Inhibition of nucleic acids synthesis in Chlorella by 2,4-dinitrophenol a unique concentration effect on DNA synthesis

Nucleic acids and protein synthesis in synchronously growing Chlorella cells were inhibited by 2,4-dinitrophenol. RNA and protein synthesis decreased gradually from about 100% at 0.1 mM to almost 0% at 10 mM dinitrophenol. DNA synthesis was strongly inhibited at 0.5 mM but less at 1 mM concentration of the inhibitor. Beyond 1 mM the inhibitory effect increased again. A transient exposure to 0.5 and 10 mM dinitrophenol was fully reversible and cell division after the inhibition proceeded normally except for a slight delay.Abbreviation DNP 2,4-dinitrophenol     

Adaptation to cycloheximide of macromolecular synthesis in Tetrahymena

Cycloheximide (CHI) at 10 ng/ml partially inhibited protein synthesis in exponential cultures of Tetrahymena Sp. At 20 ng/ml or greater, inhibition was complete. When protein synthesis was inhibited to any extent, cell division ceased immediately. In all instances where measured, synthesis of RNA and DNA also ceased. After a period of delay, cellular functions reinitiated in the order: (i) protein synthesis, (ii) DNA synthesis and, (iii) RNA synthesis and cell division. The delay in cell division was divided into three phases of: I, zero; II, low; and, III, fully recovered rates of exponential protein synthesis. The length of the three phases increased with increasing concentration of CHI Prior growth of cells for one generation in the presence of 7.5 ng/ml CHI (facilitation) eliminated phase I and slightly decreased phases II and III following subsequent challenge with an inhibitory concentration of CHI. Facilitation for six generations further decreased phases II and III. Protein synthesis and cell division were not inhibited during facilitation In the culture, succinate dehydrogenase activity did not increase during the delay but increased normally at the onset of division. In contrast, NADPH-cytochrome c reductase activity continued to increase for an hour after inhibition of protein synthesis, was constant for a period and did not increase again until an hour after reinitiatoin of cell division and RNA synthesis Inhibition of division of all cells was immediate and reinitiation of synthesis and cell division was non-synchronous.     

Growth regulation in irradiance limited marine Synechococcus sp. WH 7803

Examinations of the macromolecular components of the protein synthesizing system (RNA, DNA and protein) have been made in the marine cyanobacterium, Synechococcus sp. WH 7803. Slowly growing, irradiance limited cells have less RNA and lower rates of RNA synthesis than do those growing at rapid rates. RNA content and synthesis increase in conjunction with division rate. Protein content is variable. Protein synthesis increases up to a plateau at division rates less the maximum observed. The results imply that there is extra protein synthetic capacity produced at high, irradiance limited growth rates. Synechococcus sp. WH 7803 responds to an increase in irradiance through a rapid shiftup in macromolecular synthesis. RNA, protein and DNA increase in a sequential fashion which precedes the onset of cell division. After decreases in irradiance, protein synthesis is maintained despite reductions in RNA. This suggests that there is some degree of physiological buffering which occurs in this species. These studies indicate that, as in more extensively studied procaryotic models, the protein synthesizing system plays a central role in the global mechanisms regulating growth in Synechococcus sp. WH 7803.Abbreviations PSS protein synthesizing system - HMW high molecular weight - LMW low molecular weight - TCA trichloroacetic acid     

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.     

Inhibition of mitosis in pisum root meristems by continuous gamma radiation: the influence of temperature on the synthesis of DNA, RNA and protein during inhibition

Tritiated precursors of DNA, RNA and protein were used to measure synthesis at 10 and 20C in root meristem cells of Pisum after they were mitotically arrested by continuous irradiation with gamma rays. The experiments were designed to determine if the arrested cells accumulated in a certain part of interphase, to determine the effect on DNA, RNA and protein synthesis, to find out if the effects were temperature dependent, and finally to reveal possible relationships between growth inhibition and altered synthesis. The results showed that the incorporation of DNA and RNA precursors was impaired by irradiation and that decreased temperature further increased radiation impairment of DNA synthesis. Protein synthesis on the other hand was not impaired by irradiation at either temperature. Irradiation at 20C reduced the number of DNA-synthesizing cells; at 10C this number was reduced to near zero. Although irradiated cells synthesizing RNA showed a reduction in grain counts when compared to the controls, they still retained the ability to incorporate tritiated uridine at 10C. It was hypothesized that the combination of reduced DNA and RNA synthesis and unaffected protein synthesis resulted in precocious maturation of the arrested meristem cells. Growth which occurred in the absence of cell division was attributed to meristematic cells which precociously matured and cells which were in the region of elongation.     

Massive synthesis of ribonucleic Acid and cellulase in the pea epicotyl in response to indoleacetic Acid, with and without concurrent cell division

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.

The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.

It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

     

Molecular and cellular events during the germination of conidia of Sporothrix schenckii

Hyaline, non pigmented microconidia of Sporothrix schenckii were harvested and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 °C. These conditions supported only the development of the mycelial form of Sporothrix schenckii in a reproducible, synchronized manner which allowed further analysis of the early cellular events ocurring during the germination of the conidia. The relationship between macromolecular synthesis (DNA, RNA and protein synthesis) and nuclear division, hyphal growth and septum formation were established. Following inoculation, protein synthesis was observed after 10 minutes followed by RNA synthesis, after 1 h and DNA synthesis after 2 h. The first nuclear division was observed during the 9 to 12 h interval after inoculation. Germ tube formation slightly preceeded nuclear division and was first evidenced 9 h after the induction of germination but was not completed until 12 h after inoculation. Septation was first observed in the germ tubes 0.25 m from the mother cell-germ tube function 9 h after induction of germination.     

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.     

Effects of zinc on macromolecular synthesis and nuclear division during the yeast to mycelium transition in Sporothrix schenckii

Zinc ions (10 mM) have been reported previously to inhibit the yeast to mycelium transition in Sporothrix schenckii. Yeast cells of this fungus were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose in the presence of 10 mM zinc and the effects of this ion on protein, RNA and DNA synthesis and nuclear division recorded. All of these processes were affected by the addition of 10 mM zinc to the medium. Nevertheless, the inhibition of protein synthesis was observed earlier than that of RNA or DNA synthesis and was of a greater magnitude than that observed for both of these processes. Protein synthesis was inhibited within the first hour after inoculation, at which time this process begins in the control cells. RNA synthesis was inhibited during the 3 to 6 h interval after inoculation, that is, 3 h after the start of this process in the control cells. After 9 h of incubation, the inhibition of protein synthesis had reached its maximum at 70%, while that of RNA synthesis was only 52%. DNA synthesis was slightly inhibited, with maximum inhibition being observed 9 h after inoculation. Nuclear division in cells forming germ tubes in the presence of 10 mM zinc took place with a 3 h delay in relation to the control cells. These observations suggest that the inhibition of protein synthesis might be the most important mechanism by which zinc inhibits the yeast to mycelium transition in S. schenckii.     

Synthesis of macromolecules in the yeastCandida utilis as influenced by a specific factor inducing cell division

From the yeastCandida utilis a compound was isolated which uncouples the process of cell growth and division by accelerating the cell division without influencing outgrowth of cell mass when applied on another population of identical yeasts. This compound accelerated the initiation of DNA synthesis and had no influence on the synthesis of RNA and protein. Moreover, in the presence of division inducing factor the yeasts started multiplication before the content per cell reached the control level. The stimulating effect of division inducing factor was not obvious when the proteosynthesis of yeast cells was inhibited. We concluded that the division inducing factor regulates the formation of a protein which is synthesized in a very small amount and has a role in the initiation of DNA replication.     

Variations in biochemical parameters of Heterocapsa sp. and Olisthodiscus luteus grown in 12:12 light:dark cycles I. Cell cycle and nucleic acid composition

The division cycle of two phytoplankton species, Olisthodiscus luteus and Heterocapsa sp. was studied in relation to a 12:12 light:dark cycle. Batch cultures in exponential phase were sampled every three hours during 48 hours. Cell number, cellular volume and DNA and RNA concentrations were measured. Microscopic observations of the nuclei of Heterocapsa sp. were also performed. In both species, cell division took place in the dark. In Heterocapsa sp., DNA and RNA showed a similar diel variability pattern, with synthesis starting at the end of the light period, previously to mitosis and cytokinesis. In O. luteus. Major RNA synthesis occurred during darkness, and DNA was produced almost continuously. Both species presented different values and diel rhythmicity on the RNA/DNA ratios.     

Nucleic Acid, Protein, and Starch Synthesis in Developing Cotyledons of Pisum arvense L.

During the cell-division period of cotyledon development inPisum arvense L. cell volume increases slightly but nuclearvolume shows little variation and the DNA content remains atthe 2C to 4C level. During the main period of cell expansionthere is a close correlation between cell volume, nuclear volume,and nuclear DNA content, the nuclei of the largest storage cellsfinally attaining the 64C level. The rate of RNA synthesis increasesseveral days after the increase in DNA has begun and at thesame time accumulation of reserve protein and starch begins.RNA and starch synthesis apparently cease some time before maturationbut protein synthesis continues until the seeds are ripe. Cotyledondevelopment was found to comprise two distinct phases: an initialphase of cell division and differentiation during which DNA,RNA, and protein per unit volume of cell decline; and a phaseof reserve accumulation in which DNA per unit volume of cellremains constant but RNA and protein per unit volume increase,starch synthesis is initiated, and all the cotyledon cells assumethe properties of storage cells.     

ORIGIN,DEVELOPMENT, AND GROWTH OF DIFFERENTIATING TRICHOBLASTS IN ELODEA CANADENSIS

Root hairs of Elodea canadensis develop only from cells which undergo a particular series of developmental steps. These cells, the trichoblasts, are formed as the smaller, proximal product of an asymmetric division, and immediately enter a prolonged phase of synthesis. Histochemical tests show that large amounts of RNA and protein accumulate in the vastly enlarged nucleolus and cytoplasm, while histone increases in the enlarging nucleus. Cytophotometry shows that DNA in the nucleus reaches polyploid levels. Throughout the synthetic phase, almost to the point of root hair initiation at 9.5 mm proximal to the meristem initials, vacuolation is delayed and the trichoblasts elongate less extensively. All results suggest that this synthesis is the type which normally follows cell division, but is greatly enhanced in the trichoblast. In contrast, the initially larger atrichoblasts only accumulate RNA, DNA, and protein in the region from 1 mm to 2 mm proximal to the meristem tip, and they then enter a phase of extensive vacuolation and elongation.     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

Coupling between the initiation of DNA replication and cell division in the blue-green alga Anacystis nidulans

The effect of mitomycin C on cell mass increase, cell division, RNA synthesis and DNA synthesis in the blue-green alga Anacystis nidulans has been examined. Data suggests that the initiation of DNA replication, rather than its termination was the necessary event for cell division to occur.     

Macromolecule synthesis leading to cell division in Tetrahymena pyriformis after replacement of required amino acids

Summary Tetrahymena pyriformis W were brought to a nonmultiplying state by removal of required amino acids from their growth medium. After amino-acid replacement, the incorporation rates of H³-uridine, H³-thymidine and H³-leucine were measured by the autoradiographic method. Following amino-acid replacement, the first response was detected in RNA synthesis, then protein synthesis, then DNA synthesis and, lastly, in cell division. Amino-acid deprived cells showed a 23% net increase in DNA content, a result supporting the view of others that protein synthesis is not necessary for the initiation of DNA synthesis but is necessary for the maintainance of DNA synthesis.     

Studies on the inhibition of bacterial macromolecule synthesis by roquefortine,a mycotoxin from Penicillium roqueforti

Summary The inhibitory effect of roquefortine, a secondary metabolite of Penicillium roqueforti, on bacterial protein, RNA and DNA synthesis was studied. Similar results were obtained in colorimetric measurements and in studying the incorporation of radioactive precursors. They show that RNA synthesis was most significantly affected by roquefortine. Inhibition of protein and DNA synthesis was less pronounced and might be a result of primary inhibition of RNA synthesis.Abbreviations RNA ribonucleic acid - DNA desoxyribonucleic acid - DMSO dimethylsulfoxide - Cpm counts per min - ATCC American Type Culture Collection     

Relations between DNA, RNA and Protein Synthesis, and the Cellular Basis of the Growth Response in Gibberellic acid-Treated Pea Internodes

1. A study was made of the influence of gibberellic acid (GA₂)on nucleic acid, protein, and cell-wall synthesis in pea internodesin vivo. 2. GA₃-treated fifth internodes finally contained more thantwice as much total RNA and protein as comparable untreatedones, and the contents of RNA and protein were closely relatedto the length of internode cortical cells. 3. Cell elongation, RNA, protein, and cell-wall synthesis werestimulated 24–48 h before there was any demonstrable GA₃effect on DNA synthesis and cell division. 4. Treated fifth internodes finally contained twice as manycortical cells as control internodes, a response that was matchedby a proportionate increase in the amount of DNA. 5. Internodes treated with actinomycin D or cycloheximide failedto elongate in response to GA₃ treatment, indicating that bothRNA and protein synthesis are essential for gibberellin-stimulatedcell elongation to occur in this tissue. 6. 5-fluorodeoxyuridine at concentrations which completely blockcell division did not prevent cells from elongating in the presenceof GA₃. 7. With the possible exception of pectic substances there wasno change in the relative proportions of each of the major cell-wallconstituents in treated, as compared to control internodes.     

RELATIONSHIP BETWEEN RNA SYNTHESIS, CELL DIVISION, AND MORPHOLOGY OF MAMMALIAN CELLS : I. Puromycin Aminonucleoside As An Inhibitor of RNA Synthesis and Division in HeLa Cells

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P³² and H³-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.     

Synchronous growth and synthesis of macromolecules in a naturally wall-less volvocale,Dunaliella bioculata

Summary Dunaliella bioculata, a naturally wall-less unicellular green alga, can be induced to divide synchronously when subjected to a 12 hours light-12 hours dark cycle. This rhythmic cell division will last for at least 15 days under a subsequent constant illumination. Synchronization can be improved when cells are submitted to 8 hours light-16 hours dark cycles under bright white light (10,000 lux). In these conditions the cell division gives rise to two daughter cells: The chronology of DNA, RNA and proteins synthesis has been studied during such a synchronized cell cycle. DNA synthesis begins 4 hours before the outset of cell division and is completed after two hours in the dark; in difference, illumination seems necessary to the synthesis of RNA and proteins.