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Condensation studies of chromosomal DNA in E. coli with a tetranuclear ruthenium complex are carried out and images obtained with wide-field fluorescence microscopy. Remarkably different condensate morphologies resulted, depending upon the treatment protocol. The occurrence of condensed nucleoid spirals in live bacteria provides evidence for the transertion hypothesis.

DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

The binding of ΔΔ/ΛΛ-[{Ru(phen)(2)}(2)(μ-bb(n))](4+) {where phen = 1,10-phenanthroline, bb(n) = 1,n-bis[4(4'-methyl-2,2'-bipyridyl)]-alkane (ΔΔ/ΛΛ-Rubb(n))} to the non-self complementary oligonucleotide 5'-d(CGCGATAAGCCGC·5'-GCGGCATTACGCG) (3-DB) has been examined using a 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) displacement assay. The 3-DB oligonucleotide contains two single adenine bulge nucleotides that are separated by three base pairs. (1)H NMR spectroscopy data demonstrated that the adenine bases are intra-helical and that the segment containing the two bulge nucleotides and the three A·T base pairs between the bulges forms a destabilised segment within the stable duplex oligonucleotide. The DAPI displacement assay demonstrated that ΔΔ-Rubb(7)-bound 3-DB with higher affinity than the other members of the ΔΔ/ΛΛ-Rubb(n) series. Molecular models suggested that the seven-carbon chain length in ΔΔ-Rubb(7) was ideal to span the distance between the two bulge sites. The binding of ΔΔ-Rubb(7) to 3-DB was also studied by (1)H NMR spectroscopy and molecular modelling. The selective changes in chemical shifts for the resonances from 3-DB upon addition of ΔΔ-Rubb(7) suggested that the metal complex specifically bound at the destabilised segment between A(5) and A(19). Observation in NOESY spectra of NOE cross peaks between 3-DB and ΔΔ-Rubb(7) confirmed that one of the ruthenium centres bound at the A(5) bulge site, with the other metal centre positioned at the A(19) bulge. In addition, ΔΔ-Rubb(7) was found to bind chromosomal DNA extracted from a suspension of Staphylococcus aureus that had been incubated with the ruthenium(ii) complex. As inert dinuclear ruthenium(ii) complexes are capable of being transported into a bacterial cell and bind chromosomal DNA, it is possible that they could be developed into anti-microbial agents that specifically target destabilised segments of DNA that are recognised by essential DNA-binding proteins.

Thymine deprivation results in the loss of viability in cells from bacteria to eukaryotes. Numerous studies have identified a variety of molecular processes and cellular responses associated with thymineless death (TLD). It has been observed that TLD occurs in actively growing cells, and DNA damage and DNA recombination structures have been associated with cells undergoing TLD. We measured the loss of viability in thymine-starved cells differing in the number of overlapping replication cycles (n), and we found that the magnitude of TLD correlates with the number of replication forks. By using pulsed field gel electrophoresis (PFGE), we determined the proportion of linear DNA (DSBs) and the amount of DNA remaining in the well after treatment with XbaI (nmDNA) under thymine starvation in the absence or presence of both rifampicin (suppressing TLD) and hydroxyurea (maintaining TLD). Our results indicate that DSBs and nmDNA are induced by thymine starvation, but they do not correlate with the lethality observed in the presence of the drugs. We asked whether TLD was related to chromosomal DNA initiation. DNA labeling experiments and flow cytometric analyses showed that new initiation events were induced under thymine starvation. These new DNA replication initiation events were inhibited in the presence of rifampicin but not in the presence of hydroxyurea, indicating that TLD correlates with the induction of new initiation events in Escherichia coli. In support of this finding, cells carrying a deletion of the datA site, in which DNA initiation is allowed in the presence of rifampicin, underwent TLD in the presence of rifampicin. We propose that thymineless-induced DNA initiation generates a fraction of DNA damage and/or nmDNA at origins that is critical for TLD. Our model provides new elements to be considered when testing mammalian chemotherapies that are based on the inhibition of thymidylate synthetase., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Faithful chromosome segregation is vital to all organisms. Eukaryotic cells use the tubulin-based cytoskeleton to segregate their chromosomes during mitosis. A handful of papers have provided convincing evidence that, in bacteria, this task is accomplished by the actin homolog MreB. In particular, a recent study by Gitai et al. demonstrates that MreB specifically binds to and segregates the replication origin of the bacterial chromosome.

The effect of quinolones on the inhibition of DNA synthesis in Staphylococcus aureus was examined by using single resistance mutations in parC or gyrA to distinguish action against gyrase or topoisomerase IV, respectively. Norfloxacin preferentially attacked topoisomerase IV and blocked DNA synthesis slowly, while nalidixic acid targeted gyrase and inhibited replication rapidly. Ciprofloxacin exhibited an intermediate response, consistent with both enzymes being targeted. The absence of RecA had little influence on target choice by this assay, indicating that differences in rebound (repair) DNA synthesis were not responsible for the results. At saturating drug concentrations, norfloxacin and a gyrA mutant were used to show that topoisomerase IV-norfloxacin-cleaved DNA complexes are distributed on the S. aureus chromosome at intervals of about 30 kbp. If cleaved complexes block DNA replication, as indicated by previous work, such close spacing of topoisomerase-quinolone-DNA complexes should block replication rapidly (replication forks are likely to encounter a cleaved complex within a minute). Thus, the slow inhibition of DNA synthesis at growth-inhibitory concentrations suggests that a subset of more distantly distributed complexes is physiologically relevant for drug action and is unlikely to be located immediately in front of the DNA replication fork.

High concentration salt effects on the structure and radiation-induced damages of DNA were studied to elucidate the biochemical mechanism of the resistance of halophilic H. salinarium against DNA damaging agents. High concentration of KCl did not induce significant conformational changes in H. salinarium chromosomal DNA, but exhibited an extensive protective effect on the radiation-induced single-strand breaks of plasmid DNA.

trans-4-Guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPh'Bu), a trypsin inhibitor, dose-dependently inhibited the growth of Escherichia coli K-12 IAM1264. Growth inhibition was preceded by dose- and time-dependent inhibition of DNA synthesis. These results strongly suggested participation of a trypsin-like proteinase in DNA synthesis. To clarify this suggestion, the effects of GMCHA-OPh'Bu on the doubling time and on the uptake of [methyl-3H]thymidine into DNA were examined with E. coli K-12 IAM1264 synchronized by a modified version of phosphate starvation. The synchrony lasted for two or three cycles with a doubling time of 55 min and a cell division period of 15 min. The cell cycle of E. coli was divided into three periods, cell division period (P), the period between cell division and initiation of chromosome replication (Q) and the period between initiation of chromosome replication and cell division (R). The R period was subdivided into two periods, R1 in which the rate of thymidine uptake into DNA was increasing, and R2 in which it was constant. The addition of GMCHA-OPh'Bu at the R1 period did not affect the already-initiated round of cell division, however, it retarded the next round. The addition at P, Q or R2 retarded the cell division in the same round, causing prolongation of the R1 period. A sharp and momentary appearance of trypsin-like proteinase activity peaked at the Q/R1 boundary in one cell cycle, and inhibition of the activity prolonged the R1 period.(ABSTRACT TRUNCATED AT 250 WORDS)

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and supercoiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA, and indicate that kinks are an important intermediate step in many of the transitions.

The uptake of circular and linear plasmid RSF0885 deoxyribonucleic acids, (DNAs) obtained from Haemophilus parainfluenzae 14, in both homologous and heterologous recipients was studied and compared with that of chromosomal DNA. High concentrations of divalent cations stimulated the uptake of either circular or linear plasmid DNA in H. parainfluenzae 14 competent cells but did not affect the uptake of chromosomal DNA. The biological activity of linear plasmid DNA was similar to that of circular DNA, and the transforming efficiencies for ampicillin resistance of both molecular forms were stimulated by divalent ions. Plasmid DNA was taken up efficiently either with or without the addition of divalent ions but was not biologically active in the heterologous Haemophilus influenzae Rd recipient. Our results suggest that in H. parainfluenzae 14 some of the steps for chromosomal and plasmid DNA uptake are different.

Torsional tension in the DNA double helix can be detected in living cells of Escherichia coli from measurements of the rate of trimethylpsoralen photobinding to the intracellular DNA. Here we show that this tension is relaxed in vivo when single-strand DNA breaks are introduced by gamma-irradiation and that approximately 160 nicks per genome equivalent of DNA are required to relax greater than 95% of the tension. Chromosomes containing less than 160 nicks per genome equivalent lose only a part of the tension, depending on the number of nicks. The remaining tension is maintained during incubations of cells at 0 degrees C. Chromosomes with tension relaxed by incubation of cells with inhibitors of DNA gyrase interact with the trimethylpsoralen probe independently of the number of nicks introduced by gamma-irradiation. The results fit a model in which the chromosome in growing E. coli cells (mean generation time, 30 min) is segregated into 43 +/- 10 domains of supercoiling per genome equivalent of DNA or 120 +/- 30 domains per nucleoid. The number of domains is unchanged in cells depleted of nascent RNA by growth with rifampicin, but varies somewhat in cells growing at different rates in different media.

Coumermycin A1, a specific inhibitor of DNA gyrase, differentially changes the spectrum of proteins synthesized in wild type E. coli cells but has no effect on the protein spectrum in mutant cells with coumermycin-resistant DNA gyrase. The rpoB265 mutation affecting RNA polymerase decreases the coumermycin A1-sensitivity of bacteria while the rpoC3 mutation increases it. The interaction of wild type and mutant RpoB265 RNA polymerases with ColEl plasmid DNA in vitro is differently affected by DNA supercoiling. No such differences are observed in the case of RpoC3 RNA polymerase. The results suggest that template supercoiling may have a substantial effect on transcription in vivo, an effect which, in some cases, depends on the properties of RNA polymerase.

The inhibitor of DNA gyrase, novobiocine affects recombination between homologous plasmids in the course of plasmid transformation. Novobiocine decreased significantly the yield of recombinants when added to competent cells during the first 10 minutes. Later, after 30 to 40 min, addition of the antibiotic had no effect. Probably, recombination is completed, in general, in 10 to 20 min after DNA uptake. Plasmid recombination was depressed totally after preincubation of competent cells with the drug. Evidently, inhibition of recombination was due to the loss of superhelicity in resident plasmid DNA.

The time necessary to replicate the chromosome (C period) was measured in Escherichia coli B/r (ATCC 12407) and a low-thymine-requiring derivative of that strain. In the Thy- strain, C was measured as a function of growth rate and exogenous thymine concentration either from step-up or chloramphenicol experiments. In the Thy+ parental strain, C was measured only as a function of the growth rate and only by the chloramphenicol method. The C period was found to decrease with growth rate and, in the Thy- strain, the C period also decreased with increasing thymine concentration. It approached a value of approximately 37 min at high growth rates.

Until recently it has been assumed that the factors being responsible for the condensation of the DNA in the nucleoid of Escherichia coli are destroyed by rifampicin because it has been impossible to obtain folded chromosomes from cells treated with this inhibitor. In this paper it is shown by viscosity and sedimentation measurements that unfolding of the DNA does not take place during the process of the cell lysis as it should be predicted from this assumption, but is occurring distinctly afterwards. Since the observed unfolding process is too slow to be caused simply by molecular movements it is concluded that it is brought about by the action of salts or detergents of the lysis medium. The structure of the nucleoid is still intact in vivo despite inhibition of RNA synthesis by rifampicin.

An examination of the relationship of both nonreplicating and replicating forms of R6K plasmid DNA to the Escherichia coli folded chromosome showed that both forms cosediment with the chromosome in neutral sucrose gradients. Approximately 20% of the nonreplicatin molecules was found as freely sedimenting forms when the folded-configuration of the chromosomes was preserved. However, under the same conditions negligible amounts of the replicating forms were found as freely sedimenting molecules. Thus, it is concluded that the replicating forms, when compared with nonreplicating molecules, are preferentially associated with the folded chromosomal structure. Exposure of the folded chromosomal structure to RNase resulted in an unfolding of the chromosome and a concomitant increase in the amount of freely sedimenting replicating and nonreplicating forms of R6K DNA. Analyses of the single-stranded length of RNase-released nascent molecules suggest that they replicate in continuous association with the folded chromsome complex. Nonenzymatic unfolding of the chromosomes by progressively lowering the sodium ion concentration during lysis resulted in a progressive increase in the release of nonreplicating molecules. Replicating molecules wer not released by unfolding the chromosome in this fashion.

Donor DNA in its initially bound, single-stranded form exists in a chromosomally-unassociated complex where it is resistant to exogenous DNase I but sensitive to micrococcal nuclease. Most of the complexes are readily recuperable from the supernatant of recipients converted into spheroplasts. Subsequent to formation of this superficially located complex, donor DNA progressively associates with the recipient chromosome into which it is eventually integrated. Treatment of recipients with ethidium bromide at various times after initial DNA binding almost immediately halts translocation of whatever donor material is not yet synapsed with the chromosome. On the other hand, donor DNA that has already synapsed experiences no difficulty in becoming genetically integrated. Some degradation occurs to DNA that fails to undergo translocation as a result of ethidium bromide treatment, the acid-soluble products appearing in the culture medium. DNA in untranslocated complexes surviving treatment is not appreciably different in single-strand length from that in untreated complexes. When these surviving complexes are isolated from a cell lysate, the contained DNA can be shown by spectrofluorometry to have bound the drug.

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.

Initiation of deoxyribonucleic acid replication is absent in Bacillus subtilis cells made permeable by toluene. The absence of initiation may be (i) a temporary removal of toluene, or (ii) irreversibly lost due to damage by toluene treatment to a cellular structure or a process required for chromosome initiation. Washed cells, previously treated with toluene and subsequently washed free of detectable amounts of toluene, have the same characteristics of toluene-treated cells in which toluene is not removed. The continued absence of initiation in the washed cells indicates a permanent loss of initiation in cells treated with toluene. Protein synthesis is also inhibited irreversibly by toluene treatment, indicating damage to translation as a possible mechanism for loss of initiation of toluene-treated cells.

The antitumor agent cis-platinum(II)diamminodichloride (PDD) caused wild-type and recA+ deoxyribonucleic acid (DNA) repair-deficient mutant cells of Escherichia coli K-12 to grow as long, multinucleated filaments. At 5 micrograms/ml, the times required for reduction of viability to 37% for wild-type, polA, recB,C, uvrA, and recA organisms were > 200, 200, 120, 25, and 5 min, respectively. Only recA cells exhibited @reckless" degradation of DNA at this concentration of PDD. As shown by sedimentation in alkaline sucrose gradients, generation of single-strand breaks in DNA of the remaining organisms was a major consequence of growth in PDD. Upon incubation in fresh medium after removal of the compound and storage for 4 h at 4 degrees C, a respective lag of 3, 4, 6, and 9 h occurred before filaments of wild-type, polA, recB,C, and uvrA cells commenced cell division. Maintenance at 4 degrees C, which evidently delayed postshift initiation of chromosome replication, was only essential for fragmentation of uvrA filaments. In all cases, these periods of division delay corresponded to those required for restoration of normal chromosomal molecular weight as determined in alkaline sucrose gradients.

The existence of nonintegrated plasmid-chromosome complexes has been deduced in previous work from the cosedimentation of covalently closed, circular plasmids with host folded chromosomes. In the present work, it is shown that about 70 to 90% of the covalently closed, circular F deoxyribonucleic acid could be released in vitro from chromosome complexes by ribonuclease treatment but not by protease, Sarkosyl, or ethidium bromide. Consistent with the in vitro studies, Escherichia coli cells treated for 5 min with rifampin, an inhibitor of ribonucleic acid initiation, released upon lysis 90% of their plasmid deoxyribonucleic acid as freely sedimenting molecules.

Vaccine regimens which mimic actual infection with bacterial enteropathogens should offer the best opportunity for successful long-term immunoprotection against diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC) or Vibrio cholerae. Based on this principle, we designed and tested an oral whole cell anti-ETEC vaccine consisting of intact cells of ETEC strain H-10407 (ST+LT+; O78:H11:CFA/I) which were rendered incapable of replication by treatment with a potent DNA endonuclease, colicin E2. Young healthy volunteers were administered two oral doses of either placebo or approx. 3 X 10(10) vaccine cells. In a double-blind study, 9 of 10 vaccinees responded with an increase in CFA/I-specific intestinal IgA antibody, determined as percent of total IgA. Challenge with virulent strain H-10407 (5 X 10(9) living cells) produced diarrhea in 8 of 9 (89%) of the placebo-treated volunteers and in 2 of 10 (20%) of the vaccinees. Thus, the colicin E2-killed whole cell vaccine afforded both a significant intestinal immune response and significant protection against challenge with the virulent organism. The data presented here suggest that for this vaccine preparation an intestinal anti-CFA/I IgA response is a good indicator of a protective immune response, which most likely involves antibody responses to a number of antigens in addition to CFA/I. We conclude that the colicin E2 method for preparing an oral anti-ETEC vaccine merits further study and that this method may also be applicable to other enteropathogens.

Chromosomal DNA in 5 hereditary variants occurring in Photobacterium leiognathi population was subjected to restriction analysis. The variants differed in the levels and regulation of luminescence and colony morphology. Agarose electrophoresis of DNA fragments isolated after exposure to Hind II, Bam HI, Bgl I and Pst I restriction endonucleases revealed respectively 38, 28, 35 and 29 fragments equally distributed by their molecular weights. Electrophoregrams of the 5 strains were absolutely identical. After exposure of DNA of all the strains to PVu II, Xho II, Sal GI and Eco RI restriction endonucleases there were detected no fragments. The pleoiotropic genetic variation in these strains was not associated with large deletions or amplification of chromosomal DNA regions.

This paper describes the results of treating plasmid DNA in vitro with mutagens, to obtain mutations both in plasmid genes and chromosomal genes comprised within the plasmid, thus avoiding disorganization characteristic of in vivo mutagenesis. The model system is represented by DNA of RSF2124 responsible for colicine E1 synthesis and resistance to ampicillin. Col- mutants were looked for after exposure to UV- and gamma-irradiation. The lethal effect was estimated as inactivation of the ampicillin resistance marker. After reisolation from mutant transformant of the plasmid DNA, the novel character and resistance to ampicillin proved to retain in the course of subsequent transformations and passages of transformed colonies, suggesting the mutational nature of the changes. Exposure of RSF2124 to short-wave UV-irradiation (lambda = 254 nm) produced a pronounced mutagenic effect: the relative quantity of Col- mutants under optimal conditions of mutagenesis increased about 10 times. In the case of W-reactivation (additional UV-irradiation of C600 wild type cells) of lethal lesions, a 95% reliable increase in mutagenic effect was observed. Significant enhancement of mutagenesis (about 4-fold) was detected when only recipient cells were exposed to low doses of UV (the so-called indirect UV mutagenesis). Thus, with regard to W- and indirect UV mutagenesis, the plasmid DNA behaves like DNA of temperate phages which suggests their evolutionary relationship. Treatment of plasmid DNA with acridine orange prior to UV, only protected from lethal lesions. Gamma-irradiation (60Co) at the dose producing 100-fold inactivation, increased the yield of Col- mutants by one order of magnitude. The presence of RSF2124 plasmid in a cell does not affect its UV sensitivity.

To proceed the works on induced mutagenesis in plasmids, mutagenic effects of chemicals on the DNA of RSF2124 plasmid mediating colicine E1 biosynthesis and resistance to ampicillin, were studied. After exposure to mutagens, plasmid DNA was used to transform Escherichia coli C600 rk-mk-cells. The lethal effect was estimated from inactivation of the ampicillin marker, the mutagenic effect being measured by the appearance of mutants unable to synthesize colicine (Col-). The reaction of the plasmid DNA with a mutagen was stopped by 10-fold dilutions of aliquots in TEN buffer, followed by dialysis in 10 mH CaCl2 for 24 h. To select the most efficient mutagens for plasmid DNA, the compounds were predominantly tested which are known to be effective in other systems (transforming and transfecting DNA, microbial viruses). An a result, all chemicals tested by their activity were classified into 4 groups: inducing more than 100 fold increase (hydroxylamine, O-methylhydroxylamine); inducing 10 fold increase (UV-irradiation, lambda = 254 nm; W-mutagenesis, gamma-irradiation, nitrous acid, mitomycin C); inducing less than 10fold increase (indirect UV-mutagenesis, nitrous acid, beta-chloroethyldiethylamine hydrochloride, nitrosoguanidine); no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, O-beta-diethylhydroxylamine).

The effect of phenethyl alcohol on DNA synthesis was examined using several in vitro systems of Escherichia coli H560; i.e., ether-treated cells, membrane fractions and folded chromosomes fortified with DNA polymerase. In all systems, the incorporation of deoxyribonucleotides was much reduced for the phenethyl alcohol-treated cells compared with the non-treated cells. The total activity of DNA polymerases in polA1 cells (mostly DNA polymerase II) was not impaired for the phenethyl alcohol-treated cells and the reduction of the rate of DNA synthesis in vitro was ascribed to the reduction of the chromosomal template activity which was related to trypsin sensitive protein components. The analysis of chromosomes from the phenethyl alcohol-treated cells revealed the remarkable reduction of a protein component of molecular weight approx. 58 000 in contrast with a protein component of molecular weight approx. 30 000.

When a thermosensitive mutant of E. coli affected in DNA initiation is heated to 41 degrees , a protein needed for DNA initiation is irreversibly denatured. When these bacteria are incubated at 30 degrees in the presence of several concentrations of chloramphenicol, DNA synthesis is greatly stimulated at a chloramphenicol, DNA synthesis is greatly stimulated at a chloramphenicol concentration of 2.5 mug/ml, and then sharply decreases. This stimulation of DNA synthesis appears to be caused by an increased frequency of initiation. The existence of an "anti-initiator" protein controlling the frequency of chromosomal initiation is proposed.

At high concentrations (10 mug/ml), actinomycin D inhibited deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis. Inhibition occurred quickly (in less than 1 min) and was complete. In strain 23 thy his, inhibition of DNA synthesis by actinomycin D was followed by partial degradation of one of the two daughter strands to acid-soluble products. Degradation began at the replication point and proceeded over a distance equal to about 12% of a chromosome in length. Actinomycin D played some essential part in degradation, since exposure of the cells to other treatments or agents which inhibit growth did not lead to the above result.