Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

Delays in prophase induced by adenosine 2-deoxyriboside and their relation with DNA synthesis

The rate of DNA synthesis in the course of the division cycle in root meristem ofAllium cepa growing under constant temperature and aeration conditions has been studied by means of treatment with AdR, as a specific inhibitor of the synthesis, as well as by the incorporation of tritiated thymidine. The one-hour treatment with AdR or tritiated thymidine was given at various hours in the course of the interphase of a synchronous population of binucleate cells induced by caffeine. In the case of AdR, sensitivity to the inhibition of DNA synthesis was studied by recording the delays produced by the treatment in the appearance of biprophases and bitelophases. The selection by the use of caffeine, of spontaneously synchronous populations of cells going through the telophase and becoming binucleate and the detection of the first biprophases in the subsequent mitosis provide a highly synchronized system with which to study the incorporation of tritiated thymidine during the interphase. The curves representing sensitivity to the inhibition of DNA synthesis by AdR and the rate of tritiated thymidine incorporation coincide, so that we can regard the delays, under our conditions, as proportional to the rate of DNA synthesis at the moment of the AdR treatment. This rate, in the S period, was found to be variable by both methods, being higher in the first and the last thirds of the S period (S₁ and S₃) and lower in the middle third (S₂).     

Immunocytochemical localization of callose in root cortical cells parasitized by the ring nematodeCriconemella xenoplax

Summary Under hypoxia (10 and 5% partial oxygen tension) meristematic cells ofAllium cepa L. roots acquired new cycle kinetics, characterized by reduced but constant rates of root growth. Under these conditions, there was preferential lengthening of G₁ and of the last third of the S period, S₃. Since hyperoxygenation shortened S₃ but not G₁ in these cells, the high sensitivity of late replication to environmental oxygen is demonstrated. The preferential depression of the replication rate when those cells replicated the last third of their DNA was not associated with diminished cell size. Rather, the lower the oxygen level the larger the mean size of the cycling cells. Under anoxia (0% oxygen tension) the rate of growth slowed, accompanied by preferential accumulation of cells in G₁. However, steady state kinetics of root growth was not achieved under these extreme conditions.Abbreviations Mean cell length - LI labelling index or frequency of cells with labelled nuclei after [³H]thymidine - G₁, S, G₂ pre-replicative, replicative, and post-replicative periods of the interphase of cycling cells - M mitosis     

Structural changes in chromatin during interphase

The structural organization of the dense chromatin in G₁, S and G₂ of onion meristem cells has been evaluated. A naturally synchronous subpopulation of caffeineinduced binucleate cells was employed. — Fibre size is positively correlated with cell stage in interphase. Fibre size distribution is unimodal in G₁ nuclei with the peak at 12.5 nm in diameter, while in G₂ the distribution is bimodal due to a new population of thicker fibres (22.5 nm in diameter). Separation between fibre centres takes place between mid G₁ and mid S. — Using stereological principles, the length of chromatin fibres integrated in the chromatin patches could be estimated. This length remains constant from mid G₁ to mid S, when a 1.5 fold increase in total DNA takes place. On the other hand, 40 mm/hour of chromatin fibre is integrated in the chromatin patches between mid S and mid G₂. Comparable data of the relative proportion of the different nuclear components have been obtained in each interphase period. — The reported changes provide evidence of a cyclic pattern of chromatin condensation, which may be the structural support for a model of chromatin function in these cycling cells.     

INCORPORATION OF TRITIUM-LABELED THYMIDINE AND LYSINE INTO CHROMOSOMES OF CULTURED HUMAN LEUKOCYTES

The incorporation of thymidine-H³ and lysine-H³ into human leukocyte chromosomes was studied in order to determine the temporal relationships between the syntheses of chromosomal deoxyribonucleic acid and chromosomal protein. The labeled compounds were incorporated into nuclei of interphase cells. Label from both precursors became apparent over the chromosomes of dividing cells. Incorporation of thymidine-H³ occurred during a restricted period of midinterphase (S) which was preceded by a nonsynthetic period (G₁) and followed by a nonsynthetic period (G₂). Incorporation of lysine-H³ into chromosomal protein occurred throughout interphase. Grain counts made over chromosomes of dividing cells revealed that the rate of incorporation of lysine-H³ into chromosomal protein differed during various periods of interphase. The rate of incorporation was diminished during G₁. During early S period the rate of incorporation increased, reaching a peak in late S. The high rate continued into G₂. Thymidine-H³ incorporated into DNA was distributed to mitotic chromosomes of daughter cells in a manner which has been referred to as a "semi-conservative segregation." No such semi-conservative mechanism was found to affect the distribution of lysine-H³ to the mitotic chromosomes of daughter cells. Therefore, it is concluded that synthesis of chromosomal protein and its distribution to chromosomes of daughter cells are not directly influenced by synthesis and distribution of the chromosomal DNA with which the protein is associated.     

Effects of 1000 R whole-body X-irradiation on DNA synthesis and mitosis in the duodenal crypts of the BCF1 mouse

Summary Effects of 1000 R, whole-body X-irradiation on the proliferative cells of the mouse duodenal crypts, in the four phases of the generation cycle; namely, the DNA synthesis phase, S; the pre-mitotic gap, G ₂; the division phase or mitosis, M; and the pre-synthesis gap, G ₁. As pointed out by Whitmore and Till (1964) G₁ and G₂ are characterized only by the fact that no DNA synthesis is taking place in these phases.In the intestinal crypts of BCF₁ mice, a 1000 R whole-body X-ray exposure blocks cells in G₂ for approximately 18 hours, and reduces the number of cells in S to less than 1/2 that observed in control animals during the first 12 hours after exposure. Cells synthesizing DNA, and undergoing division, remain few in number for more than 48 hours. Between 48 and 72 hours a compensatory reaction begins, and the number of cells in M and S increases from 28 at 48 hours to 150 at 72 hours and reaches a mean value of 482 at 96 hours.Work supported under the auspices of the US Atomic Energy Commission.     

Early and late DNA replication in respectively condensed and decondensed heterochromatin ofAllium carinatum

Summary The structure and ultrastructure of nuclei in the S period and other phases of the mitotic cell cycle have been studied in semi- and ultrathin sections of root tips ofAllium carinatum. Significant structural differences have been found and classified by means of DNA measurements by scanning photometry of Feulgen-stained squash preparations. In G₁ and early S (S₁ and S₂) the euchromatin forms small, compact and electron-dense patches, while the heterochromatin is condensed into a number of chromocenters of the same electron-density as the euchromatin. In middle S (S₃) the euchromatic elements become larger and more thread-like. In late S (S₄) the euchromatin appears in the form of thick and uniform strands as in G₂, and the heterochromatin decondenses into strands of the same, or a little higher, diameter, as the euchromatin. DNA replication starts in the condensed heterochromatin (S₁, becomes shifted to euchromatin (S₂), continues over both eu- and heterochromatin during middle S (S₃), and is restricted to decondensed heterochromatin in late S (S₄). Quantitative data of various nuclear parameters are given for the different stages. The results are discussed in relation to the species-specific nuclear ultrastructure, its molecular basis, and its variation during the mitotic interphase, as well as with respect to the timing and structural expression of DNA replication.     

Cell cycle analysis in a multinucleate green alga,Boergesenia forbesii (Siphonocladales,Chlorophyta)*

The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G₁ period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G₂ period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G₁ stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G₂ and mitosis. In other words, the cell cycle regulation of entrance into S phase from G₁ is an important factor in the growth and morphogenesis in B. forbesii.     

The probability of G1 cells to enter into S increases with their size while S length decreases with cell enlargement in Allium cepa

The distribution of cell surface area projection (cell size) has been measured at birth and at initiation of DNA synthesis in steady-state populations of Allium cepa root meristems. The conditional probability, P(I/G₁), that initiation occurs given that the event of being in g₁ also occurs has been estimated from these data. p(I/G₁) was found to increase when cells became larger. The distribution of G₁ duration has been constructed from indicated cell size distributions. The absolute frequencies of G₁ times showed a maximum in the zone of cells with short G₁ periods; about 14% of cells appear to enter into S with G₁ - 1 h. These results suggest that the increase of p(I/G₁) was due to cell enlargement and not to cell aging. By comparing the cell size distribution at initiation of S and at the end of this period, a drastic reduction of cell size variability during DNA replication was observed and both curves were seen as rather similar in shape although they obviously had different modal points. These observations support that there is a negative correlation between the initiation size and the duration of genome duplication, and that cells which initiate DNA synthesis with the same size have a similar replication time. From this hypothesis, a plot of S duration versus cell size at initiation of this period was constructed by comparing the distributions of cell size at start and end of replication; this plot was also consistent with the existence of a negative correlation between cell initiation size and S length.     

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.     

Change of chromatin morphology during the cell cycle detected by means of automated image analysis

Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G₁, middle S and G₂ phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G₁ through S to G₂ phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G₁, S and G₂ phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.     

Regulation of DNA synthesis in cytochalasin B (CB)-induced binucleate HeLa cells

The effects of timing and duration of cytochalasin B (CB) treatment on the kinetics of the initiation of DNA synthesis in mono- and binucleate HeLa cells, synchronized in the G1 phase of the cell cycle by the reversal of a mitotic block (N₂O at 80 PSI), were studied. In the control, bi-, tri- and tetranucleate cells entered S phase slightly earlier than the mononucleate cells at a rate proportional to the number of their nuclei. The difference between any two adjacent sub-populations was less than 0.5 h. However, the binucleate cells produced by a 90 min CB treatment immediately after the reversal of the mitotic block exhibited a considerably shorter G1 period as compared to mononucleate cells (a difference of 1.5 h). This exaggerated difference in the duration of G1 period between mono- and binucleate cells disappeared when the CB treatment was delayed by 75 or 90 min indicating that it was an experimental artifact. From this study, we conclude that there is naturally some degree of nuclear cooperation in the multinucleate systems, particularly with regard to the initiation of DNA synthesis, which is not influenced by CB treatment.     

An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data

Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G₁ and G₂ cells, was calculated. RM was then used to calculate DNA synthesis time (T_S), at all post-labelling times (t). Since labelled cells in G₂ and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G₂ and M (T_g2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of T_S yields results that are affected by t. We found that RM data can be corrected with regard to T_G2+M resulting in the derivation of a non-linear T_S formula. This non-linear T_S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G₁, S and G₂+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. T_S obtained in this way was in agreement with T_S obtained with the corrected RM method. In conclusion, we present a method to calculate T_s which theoretically first makes the determination of RM independent of T_G2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G₂+ M.     

ANALYSIS OF GROWTH OF TETRAPLOID NUCLEI IN ROOTS OF VICIA FABA

Growth of nuclei of a marked population of cells was determined from G₁ to prophase in roots of Vicia faba. the cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G₁ and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. of the marked population of cells, about 65% had completed a cell cycle 14–15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.     

NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G₁) has been investigated in synchronized HeLa S₃ cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G₁ was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope.     

Cytochemistry of histones throughout the division cycle in meristematic cells

Summary We have been able to follow the increase in nuclear histones along the division cycle, using a natural synchronous meristematic cell population labelled by caffeine as binucleate. We have thus found a parallel increase in DNA and histone along the S period. Some increase in nuclear histones during the G₁ and G₂ has also been detected. At the same time a high rise of stainable histone in the nuclei in prophase has been found.Changes in the composition of nuclear histones have been followed counting the percentage of arginine-rich histones in the total histone content. Two peaks located at the beginning and at the end of the S period as well as a sharp descent of the percentage of arginine-rich histone in the prophasic nucleus have been found.     

The chromosomes of Puschkinia libanotica during mitosis

The chromosome complement of Puschkinia libanotica is described. In addition to five pairs of A chromosomes plants may possess up to 7 B chromosomes. Part of the long arm of the B chromosome gives rise to a heterochromatic mass in interphase nuclei and this can be seen to be a double structure in G₁ nuclei and a quadruple structure in G₂ nuclei. It is believed that these configurations represent the pre- and post-replication forms of subchromatids in the heterochromatic segment of the B chromosome. Microdensitometry of metaphase chromosomes shows that the segment of the B chromosome that is heterochromatic during interphase has no more DNA per unit volume than any of the euchromatic A chromosomes.     

The time and duration of the premeiotic inteprhase in microsporocytes ofTrillium kamtschaticum

The time and duration of each phase of the premeiotic interphase were determined in microsporocytes of two clones (S and K clones) ofTrillium kamtschaticum. After collectionTrillium plants were stored at 3 C or 7 C prior to completion of premeiotic mitosis in archesporial cells. For autoradiography, cells were explanted in the presence of³H-thymidine to identify the interval of the premeiotic DNA synthesis. Approximate durations of the G₁, S and G₂ phases for the K clone stored at 3 C were estimated to be 12, 12 and 14 days, respectively. The interval of premeiotic development was markedly different between clones. A high degree of synchrony in meiotic development, which is usually observed within anthers up to late meiotic prophase, was confirmed at the S phase, suggesting that synchrony is established during the G₁ interval.     

Some effects of 2,4,5-trichlorophenoxyacetic acid on the mitotic cycle of lateral root apical meristems of Vicia faba

Roots of Vicia faba were given a one hour pulse label with. ³H-TdR (1 C/ml), either before or after a three hour treatment with a 10^–5 M solution of 2,4,5-trichlorophenoxyacetic acid (TCPA). The durations of the various phases of the mitotic cycle were derived from labeled prophase curves, prepared from autoradiographs of lateral root apical meristems. — TCPA was found to lengthen the duration of the mitotic cycle, primarily because it extended the duration of the period of DNA synthesis (S), though post-synthetic interphase (G₂) was also longer. No measurements could be made with respect to the duration of presynthetic interphase (G₁), because of rapid changes in the lengths of the G₂ and S periods following treatment. — As well as extending the duration of S, TCPA treatment also resulted in at least an initial increase in the rate of DNA synthesis and a decrease in the actual number of cells in S. These results have been discussed with respect to the control of the organization of the root apical meristem.Supported by a grant from the Assistant Professor Research Fund of the University of Missouri.