Biosynthesis of eideine:: II. Localization of edeine synthetase within Bacillus brevis Vm4

Edeine-synthesizing polyenzymes, associated with a complex of cytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from cells intensively synthesizing edeines (18–20 h culture) contained edeine B. Edeine B was found to be bound covalently to the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1–0.3 μmol/mg protein, depending on the age of cells.Detachment of edeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH₄)₂SO₄ at 45–55% saturation or by DEAE-cellulose colum fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to protein fraction of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16–22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine b of specific activity: 80 units/μmol was released.The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associatd with this complex did not effect the DNA-synthesizing activity.     

Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.     

Characterization of versilin-sensitive sites in self-sensitive producer and sensitive non-producer or unrelated organism

Studies on self-sensitivity of producer mutant vs. sensitivity of non-producer parent and unrelated organism showed that versilin inhibited spore germination and sporulation in the self-sensitive producer mutant, non-producer parent Aspergillus versicolor N₅ and the unrelated sensitive Trichophyton rubrum . Sporulation appeared to be more sensitive than spore germination. The inhibition of in vivo synthesis of protein was very marked, but inhibition of RNA and DNA was slight and moderate, respectively. Thus versilin was not specific in its action, but the principal sensitive site was protein synthesis, as further suggested by inhibition of polyU-directed in vitro synthesis of polyphenylalanine. The activation of leucine was unaffected, but the formation of leucyl-tRNA was severely inhibited in all three strains. The differences in sensitivities between the strains were the same, whether as whole cells or as cell-free extracts. Thus the nature of the sensitive site appeared to be identical in the self-sensitive producer and sensitive non-producer or unrelated organism.     

Characterization of versilin-sensitive sites in self-sensitive producer and sensitive non-producer or unrelated organism

Studies on self-sensitivity of producer mutant vs. sensitivity of non-producer parent and unrelated organism showed that versilin inhibited spore germination and sporulation in the self-sensitive producer mutant, non-producer parent Aspergillus versicolor N5 and the unrelated sensitive Trichophyton rubrum. Sporulation appeared to be more sensitive than spore germination. The inhibition of in vivo synthesis of protein was very marked, but inhibition of RNA and DNA was slight and moderate, respectively. Thus versilin was not specific in its action, but the principal sensitive site was protein synthesis, as further suggested by inhibition of polyU-directed in vitro synthesis of polyphenylalanine. The activation of leucine was unaffected, but the formation of leucyl-tRNA was severely inhibited in all three strains. The differences in sensitivities between the strains were the same, whether as whole cells or as cell-free extracts. Thus the nature of the sensitive site appeared to be identical in the self-sensitive producer and sensitive non-producer or unrelated organism.     

The uptake and subcellular localization of the peptide antibiotic edeine a in HeLa cells in suspension culture

The uptake of [¹⁴C]thymidine, [¹⁴C]uridine and [¹⁴C]leucine by HeLa cells incubated in the presence of 1.52 μg/ml edeine A is inhibited by 7.5, 0 and 4%, respectively. Though edeine A has no gross cytopathic effect on HeLa cells, the peptide antibiotic enters the cells and h after addition to cell cultures is found in the nuclei. After 6 h of incubation, the highest intracellular concentration of edeine is located in the nuclear fraction, but, after 12 h, a higher proportion is in the post-mitochondrial supernatant fraction where it is associated with protein components in the range of molecular weights of 20 000 and 9 500 D. In the nucleus most of the [¹⁴C]edeine is bound to the chromatin fraction after 2 h of incubation. Exhaustive deoxyribonuclease digestion of the chromatin fraction releases all the radioactivity into one ultraviolet absorbing peak, which sediments to a density of 20% sucrose. Exhaustive ribonuclease digestion of the chromatin fraction releases all the radioactivity into two ultraviolet absorbing peaks which sediment to a density of 20 and 40%, respectively; subsequent proteolytic digestion of the RNAse-treated chromatin fraction frees about 70% of the edeine A from the ultraviolet absorbing peaks. This suggests that intranuclear edeine A associates with proteins in the chromatin. The radioactivity was recovered from the enzymatically digested chromatin fractions and characterized as biologically active edeine A.     

Treatment of Chinese hamster ovary cells with the transcriptional inhibitor actinomycin D inhibits binding of messenger RNA to ribosomes

Inhibitors of RNA synthesis such as actinomycin D, MPB, and cordycepin progressively inhibit the initiation of protein synthesis in intact, nucleated mammalian cells. This inhibition is not dependent on the levels of mRNA, ribosomes, or tRNA. Lysates prepared from CHO cells treated with actinomycin D do not incorporate labeled globin mRNA or ovalbumin mRNA into 80S initiation complexes at the rates of untreated control extract. The ability of the extracts to produce and accumulate 48S preinitiation complexes was assessed using the 60S subunit joining inhibitors edeine and 5'-guanylyl imidodiphosphate. Control extracts were able to accumulate both the 48S preinitiation complexes and the migration-related intermediates in the presence of both inhibitors. However, lysates derived from CHO cells treated with actinomycin D were unable to produce these complexes. This was also true at low temperature, a condition that does not inhibit mRNA binding but prevents migration of the 43S complex along the mRNA. Mixing experiments with extracts from untreated control or AMD-treated CHO cells provided no evidence for a translational inhibitor. Thus, our data are consistent with the hypothesis that treatment of whole cells with actinomycin D inhibits protein synthesis initiation at the level of mRNA binding and not at migration or 60S subunit joining.     

Edeine inhibition and resistance in Neurospora

To obtain data on the biochemical effects of edeine in the fungus Neurospora crassa, in vivo protein synthesis, in vitro protein synthesis, as well as in vivo RNA and DNA synthesis of the wildtype and an edeine resistant mutant were measured.—Incorporation of ³H leucine into conidia of both strains, which served as a measure for in vivo protein synthesis, was inhibited by 200 g edeine/ml as follows: Wildtype approx. 40%, mutant approx. 6%.—Incorporation of ¹⁴C phenylalanine into polyphenylalanine in a cell free system with ribosomes from either the wildtype or the mutant, was inhibited between 74 and 95% by edeine at a ratio of 2 molecules edeine per ribosome.—Incorporation of ³H adenosine into conidia, serving as a measure for in vivo RNA synthesis, was inhibited in the wild-type (approx. 30% inhibition by 200 g edeine/ml). It was, however, not influenced in the ed ^r mutant. Similarly, in vivo DNA synthesis was decreased in the wildtype, but not in the mutant.—These results suggest that edeine acts at more than one site. The resistance of the mutant ed ^r -29 (ed ^r -2 locus) is tentatively interpreted as due to a block in edeine uptake.     

Early changes in the synthesis of proteins with affinity for single-stranded DNA during the onset of transformation in NRK cells.

Early alterations in the synthesis of proteins which bind to single-stranded DNA have been examined following the onset of transformation in NRK cells transformed by a heat-sensitive mutant (ts339) of Rous sarcoma virus. Transformation was initiated by shifting quiescent cultures from nonpermissive to permissive temperatures. Cultures were prelabelled with [3H]leucine for several generations at the non-permissive temperature, and with [35S]methionine at times after shift to the permissive temperature. Cytosol extracts were passed through sequential columns of double-stranded and single-stranded DNA bound to cellulose. Within the first hour of transformation there was an increase in the synthetic rate of proteins binding tightly to single-stranded DNA, but not to double-stranded DNA. More loosely bound protein fractions showed no such early synthetic increase. Electrophoresis of the fraction eluted from single stranded DNA-cellulose with 2 M NaCl demonstrated the presence of a major protein of 93 000 daltons, which comprised more than 0.1% of the cytosol protein. The synthesis of the 93 000 dalton protein increased continuously over the first 4 h interval after the onset of transformation. The synthetic rate of a 35 000 dalton protein, a major DNA-binding polypeptide found in mammalian cells, began to increase after a 1-h lag, following the onset of transformation. The protein fraction containing the 93 000 dalton protein had considerable unwinding activity, depressing the melting temperature of poly(dA-dT) by 39 degrees C. The protein fraction containing the bulk of the 35 000 dalton protein did not have unwinding activity. Transformation-induced DNA synthesis was measured in cells made permeable to deoxyribonucleoside triphosphates at times after shift to the permissive temperature. It was determined that synthesis of DNA began within the first 1--2 h after the onset of transformation. We conclude that the early transformation-associated synthesis of SS93 and perhaps other proteins binding to single-stranded DNA may be related to early transformation-associated changes preparatory to DNA replication and subsequent growth.     

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.     

Quantity of compentent cells in a population of Escherichia coli treated with Ca2+ cations]

An attempt was made to estimate the number of Escherichia coli K-12 cells rendered permeable to antibiotics under Ca2+ treatment. The effect of cold factor and Ca2+ alone as well as the cell age on the induction of permeability and the energy dependence of the latter were also investigated. About 70-75% and more exponentially growing cells as a result of Ca2+ treatment became sensitive to actinomycin, rubomycin and olivomycin. This number was somewhat lower (40-50%) in sationary phase culture. A fraction (20-30%) of stationary phase cells appeared to be sensitive to antibiotics even without Ca2+ pretreatment. Preincubation of the cells in cold in the absence of Ca2+ cations did not induce the cell permeability. The transport of antibiotics inside the cell was not prevented by an uncoupler of oxidative phosphorylation --carbonylcyanid-m-chlorophenylhydrazone (CCCP). It is suggested that the cells which are rendered permeable to tested antibiotics represent the "compentent" cells capable to uptake molecules of exogenous DNA as well.     

Effects of 2',3'-dideoxythymidine triphosphate on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells

2',3'-Dideoxythymidine triphosphate differentially inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells and unscheduled DNA synthesis in bleomycin-treated permeable cells or in isolated rat liver nuclei. The mode of inhibition of 2',3'-dideoxythymidine triphosphate was competitive with respect to deoxythymidine triphosphate. 2',3'-Dideoxythymidine triphosphate inhibited replicative DNA synthesis with a Ki of 8 microM, whereas unscheduled DNA synthesis was more sensitive, the Ki being 0.5 microM. Referring to the differential sensitivity of DNA polymerases alpha and beta to 2',3'-dideoxythymidine triphosphate and to other related information reported previously, the present results suggested that DNA polymerase alpha is playing a major role in replicative DNA synthesis, and DNA polymerase beta in unscheduled DNA synthesis.     

Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

DNA synthesis and repair in permeable cells of Micrococcus radiodurans.

Cells permeable to deoxyribonucleoside triphosphate were prepared from Micrococcus radiodurans, and DNA synthesis and rejoining of strand scissions induced by gamma-rays were investigated. DNA synthesis was stimulated by ATP at an optimal concentration of 1mM. This reaction requires four deoxyribonucleoside triphosphates and MgCl2. NAD inhibited the reaction, but no rejoining of primer DNA was observed. Even in the presence of NAD, DNA which was synthesized in the unirradiated permeable cells had a peak molecular weight of only 1.3 - 10(6). DNA synthesis was stimulated by irradiation of the permeable cells with gamma-rays, but this stimulatory effect was eliminated by the addition of NAD. Both primer and synthesized DNA in the irradiated permeable cells were rejoined in vitro in the presence of NAD and deoxyribonucleoside triphosphates, while those in the unirradiated permeable cells were not rejoined.     

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.     

Interstrand Cross-Links Induce DNA Synthesis in Damaged and Undamaged Plasmids in Mammalian Cell Extracts

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.     

Macromolecular synthesis in permeable liver cells

Macromolecular synthesis was studied in individual liver cells rendered permeable to macromolecules and charged molecules by treatment with toluene. Toluene-treated cells were compared to intact cells with regard to their ability to synthesize protein, RNA, and DNA. The permeable cells catalyzed the incorporation of amino acids into protein in a system which was sensitive to cycloheximide. Maximal incorporation required the addition of tRNA, ATP, GTP, an energy source and various cations. RNA synthesis also took place in these cells and was inhibited by actinomycin D. Maximal incorporation required all four ribonucleoside triphosphates, an energy-generating system, and Mn²⁺, K⁺, and F^?. The toluene-treated cells also were active for DNA synthesis when Ca²⁺ was present to induce endonucleolytic cleavage of the endogenous DNA. For maximal synthesis, all four deoxyribonucleoside triphosphates, ATP, K⁺, Mg²⁺, polyamines, and mercaptoethanol were required. These studies serve to emphasize the potential usefulness of toluene treatment for studying biosynthetic processes in mammalian cells.     

DNA synthesis in detergent-treated mouse ascites sarcoma cells.

Mouse ascites sarcoma cells (SR-C3H/He cells) were made permeable to nucleoside triphosphates by treatment with nonionic detergents in a nearly isotonic condition. The permeable cells synthesized DNA in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. The optimum detergent concentration for DNA synthesis was 0.015--0.020% with Triton X-100, 0.020% with Nonidet P-40, and about 0.0025% with Brij 58. Higher concentrations of detergents were rather inhibitory to DNA synthesis. DNA synthesis in Triton-permeabilized cells was thought to be replicative, and the activity in the optimum conditions was much higher than that measured in hypotonic permeable cells or in isolated nuclei. These studies show the potential usefulness of detergent treatment for examining DNA replication in mammalian cells in vitro.     

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.     

Response of Cultured Mammalian Cells to Diphtheria Toxin III. Inhibition of Protein Synthesis Studied at the Subcellular Level

Diphtheria toxin inhibited protein synthesis in intact KB cells. The action of the toxin upon the cell did not result in disaggregation of polyribosomes, or in impairment of their ability to function in protein synthesis. A reduction in single ribosomes and a concomitant increase in polyribosomes did result from the action of toxin. Nascent peptides were not cleaved from polyribosomes by the action of toxin, but treatment of fully intoxicated cells with puromycin resulted in cleavage of these peptides, and caused accelerated polyribosome breakdown. Our data indicated that the toxin must enter the cell to exert its effect. The component or components sensitive to toxin were localized in the 100,000 x g supernatant fraction of cytoplasmic extracts. When extracts from intoxicated cells were treated with nicotinamide, a significant proportion of their capacity to synthesize protein was restored. The specificity of this reaction suggested that nicotinamide adenine dinucleotide is involved in the action of toxin in the intact cell, and that one component inactivated by toxin is soluble transferase II.     

Mechanism of conjugation and recombination in bacteria

Summary For inhibition of DNA synthesis an antibiotic, edeine, acting specifically on DNA replication, was used. The inhibition of DNA synthesis in F^– cells caused only small decrease (three to four-fold) in recombination frequency. On the other hand a full inhibition of DNA synthesis in Hfr cells affected the recombination to the high extent, lowering its frequency 20–40 fold, at the same time lowering to the similar degree chromosome transfer (measured by zygotic induction frequency). However, the partial inhibition of DNA synthesis in Hfr cells, amounting to about 10 per cent of the control, permitted normal chromosome transfer and normal level of recombination. The results do not agree with Jacob and Brenner's model of chromosome transfer, yet they do not unequivocally confirm Bouck and Adelberg's model. It is possible that the limited DNA synthesis is necessary for other processes, and not for completing of the replication round. The results do not exclude also, that some residual DNA synthesis in female cells is of importance in mating.