclear migration. On the basis of these observations and the results of flow microfluorimetric analysis of exponential-phase cells, a map of the yeast-phase cell cycle was constructed. The cycle appears to consist of two independent sequences of events, a budding growth sequence and a DNA division sequence. The nuclear division cycle of yeast-phase cells growing exponentially with a 4.5-h generation time is composed of a G1 interval of 148 min, as S phase of 16 min, and a G2 plus M interval of 107 min.     

Flow Cytometric Determination of Nuclear Replication Stages in Tomato Seeds during Priming and Germination

Flow cytometric determination of DNA levels in embryos of fullymatured dry tomato (Lycopersicon esculenium) seeds revealedlarge amounts of 2C DNA signals, indicating that most cellshad arrested in the cell cycle at the presynthetic G₁ phaseof nuclear division. After imbibition in water, an augmentationof the 4C signal in the embryonic root tip region was found.This increase could be ascribed to cells entering the syntheticphase of nuclear division leading towards the doubling of chromosomalmaterial. In the root tip cells, 4C:2C ratios increased I dafter imbibition in water though radicle emergence started 2d later. Apparently, DNA synthesis preceded germination. Onlya small increase in the number of cells with 4C DNA levels wasfound in the rest of the embryonic tissues. In whole dry seeds,DNA histograms revealed both a 2C signal and a considerable6C peak, the latter originating from the endoreduplicated endosperm. A priming period of 14 d in PEG-6000 considerably enhanced therate and uniformity of germination. In the ungerminated seeds,the 4C DNA signal of root tip cells started to increase after3 d incubation in PEG. The ratio of 4C:2C steadily increasedduring the 14 d priming period, though did not reach the levelobtained after hydration in water. Upon priming, the 4C:2C ratiowas constant after redrying the seeds towards the original moisturecontent, indicating that the chromosomal material in the rootcells had stably ceased cell cycle activity at the G² phase.The present results indicate that the beneficial effects ofpriming on seedling performance are associated with the actionof replicative DNA synthetic processes prior to germination. Lycopersicon esculeniumMill, tomato, DNA content, flow cytometry, priming, seed, nuclear replication stage, C levels     

Regulation of mating in the cell cycle of Saccharomyces cerevisiae

The capacity of haploid a yeast cells to mate (fuse with a haploid strain of alpha mating type followed by nuclear fusion to produce a diploid cell) was assessed for a variety of temperature-sensitive cell division cycle (cdc) mutants at the permissive and restrictive temperatures. Asynchronous populations of some mutants do not mate at the restrictive temperature, and these mutants define genes (cdc 1, 4, 24, and 33) that are essential both for the cell cycle and for mating. For most cdc mutants, asynchronous populations mate well at the restrictive temperature while populations synchronized at the cdc block do not. Populations of a mutant carrying the cdc 28 mutation mate well at the restrictive temperature after synchronization at the cdc 28 step. These results suggest that mating can occur from the cdc 28 step, the same step at which mating factors arrest cell cycle progress. The cell cycle interval in which mating can occur may or may not extend to the immediately succeeding and diverging steps (cdc 4 and cdc 24). High frequency mating does not occur in the interval of the cell cycle extending from the step before the initiation of DNA synthesis (cdc 7) through DNA synthesis (cdc 2, 8, and 21), medial nuclear division (cdc 13), and late nuclear division (cdc 14 and 15).     

Computerized video time-lapse (CVTL) analysis of cell death kinetics in human bladder carcinoma cells (EJ30) X-irradiated in different phases of the cell cycle

The purpose of this study was to quantify the modes and kinetics of cell death for EJ30 human bladder carcinoma cells irradiated in different phases of the cell cycle. Asynchronous human bladder carcinoma cells were observed in multiple fields by computerized video time-lapse (CVTL) microscopy for one to two cell divisions before irradiation (6 Gy) and for 6-11 days afterward. By analyzing time-lapse movies collected from these fields, pedigrees were constructed showing the behaviors of 231 cells irradiated in different phases of the cell cycle (i.e. at different times after mitosis). A total of 219 irradiated cells were determined to be non-colony-forming over the time spans of the experiments. In these nonclonogenic pedigrees, cells died primarily by necrosis either without entering mitosis or over 1 to 10 postirradiation generations. A total of 105 giant cells developed from the irradiated cells or their progeny, and 30% (31/105) divided successfully. Most nonclonogenic cells irradiated in mid-S phase (9-12 h after mitosis) died by the second generation, while those irradiated either before or after this short period in mid-S phase had cell deaths occurring over one to nine postirradiation generations. The nonclonogenic cells irradiated in mid-S phase also experienced the longest average delay before their first division. Clonogenic cells (11/12 cells) divided sooner after irradiation than the average nonclonogenic cells derived from the same phase of the cell cycle. The early death and long division delay observed for nonclonogenic cells irradiated in mid-S phase could possibly result from an increase in damage induced during the transition from the replication of euchromatin to the replication of heterochromatin.