Promotive and inhibitory effects of diverse arabinogalactan proteins on Daucus carota L. somatic embryogenesis

In the 3-d-old 2-mm root tip of Pisum sativum L. cv. Lincoln the percentage of actively proliferating cells is estimated to be 70%. The remaining cells are non-cycling and arrested with 2C and 4C DNA content in G₀ and in G₂Q, respectively. In this work we studied the kinetic significance of these quiescent cells, using the sorting capabilities of flow cytometry and immunofluorescence techniques to detect the proliferation marker PCNA (proliferating cell nuclear antigen) inside cells within the different cell-cycle compartments. While in animal cells, PCNA is present at a high level only in actively proliferating cells, in 3-d-old pea root tips 95% of the cells are PCNA-positive. After flow cytometry and sorting of pea non-cycling nuclear populations, all G₂Q nuclei appeared strongly PCNA-positive, indicating that these cells had recently left the cell cycle. By contrast, most G₀ nuclei showed a low level of PCNA immunofluorescence intensity, as measured by image analysis, with about 25% of the nuclei being PCNA-negative. This small percentage was found to correspond to root cap cells, as could be observed in the root tip section. These are the only cells in the root apical region which are fully differentiated and which, therefore, lack the competence to enter the cell cycle. In contrast, the more or less PCNA-positive G₀ nuclei could represent a kinetically heterogeneous population of cells competent to proliferate, but which have either recently left the cell cycle or are progressing to the G₀-G₁ transition. Received: 6 November 1996 / Accepted: 14 January 1997     

Control of cell-cycle timing in early embryos of Caenorhabditis elegans

A technique has been developed for extruding either substantial amounts of cytoplasm without nuclei or individual nuclei with small amounts of cytoplasm from early embryos of C. elegans after perforating the eggshell with a laser microbeam. This technique, in conjunction with laser-induced cell fusion, has allowed the altering of nuclear/cytoplasmic ratios and the exposing of the nucleus of one cell to cytoplasm from another. Using these approaches the roles of nuclei and cytoplasm in determining the different cell-cycle periods of the several blastomere lineages in early embryos have been examined. It was found that nuclei in a common cytoplasm divide synchronously; enucleated blastomeres retain a cycling period characteristic of their lineage; cycling period is not substantially affected by changes in the ratio of nuclear to cytoplasmic volumes or the DNA content per cell; the period of a cell from one lineage can be substantially altered by introduction of cytoplasm from a cell of another lineage with a different period; and short-term effects of foreign cytoplasm on the timing of the subsequent mitosis differ depending on position of the donor cell in the cell cycle. These results are discussed in connection with models for the action of cytoplasmic factors in controlling cell-cycle timing.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.     

芍药胚和胚乳早期发育的研究

本文报道芍药(Paeonia lactiflora)的胚和胚乳的早期发育,主要结论是:1,开花后4—20天,合子核反复分裂形成多核原胚。2.原胚及胚乳游离核分裂均以有丝分裂为主,晚期有无丝分裂和多倍体核;3,细胞壁形成可由纺锤丝间和自由壁两种方式进行。原胚壁形成由合点端向珠孔端进行,胚乳则由胚附近向合点端发展。我们支持 Cave 等基于多核原胚提出的 Paeonia 与裸子植物平行演化的意见,同时认为 Paeonia 的多核原胚演化趋势是原胚由珠孔端至合点端功能分区的特化。     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

CULTURE STUDIES ON THE MARINE GREEN ALGA HALICYSTIS PARVULA—DERBESIA TENUISSIMA. II. SYNCHRONY AND PERIODICITY IN GAMETE FORMATION AND RELEASE

Unialgal cultures of the macroscopic, vesicular, coenocytic gametophyte (Halicystis parvula Schmitz) of Derbesia tenuissima (DeNotaris) Crouan fr. were grown under various environmental regimes to elucidate the cytology of gamete formation and the factors controlling synchronous gamete formation and release. No synchrony of nuclear division was observed in vegetative plants or during the early stages of gamete formation. In the later stages of gamete formation in plants in a light-dark cycle, nuclear divisions within any gametangium were synchronous, and the stages of gamete formation were synchronous for the population. This synchrony was not as great for plants in continuous light. Gametes of plants in a light-dark cycle were released explosively immediately following the dark-to-light transition. Release was random and much less forceful for plants in continuous light. After a certain stage of gamete formation, gamete release was timed to occur after a particular interval of darkness, but release could be triggered by light during the last portion of this interval. The length of the dark interval was shorter for male plants than for females, but the period of light sensitivity was longer for females. Formation of gametangia by series of isolated plants was also synchronous and sometimes periodic under certain conditions. Intervals between gametangia on the same plant varied from 2 to 14 days but were usually 4 or 5 days (unlike plants in nature, which show a bi- or tri-weekly periodicity). Male and female plants did not differ in synchrony or periodicity. Different media affected the number of gametangia formed over a period of time but not the synchrony of formation. Under some conditions changing the medium had a stimulating or synchronizing effect. Non-repeated temperature changes also synchronized gamete formation. Optimum temperature for continued gamete formation was about 21 C. Regular daily light and temperature variation together maintained synchronous and periodic gamete formation in populations of isolated plants. Reproduction diminished and became less synchronous at constant temperature either in continuous light or under a light-dark schedule, although in the light-dark regime steps in the formation of any given gametangium remained synchronous with the light-dark cycle. Length of times between gametangial formation on individual plants showed a tendency to occur in multiples of the usual period lengths; e.g., plants sometimes tend to “skip” intervals, thus maintaining the synchrony of the population. These results suggest that interaction between daily environmental cycles and an endogenous physiological cycle may maintain periodic reproduction.     

Cell synchrony techniques. I. A comparison of methods

Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G1, S, or G2 + M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G1 phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G1 peak yielded a relatively pure but heterogeneous G1 population (i.e. early to late G1). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

Expression in an arbuscular mycorrhizal fungus of genes putatively involved in metabolism,transport, the cytoskeleton and the cell cycle

Arbuscular mycorrhizal (AM) fungi are multinucleate, coenocytic, obligate symbionts with no known sexual stages and very wide host and habitat ranges. While contributing vitally to the growth of land plants they face unique challenges in metabolism, transport, growth and development. To provide clues to the strategies that AM fungi have adopted, random sequencing of cDNA's from Glomus intraradices was undertaken. Putative genes for enzymes, transporters, structural proteins and cell-cycle regulatory factors were discovered. Among the EST's of particular interest are sequences with homology to known trehalase, arsenite transporter, cysteine synthase, tubulins, actin, dynein, cell cycle regulatory proteins, and three meiosis-related proteins. The significance of these sequences is discussed in the context of what is known about AM metabolism, transport, growth and phylogeny.     

The Early Development of Embryo and Endosperm of Paeonia lactifiora

The present paper reports the early development of embryo and endosperm of Paeonia lactiflora. The main conclusions are as follows: 1 The zygote nucleus divides directly to form a coenocytic proembryo with different number of free nuclei. The result confirms the conclusion of Yakovlev[6] as well as Yakovlev and Yoffe[7] On the occurnce of coenocytic proembryo in Paeonia suffruticosa Andr. (P. moutan Sims), P. albiflora Pall. (P. lactiflora Pall), P. officinalis L., P. tenuifolia L., P. anomala L., P. veitchii Lynch (P. beresowskii Komarov), P. wittmanniana, also it agrees with the report of Cave et al.[4] on the occurence of the coenocytie proembryo in Paeonia californica and P. brownii. From the result of our investigation, there is no basis to support Murgai's conclusion obtained in certain species of Paeonia, i.e., the first division of zygote nucleus is accompanied by wall formation and the coenocyte is a suspensor. The primary endosperm nucleus of P. lactiflora divides often earlier than zygote nucleus, or almost at the same time or later in a few cases; 2 Both mitosis and amitosis occur in the free nuclei in the coenocytic proembryo, and mitosis is dominant. In the later stage of development the amitosis occurs at the micropylar region of the coenocytic proembryo, in the free nuclear endosperm at the chalazal region and the appressed part of the chalazal end of the coenocytic proembryo. In addition, in the region of conenocytic proembryo and endosperm polyploid nuclei, irregular nuclei are also frequently met with; 3 Cell wall formation in the coenocyte and the endosperm initiates by means of both cell plate and freely growthing walls, but in the coenocyte wall formation is earlier than in the free nuclear endosperm. At first, wall formation of the coenocytic proembryo begins at the chalazal end, and then extends toward the micropylar region, and in endosperm it begins at the part appressed to the part of the chalazal end of coenocytic proembryo, and then extends toward the chalazal end. We support Cave’s suggestion of the parallel evolution for Paeonia possessed the coenocytic proembryo has no relation to the coenocytic proembryo of the gymnosperms. Further we consider the evolution tendency of the coenocytic proembryo of Paeonia toward a functional specialization.     

Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11–13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.     

Flow cytometric determination of nuclear DNA content during differentiation (spherulation and germination) of the myxomycete Physarum polycephalum

Summary Using flow cytometry, spherulating nuclei of Physarum isolated at the beginning of spherule wall formation were found to exhibit a DNA content corresponding to the G₂ phase of the cell cycle, although 8% lower. Before the first mitosis after spherule germination, a very slight incorporation of ³H thymidine into DNA was observed that was too weak to correspond to S phase, strongly suggesting that nuclei are stopped in G₂ phase inside the spherules. The lower value of nuclear DNA content found using flow cytometry of germinating spherules may not be related to DNA quantity, but may be due to a difference in chromatin organization during growth or spherulation, resulting in interference with the staining.     

Cell-cycle analysis of fission yeast cells by flow cytometry

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.     

Flow cytometric cell division tracking using nuclei

BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle.     

Synchronization of murine erythroleukemic cells. Nuclear volume measurements for monitoring cell cycle traverse

Murine erythroleukemic cells (T3C12 clone) were synchronized either by using two 8-h thymidine (2 mM) blocks separated by a 7-h release period, or by centrifugal elutriation. To monitor the synchrony induction and cell cycle traverse, the cells were lysed with NP40 for 20 min and the resulting nuclei fixed with glutaraldehyde. These nuclear volume measurements were performed with a Coulter Electronics H4 cell volume spectrometer and compared with flow cytometric determinations of nuclear DNA content on a Coulter Electronics TPS-1 cell sorter. The results indicate that the nuclear volume measurements can be used as a simple precise method to monitor the cell cycle position and traverse of synchronized erythroleukemic cells.     

Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity

Synchronized suspension cultures are powerful tools in plant cell-cycle studies. However, few Arabidopsis cell cultures are available, and synchrony extending over several sequential phases of the cell cycle has not been reported. Here we describe the first useful synchrony in Arabidopsis, achieved by selecting the rapidly dividing Arabidopsis cell suspensions MM1 and MM2d. Synchrony may be achieved either by removing and re-supplying sucrose to the growth media or by applying an aphidicolin block/release. Synchronization with aphidicolin produced up to 80% S-phase cells and up to 92% G2 cells, together with clear separation of different cell-cycle phases. These synchronization procedures can be used for analysis of gene expression and protein activity. We show that representatives of three CDK gene classes of Arabidopsis (CDKA, CDKB1 and CDKB2) show differential expression timing, and that three CDK inhibitor genes show strikingly different expression patterns during cell-cycle re-entry. We propose that ICK2 (KRP2) may have a specific role in this process.     

Malaria and the cell cycle

The current model of cell cycle control features a succession of active cyclin-CDK (cyclin-dependent kinase) complexes, where accumulation of each successive cyclin leads to activation of its associated kinase. Cell fusion experiments have shown that nuclei sharing common cytoplasm progress through the cell cycle in synchrony. During schizogony of Plasmodium falciparum, nuclear division occurs asynchronously, and thus cannot be regulated by synthesis and accumulation of cyclins in the cytoplasm. We suggest that schizonts must have a ready pool of cyclins for activating all stages of the cycle, and that the cell cycle is regulated independently in each nucleus.     

Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum

Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed.     

RNAi of mitotic cyclins in Drosophila uncouples the nuclear and centrosome cycle

BACKGROUND: Successful cell duplication requires orderly progression through a succession of dramatic cell-cycle events. Disruption of this precise coupling can compromise genomic integrity. The coordination of cell-cycle events is thought to arise from control by a single master regulator, cyclin:Cdk, whose activity oscillates. However, we still know very little of how individual cell-cycle events are coupled to this oscillator and how the timing of each event is controlled. RESULTS: We developed an approach with RNA interference (RNAi) and real-time imaging to study cyclin contributions to the rapid syncytial divisions of Drosophila embryos. Simultaneous knockdown of all three mitotic cyclins blocked nuclei from entering mitosis. Despite nuclear arrest, centrosomes and associated myosin cages continued to divide until the midblastula transition. Centrosome division was synchronous throughout the embryo and the period of the uncoupled duplication cycle increased over successive divisions. In contrast to its normal actions, injection of a competitive inhibitor of the anaphase-promoting complex/cyclosome (APC/C) after knockdown of the mitotic cyclins did not interfere with the centrosome-duplication cycles. Finally, we examined how cyclin knockdown affects the onset of cellularization at the midblastula transition and found that nuclear cell-cycle arrest did not advance or delay onset of cellularization. CONCLUSIONS: We show that knockdown of mitotic cyclins allows centrosomes to duplicate in a cycle that is uncoupled from other cell-cycle events. We suggest that high mitotic cyclin normally ensures that the centrosome cycle remains entrained to the nuclear cycle.