THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

Variety of cell cycle patterns in the alga Scenedesmus quadricauda (Chlorophyta) as revealed by application of illumination regimes and inhibitors

In the course of the cell cycles of synchronous cultures of the chlorococcal alga Scenedesmus quadricauda, the following were monitored: total protein and RNA accumulation as a measure of growth processes, the timing of the commitment points at which the cells trigger the sequence of reproductive processes (DNA replication, nuclear and cellular division) and the course of the reproductive processes. The synchronous cultures were grown either under various lighting regimes, or in the temporary presence of specific inhibitors of either proteosynthesis (cycloheximide) or DNA replication (5-fluorodeoxyuridine). By adjusting the length of the light period, the cell cycle could be manipulated. Cell cycle patterns could be altered to give different numbers of sequences of reproductive processes. The extent of their mutual overlap could be influenced and the number of daughter cells produced could be altered. Schematic illustrations of various cell cycle patterns and comparisons with those of higher plants and other algal species are presented.     

High temperature enhances cytotoxicity of mercury (HgCl2) on Hela S3 cells

The combined effect of mercury (HgCl₂) and high temperature on the growth and synthesis of nucleic acid and protein, and on the cell cycle of HeLa S₃ cells was investigated. The subsequent growth of the cells was dose-dependently inhibited by mercury at 37.2° and 41.2°C. The inhibitory effect of mercury on subsequent growth was enhanced at the higher temperature. IC₅₀ values for DNA and RNA synthesis but not protein synthesis, at 41.2°C, were significantly lower than those at 37.2°C (P<0.05,P<0.01, respectively). Flow cytometric analysis using synchronous cells indicated the possibility of blocking of cell cycle progression in the early part of S phase by the combined treatment. These results suggest that the cytotoxicity of mercury to cell growth was enhanced at the higher temperature and that this enhancement is related to the increased inhibitory effect of mercury on DNA and RNA synthesis and on the cell cycle at high temperatures.     

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

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

CHEMICAL ESTIMATIONS OF DNA CHANGES DURING SYNCHRONOUS GROWTH OF EUDORINA ELEGANS (CHLOROPHYCEAE)12

Analysis of synchronous populations of Eudorina elegans Ehrenberg reveals that DNA replication occurs coincidentally with the very rapid sequence of cell divisions. The average doubling time for DNA in such synchronous populations is 40 min. The basal amount of DNA/E. elegans cell is estimated to be 0.5 × 10^?12 g.     

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

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

Effect of irradiance on the course of RNA synthesis in the cell cycle ofScenedesmus quadricauda

In synchronous populations ofScenedesmus quadricauda the RNA amount in the cells increases in waves: periods of a high rate of RNA synthesis alternate with periods of a low rate in the course of the cell cycle. Each wave usually leads to the doubling of the RNA amount per cell. In cells growing under normal conditions the waves of RNA synthesis seem to be linked with consecutive rounds of DNA replication. The pattern of RNA synthesis in the course of the cell cycle, however, does not change, if DNA replication is prevented by application of 5-fluoro-deoxyuridine. In darkness the rate of RNA synthesis drops to zero and thereafter the RNA amount per cell decreases. In cells which have been induced to cellular division RNA synthesis may become restored in the dark in newly formed daughter cells. The lowering of RNA amount and its new increase during the dark period become more pronounced with increasing irradiance in the previous light period as well as with its increasing length. In the period of protoplast fissions RNA synthesis is arrested even if the cells divide in the light; whether a similar inhibition occurs during mitoses is not clear.     

Basal-Cell Subpopulations and Cell-Cycle Kinetics In Human Epidermal Explant Cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different ³H-thymidine (³H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that ³H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G₂-phase cells were cycling, whereas some 20% of the cells stayed in G₁-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. the difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G₁- and G₂-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G₂-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated ³H-dThd at a higher rate than cells with high RNA contents.     

Erythropoietic progenitors capable of colony formation in culture: response of normal and genetically anemic W-W-V mice to manipulations of the erythron

The macromolecular reguirements for the initiation and maintenance of macronuclear DNA replication were studied in heat synchronized Tetrahymena pyriformis GL-C. Previous work had established that macronuclear S periods could occur in a consecutive fashion without intervening cell divisions during a multiple heat shock treatment, as well as immediately following the synchronized cell divisions. Cycloheximide treatment prior to or during the S period which follows the first synchronized cell division resulted in abolition of the initiation of DNA synthesis or an almost immediate cessation of DNA synthesis in progress. Temporary inhibition of DNA synthesis occurred when cycloheximide was added late in the S period. Treatment with actinomycin D was found to block the initiation of DNA synthesis but did not appreciably affect the continuation of the S period. It was concluded that RNA synthesis was required for the initiation but not the maintenance of DNA replication, whereas protein synthesis was necessary for both processes. The dependency of the initiation of an S period on prior RNA and protein synthesis was also shown to exist when a second consecutive S period was initiated without a preceding cell division. Treatment with actinomycin or cycloheximide prior to a supernumerary S period during a multiple heat shock treatment completely abolished the initiation of DNA synthesis. In T. pyriformis the synthesis of RNA and protein related to the initiation of the S period is tightly coupled to each cycle of DNA replication.     

Endomitosis and the effect of gibberellic acid in different Pisum sativum L. cultivars

The evolution of the total amount of DNA in epicotyls and of the amount of DNA per cell nucleus in epicotyl cortex cells during germination was followed in two closely related pea varieties, Pisum sativum cv. Finale and Pisum sativum cv. Rondo. Under etiolating conditions, growth of the cv. Rondo occurs only by cell elongation which is preceded by endomitotic DNA synthesis, while in the cv. Finale growth is the result of cell elongation accompanied by endomitotic DNA synthesis and cell division. The maximum C-level attained in both cultivars under etiolating conditions is 8 C (C=haploid amount of DNA in a gamete cell). Both the maximum C-level reached and the percentage of cells reaching this C-level seem to be under strict genetic control. In both cultivars, light inhibits the endomitotic DNA replication.Neither gibberellic acid (GA₃), nor AMO 1618 alter the maximum C-level or the percentage distribution of the C-classes. Both growth regulators are effective, although in an opposite way, only in tissues where cell division occurs or where endomitotic DNA synthesis is blocked, as in light-grown pea epicotyls.     

A CYTOPHOTOMETRIC STUDY OF NUCLEIC ACIDS AND PROTEINS IN THE SHOOT APEX OF WHITE SPRUCE

During the annual three-phase growth cycle of white spruce [Picea glauca (Moench) Voss] the vegetative shoot apex changed in anatomical configuration. Relative amounts of DNA, histones, RNA, and proteins were measured in three cytohistological zones and were related to the anatomical changes during ontogeny. An extended period of DNA synthesis (S) and G₂ preceded an increase in the number of apical initial cells which were part of the mammillary apex. While DNA and histones were generally synchronous during ontogeny, the ratio of DNA to histone increased on June 20. This loss of histone and subsequent increases in RNA and cytoplasmic proteins preceded the appearance of needle primordia on next year's apex. We propose that induction of the apex to reorganize and form needle primordia occurred when the DNA was in a 2C condition, following the loss of histone on June 20.     

Controls over the timing of DNA replication during the cell cycle of fission yeast

The controls acting over the timing of DNA replication (S) during the cell cycle have been investigated in the fission yeast Schizosaccharomyces pombe. The cell size at which DNA replication takes place has been determined in a number of experimental situations such as growth of nitrogen-starved cells, spore germination and synchronous culture of wee mutant and wild-type strains. It is shown that in wee mutant strains and in wild type grown under conditions in which the cells are small, DNA replication takes place in cells of the same size. This suggests that there is a minimum cell size beneath which the cell cannot initiate DNA replication and it is this control which determines the timing of S during the cell cycle of the wee mutant. Fast growing wild-type cells are too large for this size control to be expressed. In these cells the timing of S may be controlled by the completion of the previous nuclear division coupled with a requirement for a minimum period in G1. Thus in S. pombe there are two different controls over the timing of S, either of which can be operative depending upon the size of the cell at cell division. It is suggested that these two controls may form a useful conceptual framework for considering the timing control over S in mammalian cells.     

Interfacial spin trapping in model membrane systems

The nature of mitochondrial DNA replication during the synchronous cell cycle in the yeast, $\text{Saccharomyces}\text{cerevisiae}$ has been investigated by examining the rate of labeled DNA precursor incorporation into specific segments of the mitochondrial genome at defined points during synchronous growth. The movement of label uptake from one area of the DNA to another at different times during the synchronous cell cycle indicates mitochondrial DNA replication to be a synchronous process during this time with most or all molecules at the same point in replication at any given time during the cell cycle.     

NUCLEIC ACID CONTENT OF HeLa S3 CELLS DURING THE CELL CYCLE: VARIATIONS BETWEEN CYCLES

The process of continuous resynchronization with excess thymidine provides sufficient cell material for accurate chemical determination of DNA and RNA in HeLa S3 cells at hourly intervals during the cell cycle. Total DNA is constant during the non-S phase portion of the cell cycle but varies widely among cycles of synchronous growth. Total cellular RNA content increases linearly in the G₁ phase and accelerates to a higher linear rate of accumulation, which remains constant during most of the S and G₂ phases. The ratios of early and late cycle rates of RNA accumulation are not constant among cycles.     

蛋白质起始的DNA病毒复制机制

DNA聚合酶在DNA合成过程中需要的引物包括RNA引物、DNA自我引物和蛋白质引物3种类型。新DNA链(如冈崎片段)的复制多是在DNA模板上合成一段RNA引物,细小病毒利用其基因组末端的反向末端重复序列(ITRs)自我折叠成DNA引物,而一些DNA、RNA病毒及真菌质粒起始复制反应的引物则是蛋白质。以感染原核生物的噬菌体Phi29和真核DNA病毒腺病毒为例,从复制过程所涉及的蛋白质、对复制原点的识别、复制起始反应、新链的延伸、复制终止过程等方面详细阐述DNA病毒由蛋白质引发的复制机制,并对已商品化的Phi29 DNA聚合酶产品多重置换扩增及单细胞测序等的应用以及基于噬菌体Phi29蛋白质起始的最小复制系统体外扩增异源DNA等最新的应用研究作相关总结介绍。     

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum

Summary Tritiated -amanitin has been used as a specific and sensitive probe to estimate the number of RNA polymerase B molecules in isolated nuclei, chromatin and nucleoids, obtained from macroplasmodia ofPhysarum polycephalum. During mitosis (metaphase±10 min) there is at least 10-fold less RNA polymerase B than at all phases of the cell cycle, even if DNA replication has been blocked in vivo. It is concluded that many of the RNA polymerase B molecules leave the chromatin during decondensation of the chromosomes in telophase of the synchronous nuclear division ofPhysarum.     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.