Search

cially diagnosed cases of CRS that meet a more rigid set of diagnostic criteria: (1) Exposure for at least 3 years with individual measurements confirming at least 1 Gy of marrow dose received; (2) medical follow-up confirming the clinical manifestations considered by Guskova and Baysogdolov to be the criteria of CRS; and (3) absence of other illness with similar symptomatology. When one focuses on this subset of 66 cases, the dose-response relationship between CRS severity, hematopoietic depression and chromosomal aberrations is clearer. Of note is that of

I welcome the comments and thoughts of Drs. K. F. Baverstock and J. F. Ward concerning the significance ofspontaneous DNA damage in the "negligible dose" controversy (1). My intent in raising this issue was to draw attention to a fundamental biological aspect of radiation protection that seemed to be neglected and in need of further thought and study. The comments of Dr. Baverstock are relevant. I agree with his statement that "the concept of 'negligible dose' can be clearly seen for what it is, a judgment on acceptability of risk." This judgment can be strongly influenced if we understand the natural events influencing the stability of DNA. While in theory any DNA damage induced by radiation, no matter how limited, may have biological impact, the issue is the practical consequences. Here I have directed my discussion to very low and protracted doses of low-LET radiation of the order of 0.01-1.0 mSv/year. Any modification of the genome is potentially mutagenic. Proper DNA repair and/or replication is a major factor in determining the biological outcome. Dr. Ward suggests that DNA double-strand breaks (DSBs) are of singular importance in the DNA damage produced by ionizing radiation. Certainly the contribution of the DSB to lethality is emphasized in the current literature. However, the low doses of concern in my discussion are unlikely to result in direct lethal action. At protracted doses of less than 1 cGy/ year it is more likely that mutagenesis and/or cellular transformation will result. Induced mutation or transformation frequency is conventionally measured against a control or "baseline rate." For relative risk estimates of cancer induction by radiation, the excess observed is compared to the baseline rate. The doubling dose (DD) is that amount of radiation needed to double the control or baseline incidence [definitions drawn from the BEIR V report (2)]. If the spontaneous or baseline rate of mutagenesis (and cell transformation) is due primarily to endogenous DNA damage either unrepaired or misrepaired (3) then the net lifetime effect of endogenous DNA damage is equivalent to -1.0 Gy of acute exposure. Using the number of DNA damage events estimated earlier (1) and approximating the human life span at 75 years then (7 x 10')(75) or --5 X 109 DNA events/cell take place during a lifetime. Since, for mutagenesis, this is equivalent to --1.0 Gy or 100 x 102 DNA damage events/cell for low-LET radiation, it would

One of the crucial problems in radiation protection is the reality of the negligible dose or de minimus concept (1-4). This issue of a "practical zero" and its resolution is central to our understanding of the controversy concerning the existence of a "safe" dose in radiological health. However, for very low levels of environmental mutagens and carcinogens including low doses of low-LET radiations (less than I cGy or 1 rad), spontaneous or endogenous DNA damage may have an increasing impact on the biological consequences of the induced cellular response. It is this issue that is addressed in this communication.

Successive radioisotopic labeling of the bacterial chromosome has been employed to determine whether the position of the label at the time of exposure to X-rays influences its pattern of replication subsequently. Following exposure to 5000 R of X-rays the chromosome region in front of the ‘replication-fork’ at the time of exposure is replicated to a greater extent than the region in or immediately behind the growth point. The latter region is prematurely replicated. If after X-ray exposure prevention of new cycles of DNA synthesis is accomplished by incubation of the cells in medium lacking required amino acids then the replication of all regions is markedly depressed. In addition an enhanced, non-selective degradation of chromosome regions is observed when irradiated cells are incubated without amino acids. Thus DNA in the vicinity of the replication-fork is degraded to the same extent as DNA located elsewhere. The data are interpreted as showing that following irradiation there exist two populations of chromosomes, the proportions of which are dose dependent. One population responds normally in regard to replication sequence and results from the resumption of semiconservative replication by the original replication-fork. The other, having lost its integrity, shows an aberrant type of replicative synthesis in which new sites of replication are initiated at random locations.

Injection of mouse marrow preserved in glycerol at -80 C. for six weeks has been shown to be quantitatively similar to injections of fresh marrow in inducing recovery in mice after lethal radiation. The effect of the preservation procedure on subsequent rates of DNA synthesis has been determined in vitro for both preserved human marrow and preserved mouse marrow. Since DNA synthesis is a satisfactory measure of rates of cell multiplication and since preservation is shown to affect DNA synthesis in human marrow to a lesser degree than in mouse marrow, it may be concluded that the procedure used (-80 C. plus 15% glycerol) is satisfactory for the maintenance of viability in human marrow.

Escherichia coli strains 15T - (555–7) and B/r were grown in the presence of thymine- 14 C to label all DNA. The ability of these parental DNA's to undergo cycles of replication subsequent to cellular irradiation with either X-ray or ultraviolet light (UV) was followed with density labels. Exposed cells were shifted into the density medium at times which were approximately multiples of normal rounds of DNA replication. A portion of the parental DNA, replicated semiconservatively once during an initial cycle following UV or X-irradiation in E. coli , failed to replicate again within the time studied. The time course of semiconservative parental DNA replication is altered.

Measurements were made of DNA methylation during the cell cycle of Chinese hamster ovary cells collected in mitosis by the mitotic selection procedure. It was observed that a rapidly reannealing DNA component constituting approximately 10 % of the DNA synthesized during the cyle was enriched in 5-methylcytosine. The rapidly reannealing DNA was synthesized throughout S phase. However, the rapidly reannealing DNA synthesized early in the S phase was especially methyl rich and may reflect the onset of initiation of replication synthesis as the cells enter S phase.

(1974). Inactivation of Dark-repair-deficient Mutants of Escherichia Coli by Sunlight. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine: Vol. 26, No. 1, pp. 73-76.

Billen, Daniel (The University of Texas, Houston), and Roger Hewitt . Physiological aspects of modification and restoration of chromosomal synthesis in bacteria after X irradiation. J. Bacteriol. 90: 1218–1225. 1965.—A study was made of the effect of amino acid deprivation or chloramphenicol on the character of postirradiation deoxyribonucleic acid (DNA) replication in bacteria with the use of radioisotopes and 5-bromouracil as a density label. CsCl density-gradient studies of DNA showed that postirradiation incubation of amino acid-requiring Escherichia coli in an amino acid-free medium interfered with continued linear chromosomal replication. In the presence of the required amino acids, linear chromosomal replication was shown to resume. Addition of chloramphenicol was found to prevent this resumption. Deletion of the required amino acids or the presence of chloramphenicol in a fully supplemented medium allowed the detection of altered DNA synthesis in bacteria at X-ray doses as low as 500 r. The character of the limited DNA made in the presence of the density label after irradiation is described. The results are interpreted as showing that the synthesis of a protein(s) is required for restoration of linear chromosomal replication in the irradiated cells.

Bacillus subtilis was exposed to ultraviolet light (UV) or X rays, and gene frequency analysis was used to study the location of initiation sites of postirradiation deoxyribonucleic acid (DNA) synthesis. It was found that DNA synthesis resumes primarily from the origin after UV exposure. With X irradiation, the origin is not selectively replicated. Elevated origin-to-terminal marker ratios observed after UV exposure of exponentially growing cells were interpreted as evidence for selective UV resistance of the replicative origin region of the bacterial chromosome.

A comparison of repair synthesis after ultraviolet light (UV) or X-ray exposure was made in Escherichia coli strains 15T − (555–7) and B/r by use of a D, 15 N, 13 C density labeling system. During the initial 15 min of incubation after UV irradiation, both a “repair” synthesis and a reduced semiconservative deoxyribonucleic acid (DNA) synthesis occurred. In the so-called “physiological” dose range used, the latter was greater than the former. X-irradiation of cells, at doses producing similar levels of cell death as in the UV-exposed cultures, did not lead to a similar repair replication process. However, a density heterogeneity of the DNA synthesized in the initial 10 min after exposure was observed. This is interpreted in terms of X ray-induced DNA degradation. Normal cells showed only a semiconservative type of replication and, therefore, within the limits of resolution of the system used (the incorporation of 1,000 to 5,000 nucleotides per replicating chromosome could be measured), DNA in normal cells did not appear to undergo a repair synthesis involving thymine exchange. These results indicate that not all repair mechanisms mimic that found after UV exposure.

The effect of x-rays on adenosine triphosphate (ATP) metabolism of Escherichia coli B r has been investigated. ATP was assayed for by use of the firefly luminescent system. The following results were obtained: 1. 1. Exposure to 60,000 r. did not alter the initial ATP content of the cells. 2. 2. The irradiated cells, when incubated at 37 °C. in a nutrient broth containing glucose, appeared to be capable of forming ATP at a rate approximately equal to that of the control cells. 3. 3. Irradiated cells, separated from the incubating medium, showed a decreasing ATP content with increasing incubation time. An analysis of the supernatant from these cultures revealed an increasing concentration of ATP with increasing time of incubation. No such release of ATP was noted from the nonirradiated cells. 4. 4. The presence or absence of metabolites in the incubating medium markedly influenced the degree of ATP loss into the medium. The loss of ATP was noted only under those conditions in which a turnover of ATP might be expected. 5. 5. The rate of release of ATP by the irradiated cells incubated in nutrient broth plus glucose was decreased if the temperature was lowered below 37 °C. Irradiated cells incubated in phosphate buffer plus glucose exhibited a lag in ATP loss, the duration of this lag increasing as the temperature was decreased below 37 °C. 6. 6. The amount of ATP lost by irradiated cells during 90 min. incubation at 37 °C. was found to increase with increasing x-ray dose. 7. 7. On the basis of evidence obtained by turbidity measurements, microscopic observations, and cell counts, the possibility that gross lysis of the irradiated cells is responsible for the release of ATP into the medium is eliminated. However, the evidence does not eliminate the possibility of a subtle form of lysis or structural change, rather than a true permeability change in the cell membrane, being the cause of the ATP leakage.

Several recent reports bearing on the elucidation of the mechanism by which transfer of the chromosome is effected from donor to recipient cell in the Escherichia coli Kl2 mating system have suggested that DNA synthesis plays a role in the process. Jacob et al (1963) have proposed a model which explains the transfer process as a result of concomitant replication of the donor chromosome; Bouck and Adelberg (1963) have suggested that the initiation of DNA replication opens the donor chromosome at the F-attachment site, and if conjugation with a recipient occurs, the open chromosome is transferred as soon as the cycle of DNA replication is completed. The present report presents evidence to show that normal chromosome transfer in one Hfr strain occurs without concomitant DNA synthesis.

Using [3H]thymine or [3H]thymidine as a pre-irradiation label and the thymine analogue 5-bromo-[14C]uracil as a combined density and radioisotopic label for observing post-irradiation DNA synthesis, it has been determined that relatively low doses of X-rays can induce changes in the normal sequence of chromosome replication. Changes are noted at doses of 2000 R or higher, with the effects becoming increasingly manifest with higher doses. The findings may be summarized as follows: (1) DNA synthesis immediately following irradiation occurs in limited regions of the chromosome. (2) Resumption of chromosome replication occurs at a point(s) apparently differing from the original growth point. (3) An early temporal appearance of a second “generation” of DNA is observed. (4) A portion of the DNA does not appear to participate in post-irradiation chromosomal replication. A feature of the experimental method was that at least 98 % of the cellular DNA was recovered in the CsCl gradients.

A study has been made of the effects of unbalanced DNA synthesis on subsequent cell physiology and X-ray sensitivity of the polyauxotroph, Escherichia coli strain 15T-A-U- (thymine, arginine, and uracil dependent) with the following results: 1. 1. DNA replication, proceeding to completion in the absence of concomitant protein and RNA synthesis, resulted in a cell population with an enhanced DNA content and with increased resistance to the lethal effects of X-ray exposure. This resistance was expressed as an increase in the shoulder of the dose-survival curve and an increase in the mean lethal dose. 2. 2. Renewal of both protein and RNA synthesis after completion of unbalanced DNA synthesis reversed the resistant state. The presence of both arginine and uracil were required for maximum reversal. When only uracil was added, no change in sensitivity was seen. Addition of only arginine resulted in partial resensitization. 3. 3. Incubation of exponential cells in a medium lacking thymine as well as arginine and uracil led to thymine-less death as well as a sensitization to X-rays. This was characterized by a shift from sigmoidal to exponential killing. 4. 4. Substitution of 5-bromouracil for thymine during unbalanced DNA synthesis did not interfere with the completion of DNA synthesis. Buoyant density-gradient analysis in CsCl showed that only normal and hybrid DNA were present in such cells. Equilibrium centrifugation of DNA obtained from cells incubated with bromo[ 14 C]-uracil provided confirmation that only hybrid DNA was made during the unbalanced DNA synthesis period. Such cells did not exhibit enhanced resistance to X-rays, but to the contrary were more sensitive than the control population.

The influence of unbalanced growth on subsequent radiation induced inhibition of desoxyribonucleic acid synthesis in Eccherichia coli was investigated. Results are presented which show an inhibition by chloramphenicol of phasing or synchronization of the desoxyribonucleic acid-synthesizing system and that the radiosensitivity or the synthesizing mechanism is altered by previous chloramphenicol exposure. (C.H.)

SummaryThe kinetics of formation of the inducible inhibitor of post-irradiation DNA degradation has been studied by amino-acid deprivation in four Escherichia coli strains: 15T–(555–7), B/r59, WP2 and B32. In the first three, the inducible inhibitor is absent if the required amino acid is withheld and DNA degradation following gamma radiation is considerable. After restoration of the amino acid, the inhibitor appears in 7 min and is at 50 per cent maximum in 14 min. In B32 there is no effect of witholding the amino acid. Strain 555–7 also exhibits cell leakage, probably due to the induction of a defective prophage. This leakiness appears at 14 min with a 50 per cent maximum at 22 min. Strains WP2 and B/r59 have very little post-irradiation leakiness yet show strong induced inhibition. The data suggest that there is no necessary connection between the inducible inhibitor and a prophage. Preliminary U.V. induction in WP2 produces nearly complete inhibition, yet confers very little survival potential.

In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is observed. This is an exaggerated repair synthesis which can be abruptly terminated by the addition of the DNA ligase cofactor, nicotinamide adenine dinucleotide. This communication describes experiments which bear on the relationship between measurable strand breaks, DNA polymerase I-directed, exaggerated repair synthesis, and strand-break repair.

Many mutagens and carcinogens damage DNA and elicit repair synthesis in cells. In the present study we report that alkylation of the DNA of Escherichia coli that have been made permeable to nucleotides by toluene treatment results in the expression of a DNA polymerase I-directed repair synthesis. The advantage of the system described here is that it permits measurement of only DNA polymerase I-directed repair synthesis and serves as a simple, rapid method for determining the ability of a given chemical to elicit “excision-repair” in bacteria. DNA ligation is intentionally prevented in our system by addition of the inhibitor nicotinamide mononucleotide. In the absence of DNA ligase activity, nick translation is extensive and an “exaggerated” repair synthesis occurs. This amplification of repair synthesis is unique for DNA polymerase I since it is not observed in mutant cells deficient in this polymerase. DNA ligase apparently controls the extent of nucleotide replacement by this repair enzyme through its ability to rejoin “nicks” thereby terminating the DNA elongation process. The nitrosoamides N -methyl- N -nitrosourea and N -ethyl- N -nitrosourea, as well as the nitrosoamidines N -methyl- N′ -nitro- N -nitrosoguanidine and N -ethyl- N′ -nitro- N -nitrosoguanidine, elicit DNA polymerase I-directed repair synthesis. Methyl methanesulphonate is especially potent in this regard, while its ethyl derivative, ethyl methanesulphonate, is a poor inducer of DNA polymerase I activity in permeabilized cells.

The production of strand breaks by X rays in cellular DNA can result from direct action of the radiation on DNA or from indirect action. Indirect action may depend on OH radicals, hydrogen atoms, or solvated electrons formed by the radiolysis of intracellular water [for a recent review see Ref. (1)]. From studies on the rates of reaction of chemical scavengers for the OH radical and their protective activities, it has been concluded that the OH radical is the primary cause of indirect DNA strand breakage in cells (1-3). Recently it has been proposed that certain protective organic compounds may function in a more complex way than by simple OH radical removal. Ewing (4-6), using lethality as an end point, has found that under certain conditions of irradiation there is a correlation between radiation protection and the formation of a secondary a-hydroxyl radical. For example, he has shown that t-butanol and t-amyl alcohol, both excellent OH radical scavengers that are not converted to a-hydroxyl radicals, also do not protect Escherichia coli B/r cells if they are X-irradiated in air or 100% N2 but will protect if they are irradiated in 1% 02 (5, 6). The purpose of this study was to determine whether chemical protection against single-strand breaks observed in toluene-treated E. coli subjected to X irradiation in air (7) was due to the removal of OH radicals, or resulted from the production of secondary radicals as proposed by Ewing (4). In toluene-treated cells, DNA strandbreak production can be measured without the complication of strand ligation during or immediately following X-ray exposure since such cells are deficient in DNA ligase activity (7). Details of growth and of the methods of toluene treatment of E. coli strain AB3063Fwere described earlier (7, 8). Irradiation was carried out with a TFI Corporation Gemini Industrial X-Ray Unit, equipped with a 3-mm aluminum filter, at a dose rate of about 1 krad/min. Toluene-treated cells were rapidly thawed and added to 50 mM potassium phosphate buffer, pH 7.4 (a final concentration of 5-10 X 108 cells/ml), plus the radioprotective agent. All preparations were held 10 min at room temperature in sealed 2-ml vials prior to X-ray exposure; during irradiation the samples were held in an ice bath. The 02

X-ray exposure of Bacillus subtilis strains made permeable to nucleoside triphosphates by toluene treatment stimulates a repair-type DNA synthesis and inhibits semiconservative DNA synthesis. The X-ray-stimulated repair-type synthesis is primarily due to polymerase I activity, since a mutant lacking polymerase I showed a greatly reduced response. The observed X-ray response requires the presence of the four deoxyribonucleoside triphosphates and ATP for full expression. Following irradiation, the DNA synthesized in Pol+ cells appeared to consist of longer runs than the DNA observed in Pol− cells.

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.

Publisher Summary The chapter describes the use of Tween-80 as an agent that produces a reversible permeability to nucleotides in Chinese hamster ovary (CHO) cells. Several techniques are described for rendering mammalian cells permeable to exogenous nucleoside triphosphates for the purpose of studying DNA metabolism. These include cold shock and cold shock in a hypotonic medium. The use of Tween-80 to permeabilize mammalian cells seems to have general applicability. CHO cells are reversibly permeabilized by Tween; this has obvious advantages for studies in which subsequent cell growth is important. Whether other cell types can be reversibly permeabilized probably depends on whether or not appropriate conditions for achieving it are attainable. The possibility of using this technique to introduce enzymes and other proteins into cells has not been explored, however clearly, if such could be done, the usefulness of this permeabilization process would be increased for in situ studies of cellular activities.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested). The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments. Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity. These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.

NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.

The radiosensitivity of an Escherichia coli mutant deficient in DNA polymerase I was measured in the presence of OH radical scavengers. The extreme X-ray sensitivity of the mutant could be abolished by OH radical scavengers if a sufficiently high level of radioprotector was present. There was a direct correlation between the OH radical scavenging activity of the chemicals tested (NO/sub 2//sup -/, n-butanol, glycerol, t-amyl alcohol, and t-butanol) and their protective ability. The author interprets the data as showing that the indirect actions of X rays (primarily OH radicals) result in major damage to the bacterial DNA which in large part consists of potentially lethal lesions. This potentially lethal damage is repaired through an enzymatic pathway requiring DNA polymerase I. I. In the mutant lacking DNA polymerase I, these potentially lethal lesions are expressed as cell lethality.

Calf-thymus histone fractions were found to inhibit the biosynthesis of DNA in vitro to a substantial extent. The inhibition could be explained on the basis of DNA-histone interaction. The very lysine-rich histones (Fraction 1) complexed with DNA most readily and inhibited the DNA replication at a histone-DNA ratio of 1:1 and higher. Higher ratios of the moderately lysine-rich histones (Fraction 2b) and of the arginine-rich histones (Fractions 2a and 3) were required. Complete inhibition was observed only at ratios of histone-DNA of 2:1 and higher. Histone stabilizes the helical molecule of DNA towards heat denaturation. Melting temperatures of reconstituted nucleohistones were determined. Most resistant to the heat denaturation were nucleohistones with a high content of lysine whereas the arginine-rich nucleohistones showed melting temperatures close to those of the DNA.

Escherichia coli 15T− (555-7) was irradiated with ultraviolet light and X-rays and the DNA examined for quantitative and qualitative changes in the methylation pattern. DNA synthesized following ultraviolet irradiation was under-methylated by approximately 25% as compared to DNA from unirradiated cells. The ratio of 6-methyladenine to 5-methylcytosine was unchanged. DNA synthesized following X-irradiation was methylated about 15% more than normal with a slight increase in the 6-methyladenine to 5-methylcytosine ratio. No additional methylation or turnover of methyl groups could be detected in parental DNA present at the time of ultraviolet or X-irradiation. E.coli 15T− (555-7) synthesized “unmethylated DNA” by growth in the absence of methionine. Ultraviolet irradiation of cells containing unmethylated DNA did not quantitatively change the ability of the cells subsequently to methylate this DNA when methionine was added to the culture. The ratio of 6-methyladenine to 5-methylcytosine in both control and ultraviolet-irradiated cells changed from 1.8 ± 0.2 when examined 15 minutes after addition of methionine to 1.4 ± 0.1, the normal value, when examined one hour after methionine was added.

Billen, Daniel (University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Tex.), and Roger Hewitt . Influence of starvation for methionine and other amino acids on subsequent bacterial deoxyribonucleic acid replication. J. Bacteriol. 92: 609–617. 1966.—A study has been made of the subsequent replicative fate of deoxyribonucleic acid (DNA) synthesized during amino acid starvation by several multiauxotrophic strains of Escherichia coli . Using radioisotopic and density labels and a procedure whereby total cellular DNA is analyzed, we have confirmed and extended a recent report that the DNA made during amino acid starvation behaves anomalously during subsequent DNA replication. When 5-bromouracil (BU) serves as the density lable, 40% or more of the DNA synthesized during starvation will subsequently fail to replicate during three cell generations. Selective amino acid effects were noted. In two methionine-requiring bacteria, methionine deprivation appeared to be of singular importance in influencing the subsequent replicative fate of the DNA made in its absence. When a non-BU density label (N 15 , C 13 ) was utilized, the effects of amino acid starvation were less obvious. Although the DNA synthesized during complete amino acid starvation in a methionine-requiring E. coli was subsequently more slowly replicated, most of the DNA was finally duplicated during three generations of growth. If methionine was present during starvation for other required amino acids, the subsequent replication rate of the DNA synthesized during this time was more nearly normal, and complete replication was observed. The results have been interpreted as indicating that DNA synthesized during amino acid starvation, and especially during methionine starvation, is somehow altered, and that BU substitution for thymine may interfere with the restoration of such DNA to its replicative state.

Freezing of Bacillus subtilis in liquid nitrogen results, upon thawing of the cells, in an enhanced deoxyribonucleoside triphosphate and reduced thymidine (Tdr) incorporation into cellular deoxyribonucleic acid (DNA). The DNA synthesized from thymidine triphosphate (TTP) was made by a “repair”-type system as determined by density transfer experiments. The mono- and diphosphate precursors were also incorporated by a “repair”-type synthesis. When Tdr was used as the radioactive precursor in the assay mixture, the product was only that expected from a semiconservative synthesis. Superlethal ultraviolet light exposure of the freeze-treated cells stimulated incorporation of phosphorylated precursors into DNA. Tdr uptake was greatly reduced by ultraviolet exposure, and only repair synthesis was observed. TTP and Tdr do not compete with one another in this system. The possibility that two DNA synthesizing systems exist in separate, non-mixing cellular compartments is considered.

1. 1. The role of transcription in the initiation of DNA synthesis and elongation of the DNA chain in Escherichia coli and the bacteriophage ΦX174 has been studied. 2. 2. Concomitant transcription does not appear to be required for semiconservative DNA replication in bacteria, or for conversion of single-stranded ΦX174 DNA to the double-stranded replicative form. 3. 3. The data substantiate the requirement in bacteria for messenger RNA(s) coding for protein(s) involved in the initiation of a round of DNA replication.

Several thymineless Escherichia coli strains have been examined for their ability to replicate their deoxyribonucleic acid when bromouracil is substituted for thymine. The procedure we describe was used to identify a thymineless strain with characteristics relatively favorable to its use in bromouracil labeling experiments. In addition, mutants with an “absolute” thymine requirement could be easily distinguished from one with a “leaky” thymine requirement.

Prestarvation of Escherichia coli for required amino acids results in a marked enhancement in both ultraviolet light (UV) or X-ray resistance for selective strains. Preventing protein synthesis by starvation for required amino acids results in completion of the cycle of chromosomal replication then underway. We have investigated the relationship between starvation-induced resistance enhancement (SIRE) and the excision-repair (Hcr) system in several E. coli strains including E. coli B/r hcr + and its isogenic mutant E. coli B/r hcr − . The following observations were made. (i) The Hcr system is the major component of SIRE in UV-irradiated strain B/r. By using the Hcr + strain, SIRE increases the 10% survival dose from ∼400 ergs to ∼1,200 ergs/mm 2 . With the Hcr cells, the increase is from ∼45 ergs to 60 ergs/mm 2 . (ii) Although prestarvation leads to a moderate enhancement of resistance to X irradiation, this effect is not dependent on the Hcr system. (iii) The double mutant, E. coli B s–1 ( hcr − exr − ) is completely unable to express SIRE whether studied with UV or X irradiation. It is concluded that the Hcr system is the major system responsible for SIRE in UV-treated cells, whereas Exr (resistance to X rays) may be involved to a minor extent. The Exr character appears to be required for SIRE expression in X-ray exposed cells.

The repair of ultraviolet (UV) damage in Bacillus subtilis W23T − has been studied by transformation with deoxyribonucleic acid (DNA) extracted from irradiated cells before and after repair. The extent of repair of genetic markers by donor cells after low or moderate doses of UV was found to be related only to the initial degree of inactivation. After a very high dose, further inactivation occurred, also in proportion to initial damage. In addition, the competent recipient cells were shown to repair approximately 75% of the damage in transforming DNA. The sensitivities of markers irradiated either in vivo or in vitro appeared to be related to map position, the more proximal markers showing a greater resistance to UV inactivation.

The number of survivors of E. coli strains B/r or 15T- following X-ray exposure was increased if the log phase cells had been previously grown in the presence of chloramphenicol. By omission of thymine in the growth medium of E. coli strain 15T- the chloramphenicol effect failed to materialize. Under these conditions net DNA synthesis was prevented while RNA continued to be accumulated. Since protein synthesis was inhibited by chloramphenicol under all conditions employed it would appear that the “surplus” DNA was responsible for the enhanced survival of cells so treated.