DNA synthesis inEscherichia coli B/r after UV-irradiation

When studying the kinetics of DNA synthesis, growth and cell division inEscherichia coli B/r after irradiation with different doses of UV-radiation (254 nm) we could demonstrate, by means of pulse incorporation of³H-thymidine, a lag in DNA synthesis after the irradiation. The relative rate of the restored DNA synthesis (related to the number of viable cells) was higher than in the non-irradiated culture. After 3 h the rate of DNA synthesis settled at a constant value, which was identical with the control rate up to the “critical dose” of 20 J/m². The irradiated cell population is heterogenous and contains basically two categories of cells — surviving and non-surviving. Cells of both types contribute to DNA synthesis restored after the lag period to a different extent. During the first hour after the irradiation even the nonviable portion of the population,i.e. cells that do not form colonies but are still penicillin-sensitive, is involved in the DNA synthesis.     

The effect of Waring Blendor treatment on transformation in Bacillus subtilis.

Treatment of competent Bacillus subtilis in a Waring Blendor for 10 s increases transformability of the culture about twofold while reducing the attachment of DNA to competent cells by 80%. The effectiveness of attached DNA in producing transformants is increased 10-fold by this treatment. The uptake of transforming DNA into a DNase-resistant state is progressively reduced by 70% during a 120-s blending treatment. Blending for 30-45 s diminishes transformability to about 10% of the original nonblended value without affecting the viable cell titer. No effect is produced by 30 s of blending on transformability if the irreversible uptake of DNA has been completed. Thus, the inhibition occurs at an early step in the transformation sequence. Treatment of the competent culture for 60 s or longer in the Waring Blendor reduces both the number of transformants obtained and the total number of viable cells.     

Damaging agitation intensities increase DNA synthesis rate and alter cell-cycle phase distributions of CHO cells

The effects of fluid-mechanical force (agitation) on the cell cycle kinetics of Chinese hamster ovary (CHO) cells cultured in suspension in 2-L bioreactors has been examined. A two-color flow cytometry method was used to determine the fraction rate of DNA synthesis. With increased agitation intensity, cell viability decreased as a result of increased cell death. However, increased agitation induced the viable cells of the culture to a higher proliferative state relative to a control culture. The fraction of viable cells of the high-agitation culture (250 rpm) in S phase was higher (up to 45%) and in G1 phase was lower (up to 50%) compared with the viable cells of the control culture (80 rpm). The DNA synthesis rate per viable S-phase cell of the high-agitation culture was confirmed by recovery experiments, which were conducted to measure the apparent specific growth rate and the cell cycle kinetics of the high-agitation culture upon reduction in the agitation rate from 250 rpm back to 80 rpm. The apparent specific growth rate of the test culture, calculated for the first 12 h of the recovery period, was greater than the apparent specific growth rate of the control culture. Furthermore, the proliferative state of the viable cells of the test culture, which had become higher relative to the control culture during the high agitation period, gradually approached the level of the control culture during recovery. Results also show that the magnitude of the agitation intensity; the culture agitated at 250 rpm attained a greater proliferative state than a parallel culture agitated at 235 rpm. The 250-rpm culture had a higher fraction of S-phase and a lower fraction of G1-phase cells than the 235-rpm culture. The DNA sunthesis rate per viable S-phase cell of the 250-rpm culture was greater than of the 235-rpm culture. (c) 1992 John Wiley & Sons, Inc.     

Macromolecular synthesis and cell division during morphogenesis of Arthrobacter crystallopoietes

The sphere-rod-sphere morphology cycle of Arthrobacter crystallopoietes was accompanied by changes in the rate of growth and the rates of DNA, RNA and protein synthesis. The patterns of macromolecule synthesis resembled those found in other bacteria during a step-up followed by a step-down in growth rate. During the step-up in growth spherical cells grew into rods and macromolecules were synthesized in the absence of cell division. During stepdown, successive rounds of septation produced progressively smaller cells which did not separate and remained in chains. The morphology of the cells was dependent on the growth rate and could be altered by changing the dilution rate in a malate-limited chemostat. Gradual transitions in morphology and gradual increases in macromolecule content of the cells occurred as the growth rate was increased in the chemostat. Sphere to rod morphogenesis occurred when DNA synthesis was inhibited by treatment with mitomycin C or by thymine starvation. The DNA-deficient rods did not divide and eventually lysed. DNA, RNA and protein synthesis were continuously required for the reductive division of rods to spheres.Abbreviations MS mineral salts - GS mineral salts plus glucose - CA casamino acids - GSCA mineral salts plus glucose plus casamino acids - cAMP cyclic adenosine-3,5-monophosphate - RNA ribonucleic acid - DNA deoxyribonucleic acid     

Polypeptide Synthesis by Extracts from Escherichia coli Treated with T2 Ghosts

Infection of Escherichia coli B in inorganic salts-glycerol with a multiplicity of deoxyribonucleic acid-less T2 "ghosts" just sufficient to block all protein synthesis results in both viable and killed bacteria. We enriched for the viable cells by a combination of lysozyme treatment and filtration and measured the in vitro capacity of their extracts to synthesize polypeptides. Without added template ribonucleic acid (RNA), such "ghost extracts" incorporate amino acids (endogenous synthesis) at approximately one-half the rate as do extracts from uninfected bacteria. However, they are unable to use added synthetic or natural template RNAs for peptide synthesis. Some activity can be observed but only at high concentrations of Mg(2+). These results suggest that ghost infection may result in a blockage of ribosomes during translation. Mixing experiments show that the incapacity of ghost extracts to translate added template RNA is due to a defect in the ribosomes.     

Inhibition of Host Protein Synthesis During Infection of Escherichi coli by Bacteriophage T4: III. Inhibition by Ghosts

Deoxyribonucleic acid (DNA)-less T2 "ghosts" were prepared by osmotic shock and purified by KBr density gradient centrifugation. Escherichia coli B was treated with these ghosts in inorganic salts-glycerol medium to see which features of phage infection could be elicited by ghosts. At a multiplicity that was just sufficient to block induction of beta-galactosidase (EC 3.2.1.23), 89% of the bacteria were killed and the rates of ribonucleic acid (RNA) and DNA synthesis were about 10 to 15% of normal. However, protein synthesis was almost completely blocked but resumed after 30 min. During this period, it was possible to induce messenger RNA (mRNA) from the lactose operon, although this mRNA could not be translated into active beta-galactosidase. These results suggest to us that the viable cells surviving ghost infection synthesize nucleic acids at close to a normal rate but are temporarily blocked in protein synthesis. The continued formation of untranslated host mRNA mimics the pattern of bacterial synthesis just after whole-phage infection, and is consistent with the interpretation that the immediate block in the initiation of host translation by these viruses is due to their attachment.     

Magnesium Starvation of Aerobacter aerogenes II. Rates of Nucleic Acid Synthesis and Methods for Their Measurement

The rates of synthesis of Aerobacter aerogenes nucleic acids were estimated during incubation of the bacteria in a Mg(++)-free medium. Deoxyribonucleic acid (DNA) synthesized during Mg(++) starvation, or in the preceding exponential growth, remained acid-precipitable for 2.5 hr before breaking down to acid-soluble products during a period of many hours. Rates of DNA synthesis were calculated by correcting the net amounts of DNA per milliliter to values that would have appeared had there been no decay. After the first few hours, this rate was constant, the amount of DNA present at the start of Mg(++) starvation being synthesized every 130 min. Rates of synthesis of total ribonucleic acid (RNA) were established in two ways: (i) by measurements of the incorporation of exogeneous uracil and glucose carbon into RNA, and (ii) by the accumulation of transfer RNA (tRNA), since this component is stable during Mg(++) starvation. After the first few hours, this rate was constant, the amount of RNA present at the start of Mg(++) starvation being synthesized about every 120 min. Fractionation by gradient centrifugation revealed that at all times of starvation the ratio of newly synthesized tRNA-rRNA was the same as it was during exponential growth. Furthermore, newly synthesized ribosomal RNA (rRNA) became a part of polysomal structures. Thus, in the absence of Mg(++), DNA, tRNA, and rRNA were synthesized in the same relative proportions as during exponential growth, at rates close to one-half the instantaneous rates of synthesis in the bacteria growing exponentially at the start of starvation.     

Filamentous bacterial viruses : I. DNA synthesis during the early stages of infection with fd

During the first ten minutes after infection of bacteria with fd, the rate of DNA synthesis in an infected culture becomes several-fold larger than the rate in a parallel uninfected culture. This stimulation of rate is due to the synthesis of 100 to 200 double-stranded forms of viral DNA, superimposed on continuing bacterial DNA synthesis. At the end of the ten-minute period, the rate of viral plus bacterial DNA synthesis stops increasing, and remains constant for the next 50 minutes. The abrupt decrease in acceleration of net DNA synthesis corresponds in time to the onset of synthesis of single-stranded viral DNA.     

Water flow through frog gastric mucosa

To elucidate the role of protein synthesis in DNA formation, E. coli R2 infected with phage T2 was studed as a model, employing chloramphenicol to inhibit protein synthesis. The following results were obtained. 1. Chloramphenicol inhibited protein synthesis but not synthesis of nucleic acids in uninfected bacteria. 2. Studies of the effect of chloramphenicol on phage maturation indicated a delay of 2 minutes between time of addition and cessation of phage growth. 3. The increase of DNA in phage-infected bacteria was completely suppressed by the addition of chloramphenicol within 2 minutes following infection. Addition at later times showed progressively less inhibitory action depending upon the time interval, and addition after the 10th or 12th minute showed no appreciable effect on DNA synthesis despite the cessation of intracellular phage formation and protein synthesis. 4. When chloramphenicol was added to infected cells the increase of resistance to UV stopped within 2 minutes, whether or not DNA synthesis continued. Thus evolution of resistance paralleled the rate of DNA synthesis achieved, but not the amount of DNA accumulated. 5. We conclude that in infected bacteria, protein synthesis is necessary to initiate DNA synthesis but is not essential for its continuation. The resistance to UV that characterizes infected cells near the midpoint of the latent period is not due to accumulation of DNA, but depends on some chloramphenicol-sensitive process (probably protein synthesis) completed at about the time the rate of DNA synthesis becomes maximal.     

Dissociation of histone and DNA synthesis in x-irradiated HeLa cells

Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1 000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis continued with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal.     

The effect of chloramphenicol on deoxyribonucleic acid synthesis and the development of resistance to ultraviolet irradiation in E. coli infected with bacteriophage T2

To elucidate the role of protein synthesis in DNA formation, E. coli R2 infected with phage T2 was studied as a model, employing chloramphenicol to inhibit protein synthesis. The following results were obtained. 1. Chloramphenicol inhibited protein synthesis but not synthesis of nucleic acids in uninfected bacteria. 2. Studies of the effect of chloramphenicol on phage maturation indicated a delay of 2 minutes between time of addition and cessation of phage growth. 3. The increase of DNA in phage-infected bacteria was completely suppressed by the addition of chloramphenicol within 2 minutes following infection. Addition at later times showed progressively less inhibitory action depending upon the time interval, and addition after the 10th or 12th minute showed no appreciable effect on DNA synthesis despite the cessation of intracellular phage formation and protein synthesis. 4. When chloramphenicol was added to infected cells the increase of resistance to UV stopped within 2 minutes, whether or not DNA synthesis continued. Thus evolution of resistance paralleled the rate of DNA synthesis achieved, but not the amount of DNA accumulated. 5. We conclude that in infected bacteria, protein synthesis is necessary to initiate DNA synthesis but is not essential for its continuation. The resistance to UV that characterizes infected cells near the midpoint of the latent period is not due to accumulation of DNA, but depends on some chloramphenicol-sensitive process (probably protein synthesis) completed at about the time the rate of DNA synthesis becomes maximal.     

Introduction of deoxyribonucleoside triphosphates into intact cells by electroporation

A simple method, employing high-voltage electric discharge (electroporation), was developed to introduce phosphorylated nucleosides into the cytoplasm of viable cells. HL-60 leukemia cells permeabilized by this technique remained viable and incorporated deoxyribonucleoside triphosphates into nuclear DNA. Furthermore, DNA synthesis was depressed for at least 24 h in HL-60 cells made permeable to 1-beta-D-arabinosylcytosine 5'-triphosphate by this methodology. Electroporation was found to be applicable to the permeabilization of a wide variety of cell lines in culture to nucleotides, suggesting that this methodology may be useful for the introduction into intact cells of a wide variety of molecules that are not normally transported effectively.     

Effects of the ccd function of the F plasmid on bacterial growth.

The ccd segment of the mini F plasmid containing the ccdA and ccdB genes controls the coordination between plasmid proliferation and cell physiology and fate. When the DNA replication of a thermosensitive-replication plasmid carrying the ccd segment of mini F is blocked, plasmid DNA molecules are progressively diluted through cell division until the copy number reaches 1 per cell. From this time on, there is little increase in the number of viable cells, although cells continue to divide, resulting in a mixed population of viable cells (mostly plasmid containing), nonviable but residually dividing cells, and nonviable nondividing cells. Results are presented suggesting that plasmid-containing cells are viable and continue to divide, whereas plasmid-free segregants are nonviable and form filaments after a few residual divisions, with DNA synthesis reduced or arrested in the filaments. Although the ccd functions are known to induce the SOS response when plasmid replication is blocked, the production of nonviable plasmid-free segregants is independent of the SOS cell division inhibition mechanism determined by the sfiA and sfiC genes.     

Hormonal regulation of growth and life span of bullfrog tadpole tail epidermal cells cultured in vitro

Epidermal cells were dissociated from tails of the bullfrog tadpole, Rana catesbeiana, and cultured to investigate their response to steroid and thyroid hormones. Charcoal-treated serum (CTS) was used in the growth medium when cells were to be grown in the absence of steroid and thyroid hormones. The cells could be maintained for 2 weeks with a small increase in cell number in medium that contained CTS (CTS medium). Addition of cortisol to CTS medium increased both cellular attachment to the culture dishes and the proliferation of the attached cells with an optimum concentration of 5 X 10(-7) M. The cells remained viable and attached for at least a week. Cortisol stimulated the rate of protein synthesis 1.8-fold but did not alter the rate of DNA synthesis. The cells did not proliferate in the medium containing triiodothyronine (T3) and detached themselves from the dish within 5 days, which occurred in a dose-dependent manner with a maximum effect at 10(-8) M. It drastically decreased the rate of DNA synthesis but did not influence the rate of protein synthesis. These responses of cells to cortisol and T3 may reflect growth and death of tail epidermal cells in vivo at metamorphosis.     

Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells

Erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells)-are erythropoietin (EP)-sensitive cells at the late colony forming unit-erythroid (CFU-E) and cluster forming unit stages of differentiation (Koury et al., J. Cell. Physiol. 121:526-532, 1984). We investigate here the EP requirements of FVA cells in vitro for viability, proliferation, and maturation. By delaying the addition of EP to FVA cell cultures or by withdrawing EP at early times of culture, the subsequent viability, cell numbers, and maturation were diminished. The longer the delay in EP addition or the earlier the EP withdrawal, the more diminished these parameters were when compared to cultures which contained EP throughout the 48 h of differentiation. FVA cells had a period of EP requirement in vitro that lasted for only 24 h or less after the initiation of culture. During these crucial first 24 h, EP induced an increase in the synthesis of all size classes of RNA. Protein synthesis was maintained at a stable level in cells cultured with EP, but it declined in cells cultured without it. In contrast, the synthesis rate of DNA and the content of DNA per cell were not affected by the presence of EP in the culture. However, FVA cells cultured without EP had progressive accumulation of small sized DNA due to breakage of higher molecular weight DNA. The rate of DNA breakdown was sufficient to prevent DNA accumulation and thus it probably plays a role in the abortion of cell proliferation. No such breakage was found in cells cultured with EP. Our results indicate that EP exerts an effect on FVA cells in culture which is reflected in their viability, cell number, and maturation. This effect is not mediated by a stimulation of the rate of DNA synthesis, but is accompanied by stimulation of overall RNA synthesis and maintenance of protein synthesis.     

Biochemical changes in lysogenic Bacillus stearothermophilus after bacteriophage induction

Welker, N. E. (University of Illinois, Urbana), and L. Leon Campbell. Biochemical changes in lysogenic Bacillus stearothermophilus after bacteriophage induction. J. Bacteriol. 90:1129-1137. 1965.-Cultures of Bacillus stearothermophilus 1503-4R (TP-1) continued to grow at an unaltered rate after induction with mitomycin C (MC). MC-induced cultures exhibited a 2.5-fold increase in cell number before lysis occurred. Prior to lysis, cells were observed to elongate and to contain areas of lesser density. Protein synthesis was slightly inhibited in MC- or ultraviolet light (UV)-induced cultures for a period of 5 to 10 min, and then proceeded at a rate identical to that in the noninduced culture. Ribonucleic acid (RNA) synthesis was not affected by MC induction. UV induction caused RNA synthesis to occur in two stages: in the first stage, the rate of RNA synthesis was one-third that observed in the noninduced culture and lasted for a period of 15 min; the second stage of RNA synthesis then proceeded at a rate identical to that in the noninduced culture. The synthesis of deoxyribonucleic acid (DNA) in an MC- or UV-induced culture occurred in two stages. In the first stage, DNA synthesis in induced cultures occurred at a rate of one-half (MC) and one-third (UV) of that observed in the noninduced culture. The first stage of DNA synthesis in MC- or UV-induced cultures lasted for 25 to 30 min and 15 to 20 min, respectively. In the second stage, the rate of DNA synthesis in MC- or UV-induced cultures occurred at a rate three times that of the noninduced culture. UV induction appeared to have a greater inhibitory effect than MC induction on protein, RNA, and DNA synthesis as well as phage yield. The differential rate (K) of inducible and constitutive alpha-amylase synthesis was inhibited by 75 and 100%, respectively, for a period of 20 min after MC induction. After 20 min, the K values for alpha-amylase synthesis were identical to those obtained in the absence of MC induction. The synthesis of TP-1 phage DNA occurred rapidly and was complete 25 min after MC induction, whereas bacterial DNA was degraded or its rate of synthesis was decreased. During the second stage of DNA synthesis, only bacterial DNA was synthesized, but at a rate greater than that found in the noninduced culture.     

DNA replication in asynchronous and synchronous ehrlich ascites cells in different conditions of growth

Ehrlich ascites tumour cells were labelled for DNA fibre autoradiography within the peritoneal cavity of a tumour-bearing mouse. The generation and the evaluation of the autoradiographic patterns is described and discussed. To study possible changes of the autoradiographic patterns during a natural S phase the labelling was performed in the mouse or in culture with asynchronous cells which were afterwards separated into synchronous subpopulations by zonal centrifugation. The subpopulations obtained were characterized by flow cytofluorometry in connection with the thymidine labelling index. We compared the DNA fibre autoradiographic patterns of several synchronous and asynchronous cell populations growing in the mouse or under different conditions in culture: The replicon size distributions of all populations examined were virtually the same. The fork movement rate was found to depend mainly on the metabolic condition of the cells. In culture it was significantly slower than in the mouse although a shortened S phase and therewith an increased DNA synthesis rate occurred. During a natural S phase it increased slightly, at most, while the DNA synthesis rate was considerably enhanced at the end of S. The changes in the rate of total DNA synthesis cannot account for the changes in the rate of chain growth. We conclude that the DNA synthesis rate is regulated almost exclusively by changing the replicon initiation frequency, while the fork movement rate is limited by the actual metabolic condition of the cells.     

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.