MICRONUCLEAR RNA SYNTHESIS IN PARAMECIUM CAUDATUM

In a generation time of 8 hr in Paramecium caudatum, the bulk of DNA synthesis detected by thymidine-³H incorporation takes place in the latter part of the cell cycle. The micronuclear cycle includes a G₁ of 3 hr followed by an S period of 3–3½ hr. G₂ and division occupies the remaining period of the cycle. Macronuclear RNA synthesis detected by 5'-uridine-³H incorporation is continuous throughout the cell cycle. Micronuclear RNA synthesis is restricted to the S period. Ribonuclease removes 80–90% of the incorporated label. Pulse-chase experiments showed that part of the RNA is conserved and released to the cytoplasm during the succeeding G₁ period.     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

G1 arrest and the division/conjugation decision in Tetrahymena.

The transformation from the asexual proliferative stage of Tetrahymena to the sexual stage, during which cells of complementary mating types pair and nuclear fertilization occurs, provides an opportunity to study the relationship between the division cycle and differentiation. Conjugation is induced in cells starved for at least 2 hr by mixing complementary mating types. To determine the effect of starvation on the cell cycle, dividing cells were selected from a log growth culture and stepped down to non-nutrient conditions. The G₁ stage is operationally divisible into two sectors, A and B. In the A stage, cells arrest in nutrient-free medium. In the B stage, they proceed through the division cycle. Arrested G₁A cells may conjugate directly when challenged with similar cells of a complementary mating type. It is thereby demonstrated that Tetrahymena cells in G₁A can be directed to divide (nutrient conditions) or can be directed to differentiate (non-nutrient conditions plus complementary mating type) without an intervening division cycle. This rules out a requirement for reprogramming via chromosomal replication or cell division and suggests that G₁A is a stage during which the division/differentiation decision is made in direct response to ambient conditions.     

Studies on microspore development in liliaceous plants

In microspores of angiosperm plants, the period from the end of meiosis in microsporocytes unitl the first cell division may be considered as one cell cycle, and the division is polarized, resulting in the formation of two functionally different nuclei. InLilium longiflorum, the duration of the cell cycle was measured in some detail. Identification of individual stages was based on the correlation between bud length and developmental stage. The results showed that the process begins at a bud length of 24 mm and is completed about 15 days later, at a bud length of 58 mm. By autoradiography of cells cultured in the presence of³H-thymidine, approximate durations of the G₁, S, and G₂ plus M phases were estimated to be about 12, 2, and 1 days, respectively. A detailed cytological analysis of the transitions between the various microspore stages has revealed some convenient parameters which point to ghe progression of change during the interval.     

DNA synthesis in developing two-cell mouse embryos

Mated CF1 (Carworth) female mice were sacrificed at 2 hr intervals between 29 and 43 hr after human chorionic gonadotrophin (HCG) administration. One- and two-cell eggs were incubated in [³H]thymidine for 1 hr. Labeled two-cell embryos were first observed at 31 hr and reached a maximum number at 35 hr. The S period is approximately 6 hr in duration. Although both blastomeres were labeled in most cases, embryos with only one labeled blastomere were more numerous at later times. In vitro labeling was corroborated by injecting [³H]thymidine directly into the isthmic portion of the oviduct. Embryos usually complete the second cleavage division 18–20 hr after onset of DNA synthesis. The cell cycle at the two-cell stage is thus characterized by a G₁ of close to 1 hr, a 6 hr S, and a G₂ of about 12 hr.Embryos developing in vitro frequently fail to progress beyond the two-cell stage. The block is not due to absence of DNA synthesis since these embryos were found to incorporate [³H]thymidine.     

A DELAY IN THE G2 PERIOD OF CULTURED HUMAN LEUCOCYTES AFTER TREATMENT WITH RUBIDOMYCIN (DAUNOMYCIN)

Human leucocytes were cultured for 3 days at 37°C, and during this time treated with rubidomycin (also known as daunomycin) for periods up to 48 hr. The effects of this treatment were studied by examining mitotic indices, uptake of ³H-thymidine, and patterns of DNA content (measured by microdensitometry on Feulgen-stained cells). A low concentration of rubidomycin (0.1 μg/ml) caused accumulation of cells in the G₂ period, which in turn resulted in a decrease in the mitotic index. A secondary effect was a slight drop in ³H-thymidine uptake after 12 hr. Higher doses (up to 10 μg rubidomycin per ml) caused an inhibition of DNA synthesis, with accumulation of unlabelled cells between G₂ and G₂. The probable mode of action of rubidomycin, as presented by earlier authors, is the intrusion of the drug molecule between DNA strands, forming a complex with DNA, and hindering its normal folding. This is discussed with respect to the present findings.     

Polyamines in early embryonic development: Their relationship to nuclear multiplication rate,cell cycle traverse,and nucleolar formation in a dipteran egg

Polyamine synthesis and accumulation were assessed from fertilization until gastrulation in a dipteran egg (Calliphora erythrocephala Meigen). Spermidine synthesis was activated immediately after fertilization, generating a broad spermidine peak during early cleavage. This period is characterized by the most rapid nuclear multiplication known from animal material. Cleavage consists of nuclear multiplication only, and the egg remains syncytial until gastrulation. After nine synchronous nuclear divisions with a cycle length of 10 min, the cycle length is gradually increased to 20 min during the subsequent four parasynchronous nuclear divisions. The spermidine level decreased in parallel with this decreasing rate of nuclear division. The interphase of the next nuclear cycle is remarkably prolonged and lasts for more than 90 min, i.e., until after the onset of gastrulation. It consists of an initial short S phase followed by a longer G₂ phase; G₁ is extremely short or absent. During this prolonged interphase, spermidine content showed a biphasic pattern of changes with peaks during S and late G₂. The S-phase peak also coincides with the first appearance of nucleoli during embryogenesis. The late-G₂-phase peak coincides with the period of rapid cytokinesis, during which all nuclei in the peripheral layer of the syncytium become separated by membranes forming a cellular blastoderm. The polyamine pattern is consistent with the idea that the polyamines play an important role in DNA replication and in cytokinesis as well as in nucleolar formation.     

EXPERIMENTAL CONTROL OF DNA SYNTHESIZING AND DIVIDING CELLS IN EXCISED ROOT TIPS OF PISUM

The number of dividing and DNA-synthesizing cells in excised pea roots can be regulated by eliminating the carbohydrate normally supplied in the culture medium. When the excised roots were allowed to remain for 24 hr in a medium lacking carbohydrate, the number of mitotic figures and tritiated thymidine (H³-T) labeled cells was reduced almost to zero. After an additional 24 hr in the incomplete culture medium, 15% of the interphase cells were H³-T labeled, the percentage of the cells that were dividing never exceeded 1.4, and 30% of these were H³-T labeled. When the roots remained in the deficient medium for 72 hr, neither cell division nor cells synthesizing DNA were observed. Upon addition of 2% sucrose, cell division and DNA synthesis were resumed in the roots that were maintained for 24 or 72 hr without an exogenous carbohydrate supply. It has been hypothesized that some proliferative systems consist of two cellular subpopulations which selectively stop or remain in either the pre-DNA synthetic (G₁) or post-DNA synthetic (G₂) periods of the mitotic cycle. The addition of sucrose, H³-T, and 5-aminouracil to the medium, after the roots had been maintained for 24 hr without a carbohydrate, indicated that most of the proliferative cells in the roots had accumulated in either G₁, a quasi-G₁ condition, i.e., DNA synthesis stopped sometime before completion, or G₂ periods of interphase; the majority, however, were in G₁ or quasi-G₁ conditions. The results suggested that DNA synthesis (S period) and mitosis or the onset of these processes have the highest metabolic requirements in the mitotic cycle and that G₁ and G₂ were the most probable states for proliferative cells in a meristem with a low metabolic level.     

Alterations in the cell cycle of Drosophila imaginal disc cells precede metamorphosis

Flow cytometric analyses of imaginal disc and brain nuclei of Drosophila melanogaster have been made throughout the third larval instar. In wing, haltere, and leg discs the proportion of cells in the G₂M phase of the cell cycle (tetraploid cells) increases with larval age. In contrast, in the eye disc and in brain the proportion of tetraploid cells, already low at the outset of the instar, declines further. Measurement of growth rates for disc and brain tissue during the same developmental period was carried out by the cell counting procedure of Martin (1982). Our results are consistent with the conclusion that imaginal discs grow exponentially with an apparent doubling time of 5–10 hr from the resumption of cell division (in the first or second larval instar) until about 95 hr, when the apparent doubling time increases. Cell numbers increase until at least 5 hr after formation of white prepupae (122 hr), but during the preceding 10 hr the rate of increase is low. Thus, for wing and leg discs, but not for the eye disc and brain, the declining growth rate is associated with an increase in the proportions of tetraploid cells. In conjunction with cell counts and flow cytometry, fluorometric determination of disc DNA content at 112 hr indicated that the diploid DNA content of imaginal disc nuclei is 0.45 pg.     

Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis

Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G₁, DNA synthesis, S, the second growth stage, G₂, mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4′, 6-diamidino-2-phenylindole dichloride.

Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G_q, an hypothesized side loop from G₁. All morphological stages, not only division, appear to be phased by the circadian clock.

     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

Arrest of cell division blocks the utilization of polyamines in synchronized cultures of photoautotrophically grown Euglena

A trimodal change in the cellular levels of three major polyamines: spermidine, N,N′-bis(3-aminopropyl)-1, 3-propanediamine (BAP) and 3,3′-diaminodipropylamine (DAD) was observed during two successive cell cycles in synchronously dividing cultures of the algal flagellate, Euglena gracilis Z photoautotrophically grown in a 24-h light-dark cycle. The intracellular levels of these three polyamines decreased as cells divided and then were enhanced as cells exited the G₁ phase and proceeded through the S and G₂ phases. Spermidine, BAP and DAD concentrations increased about 2.5-fold during the S phase. Putrescine and 1,3-diaminopropane levels did not vary significantly. One peak of polyamine synthesis occurred in the G₁ phase prior to DNA synthesis, followed by a second more important peak during the S-G₂ phases before cell division; both peaks were observed during the light period. A third minor peak was observed during the pre-G₁ (or G₀) phase in the dark period after mitosis had been completed. In contrast, when the cells attained the “stationary” phase of growth, there was no significant increase in the content of polyamines during the light period although spermidine and BAP increased slightly twice during the dark period (putrescine and 1,3-diaminopropane and DAD levels remained almost constant). To ascertain whether the synthesis of polyamines was merely a direct effect of the photoperiod, parallel experiments with synchronous cultures were carried out in the presence and absence of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, a photosynthetic inhibitor. Although a slight decrease in the concentration of polyamines was observed, the three maxima of polyamines synthesis were observed as in normal cultures. These results clearly suggest that polyamine biosynthesis is closely related to DNA replication and cell division in Euglena cells.     

Photosynthetic Apparatus Formation during the Cell Cycle of Chlorella

Synchronous cell division in cultures of Chlorella vulgaris Beijerinck was induced by intermittent illumination: 9 hours light, 6 hours darkness. The rate of photosynthetic O₂ evolution per cell increases 4-fold in a one-step manner at the beginning of the light period, to the same extent as the increase in cell number. Over the division cycle, the following accumulation times during the light period were found: chlorophyll a, between 2 and 8 hours, chlorophyll b, between 5 and 8 hours, reaction centers of photosystems I and II, between 2 and 6 hours; and cytochrome f, between 2.5 and 5 hours. Cytochrome f accumulation is closely followed by an increase in amplitude of the rapid phase in light-induced absorption increase at 520 nanometers and in intensity of the delayed light emission. Enhancement of the delayed fluorescence yield per flash under continuous illumination (caused by the establishment of the pH difference across the thylakoid membrane) is maximal by the first hour of the light period.     

Photoperiodic determination of the male and female sexual morphs of Myzus persicae

The chronology of the photoperiodic determination of sexual morphs in holocyclic Myzus persicae was studied in the laboratory by transfer of synchronized batches of aphids between long-day (16 hr) and short-day (10 hr) régimes at a constant temperature of 20°C. The length of exposure to the short-day régime was measured in terms of the number of long dark-periods received by the aphids. The photoperiodic response extended over four generations (P, G₁, G₂, and G₃ respectively). When P generation aphids were given short days from the fourth instar, alate viviparae and males appeared successively in generation G₂, oviparae in G₃. Increasing the number of long dark-periods received during the development of G₂ embryos had a cumulative effect on the number which developed into alate viviparae. Determination of all the first-born G₂ aphids as alatae occurred only if their mothers had been exposed to the short-day régime throughout larval development. Alate viviparae gave birth to oviparae if they received a minimum of 4 long dark-periods, starting from a late stage in their embryonic development. The critical stage for ovipara determination in the G₃ embryo was on the sixth or seventh day after ovulation, more than half-way through embryonic development. G₂ aphids were determined as males before ovulation if their parents received 4 (in some circumstances only 3) long dark-periods. In the clones studied, male determination, once initiated in the G₁ parent, could not be reversed by later back-transfer to the long-day régime.     

ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G₁. During very early G₁, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G₁, a granular component appeared which was intimately associated with the fibrous material. By the middle of G₁, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G₁ to the middle of S primarily due to the accumulation of the granular component. During the G₂ period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Cell cycles in embryos of the silkworm,Bombyx mori: G2-arrest at diapause stage

Summary In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G₁, S and G₂ phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G₁, S and G₂ were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G₂ at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G₁ from G₂, suggesting that their G₁ phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G₁ fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G₂ at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G₁. Finally, most of the cells may be arrested in G₁, while there is only a small fraction of cells continuously cycling. Offprint requests to: T. Yaginuma     

Variations in Several Responses of HeLa Cells to X-Irradiation during the Division Cycle

Several responses of synchronized populations of HeLa S3 cells were measured after irradiation with 220 kev x-rays at selected times during the division cycle. (1) Survival (colony-forming ability) is maximal when cells are irradiated in the early post-mitotic (G₁) and the pre-mitotic (G₂) phases of the cycle, and minimal in the mitotic (M) and late G₁ or early DNA synthetic (S) phases. (2) Markedly different growth patterns result from irradiation in different phases: (a) Prolongation of interphase (division delay) is minimal when cells are irradiated early in G₁ and rises progressively through the remainder of the cycle. (b) Cells irradiated while in mitosis are not delayed in that division, but the succeeding division is delayed. (c) Persistence of cells as metabolizing entities does not depend on the phase of the division cycle in which they are irradiated. (3) Characteristic perturbations of the normal DNA synthetic cycle occur: (a) Cells irradiated in M suffer a small delay in the onset of S, a slight prolongation of S, and a slight depression in the rate of DNA synthesis; the major delay occurs in G₂. (b) Cells irradiated in G₁ show no delay in the onset of S, and essentially no alteration in the duration or rate of DNA synthesis; G₂ delay is minimal. (c) Cells irradiated in S suffer an appreciable S prolongation and a decreased rate of DNA synthesis; G₂ delay is shorter than S delay.     

Changes in Rabbit Brain Cytosolic and Membrane-Bound Gangliosides During Prenatal Life

The present study deals with the developmental profile of cytosolic and membrane-bound gangliosides in rabbit whole brain from the 21st day of pregnancy, the time at which brain could be macroscopically recognized and handled, till birth. In this period of prenatal life the content of membrane-bound gangliosides showed a 2.5-fold increase, referred to fresh and dry brain weight and to membrane-bound protein; the content of cytosolic gangliosides reached a maximum at 21-22 days of pregnancy, and then underwent to birth a threefold diminution. The qualitative pattern of membrane-bound gangliosides, in the same period of life, was characterized by an increase of G_D1a and G_M1 (more marked for G_D1a), a decrease of G_T1a, G_T1b and G_Q1b and a constant level of G_D3 and G_D1b. At 21 days of pregnancy the most abundant gangliosides were G_T1b, and G_Q1b, followed by G_D1a and G_D1b; at birth it was G_D1a followed by G_T1b G_D1b, and G_M1 The qualitative pattern of cytosolic gangliosides closely resembled, during the entire period of prenatal life examined, that of membrane-bound gangliosides.