Effects of bisulfite (sulfur dioxide) on DNA synthesis and fidelity by DNA polymerase I

In an attempt to explain the mechanism of comutagenesis by bisulfite, the extent and accuracy of DNA synthesis by E. coli DNA polymerase I was examined in the presence of sodium bisulfite. Bisulfite concentration of 100 mM caused nearly complete inhibition of dNTP incorporation into activated calf thymus DNA. Other salts (NaCL, Na2SO4) at the same concentration had no effect on enzyme activity. Preincubation of the various DNA synthesis assay components in 100 mM bisulfite showed that only preincubation of DNA polymerase I caused inhibition of DNA synthesis. Exonuclease functions of DNA polymerase I were unaffected by up to 100 mM bisulfite. Accuracy of DNA synthesis in the presence of bisulfite was determined using poly (dA-dT) as a template-primer. Concentrations of bisulfite greater than 50 mM caused a progressive decrease in enzyme accuracy. At 100 mM bisulfite there was an approximate 7.5-fold decrease in the fidelity of DNA synthesis, compared to control values, as measured by the ratio of noncomplementary (dGTP) to complementary (dTTP) nucleotide incorporated. Based on the known chemistry of bisulfite, it is hypothesized that sulfitolysis of the one disulfide group in DNA polymerase I by bisulfite might be responsible for the reduced polymerase activity and accuracy. The exonuclease functions of DNA polymerase I do not seem to require the disulfide linkage. These results suggest that the effects of bisulfite on mutation frequency might be mediated by effects on the fidelity of DNA repair systems.     

Effects of Hydroxyurea on Crithidia fasciculata

SYNOPSIS Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45–50 h: if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Effects of hydroxyurea on Crithidia fasciculata.

Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45-50 h; if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Effect of inhibitors on the viability and protein and nucleic acid synthesis of Escherichia coli

The object of this work was to study how the synthesis of protein, RNA and DNA in Escherichia coli M17 and its viability were influenced by chloramphenicol (50 and 300 micrograms/ml) an inhibitor of protein biosynthesis, and sodium azide (200 and 2000 microM) and aminazine (50 micrograms/ml), inhibitors of respiration. The exposed were inhibitors with the bacteria for 60 min at room temperature and for 1-4 months at -10 degrees C. The inhibition of the E. coli viability by chloramphenicol was shown to be reversible. The respiration inhibitors stabilized its viability upon storage at -10 degrees C for one month. The inhibitors were found to produce a different effect on the synthesis of RNA and protein in E. coli. The rates of DNA synthesis hardly changed. No correlation was established between changes in the synthesis of protein and nucleic acids by E. coli after the action of the inhibitors and its viability.     

Studies on the inhibition of protein synthesis by selenodiglutathione.

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.     

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. General effects on ribonucleic acid and protein synthesis

In trimethoprim-inhibited RC(str) strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RC(rel) strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RC(str) strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RC(rel) strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RC(str) strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RC(str) or RC(rel) bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.     

Effect of the Folic Acid Analogue, Trimethoprim, on Growth, Macromolecular Synthesis, and Incorporation of Exogenous Thymine in Escherichia coli

The effect of trimethoprim [2,4-diamino-5(2',4',5'trimethoxybenzyl)-pyrimidine] in the presence of thymine on Escherichia coli B temperature-sensitive and non-temperature-sensitive Thy(') strains and a phosphodeoxyribomutase-negative mutant was studied. The inhibitory effect of 5 mug of trimethoprim per ml on the growth of E. coli B was not overcome by thymine, thymidine, or thymidylate even in the presence of one-carbon metabolites and related metabolites. Deoxyribonucleic acid (DNA) and protein synthesis were more severely inhibited than ribonucleic acid (RNA) synthesis. The inhibition of DNA synthesis was partially reversed by addition of deoxyadenosine to increase the incorporation of exogenous thymine. By contrast, the inhibition of protein was not reversed even with one-carbon metabolites present, in keeping with the requirement for formylmethionyl-transfer RNA(F) for initiation. However, the inhibition of both DNA and protein synthesis in a phosphodeoxyribomutase-negative strain by 1 mug of trimethoprim per ml with thymine present was partially reversed by deoxyadenosine and one-carbon metabolites, and nearly normal growth occurred. 5-Fluorodeoxyuridine added at the time of addition of trimethoprim prevented the inhibition. Sulfadiazine in the presence of thymine inhibited both Thy(+) and Thy(-) strains whereas trimethoprim (with thymine) did not inhibit Thy(-) organisms. The effect of trimethoprim on the incorporation of labeled thymine into DNA was also studied. These experiments support the concept that trimethoprim in conjunction with the action of thymidylate synthetase inhibits the growth of Thy(+) cells because of a depletion of tetrahydrofolate. DNA synthesis is inhibited initially by a limitation of thymine nucleotide precursor, resulting from the indirect inhibition of thymidylate synthetase and the poor incorporation of exogenous thymine.     

秦皮素对大肠埃希菌作用机制的初步研究

目的以大肠埃希菌ATCC 25922为供试菌,探讨秦皮素的抑菌活性及其作用机制。方法利用TTC法测定秦皮素对大肠埃希菌ATCC 25922的最低抑菌浓度;通过测定加药前后菌体培养液电导率和大分子的变化及观察扫描电镜和透射电镜电镜结果,分析秦皮素对其细胞膜的影响;通过SDS-PAGE测定秦皮素对供试菌株蛋白含量的影响;采用逐个检出法研究秦皮素对大肠埃希菌ATCC 25922质粒合成的抑制作用。结果秦皮素可抑制大肠埃希菌ATCC 25922的生长,其最低抑菌浓度为40μg/mL。秦皮素作用菌体5 h后,培养液中的电导率比对照组增加1.96%,但DNA和RNA大分子增加的不明显。秦皮素作用大肠埃希菌20 h后,菌体可溶性蛋白总量比对照组降低42%。秦皮素对大肠埃希菌的质粒有消除作用,药物作用48 h后,秦皮素对大肠埃希菌的质粒消除率为60.3%。结论秦皮素可抑制大肠埃希菌的生长,其抑菌作用机制与抑制菌体内蛋白质合成和消除菌体内的质粒有关,但对大肠埃希菌细胞膜的影响不大。     

Mechanism of inhibition of deoxyribonucleic acid synthesis in Escherichia coli by hydroxyurea

The effects of hydroxyurea on Escherichia coli B/5 physiology (increases in cell mass, number of viable cells, and deoxyribonucleic acid [DNA], RNA, and protein concentrations) were studied in an attempt to find a concentration that completely inhibits DNA synthesis and increase in number of viable cells but has little or no effect on other metabolic processes. These conditions were the most closely approached at an hydroxyurea concentration of 0.026 to 0.033 m. A concentration of 0.026 or 0.033 m was used in subsequent experiments to study the site(s) of inhibition of DNA synthesis in E. coli B/5 by hydroxyurea. Hydroxyurea at a concentration of 10(-2)m was found to inhibit ribonucleoside diphosphate reductase activity completely in crude extracts of E. coli. The synthesis of deoxyribonucleotides was greatly reduced when E. coli cells were grown in the presence of 0.033 m hydroxyurea. Studies on the acid-soluble DNA precursor pools showed that hydroxyurea causes a decrease in the concentration of deoxyribonucleoside diphosphates and deoxyribonucleoside triphosphates and an increase in the total concentration of ribonucleotides. Sucrose density gradient sedimentation of DNA from cells treated with 0.026 m hydroxyurea for 30 min indicated that at this concentration hydroxyurea induces no detectable single- or double-strand breaks. In addition, both replicative and repair syntheses of DNA were found to occur normally in toluene-treated cells in the presence of relatively high concentrations of hydroxyurea. Pulse-chase studies showed that deoxyribonucleotides synthesized prior to the addition of hydroxyurea to cells are utilized normally for DNA synthesis in the presence of hydroxyurea. On the basis of these observations, we have concluded that the primary, if not the only, site of inhibition of DNA synthesis in E. coli B/5 by low concentrations of hydroxyurea is the inhibition of the enzyme ribonucleoside diphosphate reductase.     

The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p-hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.     

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola.

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichia coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNA(FMet) (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA(FMet) showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4+ (as glutamate). Higher salt concentrations, especially those with Cl- as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl- ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl- ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4Cl, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl- ions inhibited the attachment of initiation factors to tRNA(FMet) but had little effect on binding of initiation factors to R17 RNA.     

The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p -hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.     

Spermidine acetyltransferase is required to prevent spermidine toxicity at low temperatures in Escherichia coli

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In Escherichia coli, the speG gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of E. coli at low temperature was examined using a speG mutant. At 37 degrees C, growth of the speG mutant was normal in the presence of 0. 5 or 1 mM spermidine. However, following a shift to 7 degrees C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7 degrees C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg(2+) restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg(2+), resulting in ribosome inactivation at low temperatures.     

Effect of acetylpolyamines on in vitro protein synthesis and on the growth of a polyamine-requiring mutant of Escherichia coli

The functions of acetylpolyamines were examined with respect to stimulation of protein synthesis and cell growth. Unlike polyamines, acetylpolyamines could not lower the optimal Mg2+ concentration of protein synthesis, and the degree of stimulation of protein synthesis by acetylpolyamines was small. The addition of N1-acetylspermine did not stimulate cell growth of a polyamine-requiring mutant of Escherichia coli MA261, although acetylspermine was accumulated in the cells. Acetylspermine did not interfere with polyamine stimulation of protein synthesis and cell growth of E. coli MA261. The binding of acetylpolyamines to RNA was very weak, and the binding of polyamines to RNA was not disturbed significantly by the presence of acetylpolyamines. When the growth of E. coli MA261 was stimulated by addition of polyamines, significant amounts of acetylpolyamines were also formed in the cells. These results suggest that acetylation of polyamines, together with polyamine excretion, may regulate the intracellular level of the parent polyamines when excess amounts of polyamines accumulate intracellularly.     

Inhibition of Protein Synthesis and Amino Acid Transport by Crystal Violet in Trypanosoma cruzi

ABSTRACT. [³⁵S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [³⁵S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [³H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.     

Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibits peptidoglycan synthesis in Escherichia coli

Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibited peptidoglycan synthesis in Escherichia coli in a concentration-dependent manner. The inhibition of peptidoglycan synthesis appears to be a cause rather than a consequence of growth inhibition as it was observed soon after the addition of the antibiotic even in E. coli cells whose growth was totally inhibited by chloramphenicol.     

Control of RNA biosynthesis in rat liver. Some features of RNA biosynthesis during prolonged protein synthesis inhibition]

A drastic inhibition of protein biosynthesis in rat liver in vivo by cycloheximide (CHI) (0.3 mg/100 g of body weight) first caused an increase of RNA synthesis (after 1 hour), which was then followed by its decrease. Partial gradual restoration of the protein synthesis level was shown to be accompanied by a repeated increase of RNA synthesis (12 hs) and its normalisation after 24 hs. The first maximum of RNA synthesis increase in the isolated nuclei system was AU-type RNA synthesis (sensitive to alpha-amanitine), the second one was due to GC-type RNA synthesis (resistant to this toxin). Purified chromatine template activity in the system with E. coli RNA polymerase (by 14%) an hour after CHI treatment, but 3 hrs later was decreased and subsequently restored (12 hrs after CHI injection). The changes of RNA biosynthesis induced by prolonged protein synthesis inhibition suggest the existence of continuous RNA synthesis control in nuclei. This control is realized by translation system using the feed back principle.     

Effect of Bacteriophage Ghost Infection on Protein Synthesis in Escherichia coli

The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.