Single-strand Breaks In Deoxyribonucleic Acid Research Articles

Effect of pivaloyl-substituted pyrrole containing heterocyclic compounds on DNA repair pathways in Ewing sarcoma cells

Aim. To examine deoxyribonucleic acid (DNA) damage repair and cell cycle regulatory mechanisms of Ewing sarcoma cells exposed to pivaloyl-substituted pyrrole containing heterocyclic compounds.
 Methods. The study was performed on A673 Ewing sarcoma cell line. The tumor cells were incubated for 48 h in the presence of pivaloyl-substituted pyrrole containing heterocyclic compounds (compounds №20 and №24). Western blot analysis was utilized to examine expression of the markers of DNA single-strand (phosphorylated forms of ATR and Chk1) and double-strand breaks (phosphorylated forms of H2AX, АТМ, DNA-PK, BRCA-1, Chk-2). Analysis of the cell cycle phases was performed by flow cytometry (BD FacsCanto, USA).
 Results. Pivaloyl-substituted pyrrole containing heterocyclic compounds substantially increased the expression of histone 2A phosphorylated on serine 138 (γ-H2AX) that indicates DNA damage (double-strand breaks). Under exposure to pivaloyl-substituted pyrrole containing heterocyclic compounds the studied cells increased expression of phosphorylated forms of ATM-kinase and BRCA-1. Also cell cycle disorders leading to substantial G2/M arrest and enhanced apoptosis of tumor cells were observed.
 Conclusion. Pivaloyl-substituted pyrrole containing heterocyclic compounds induced DNA double-strand breaks in A673 Ewing sarcoma cell line; in response to DNA damage in tumor cells, the mechanisms of DNA double-strand breaks repair were activated; despite activation of DNA repair mechanisms, A673 cells underwent cell cycle arrest in the G2/M-phase and apoptosis.

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro

Background aimsAdipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. MethodsAfter isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. ResultsDuring in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. ConclusionsThe study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell–based therapy.

Base Excision Repair Facilitates a Functional Relationship Between Guanine Oxidation and Histone Demethylation

Appropriately controlled epigenetic regulation is critical for the normal development and health of an organism. Misregulation of epigenetic control via deoxyribonucleic acid (DNA) methylation or histone methylation has been associated with cancer and chromosomal instability syndromes. The main function of the proteins in the base excision repair (BER) pathway is to repair DNA single-strand breaks and deamination, oxidation, and alkylation-induced DNA base damage that may result from chemotherapy, environmental exposure, or byproducts of cellular metabolism. Recent studies have suggested that one or more BER proteins may also participate in epigenetic regulation to facilitate gene expression modulation via alteration of the state of DNA methylation or via a reaction coupled to histone modification. BER proteins have also been reported to play an essential role in pluripotent stem cell reprogramming. One emerging function for BER in epigenetic regulation is the repair of base lesions induced by hydrogen peroxide as a byproduct of lysine-specific demethylase 1 (LSD1) enzymatic activity (LSD1/LSD2-coupled BER) for transcriptional regulation. To shed light on this novel role of BER, this review focuses on the repair of oxidative lesions in nuclear DNA that are induced during LSD1-mediated histone demethylation. Further, we highlight current studies suggesting a role for BER proteins in transcriptional regulation of gene expression via BER-coupled active DNA demethylation in mammalian cells. Such efforts to address the role of BER proteins in epigenetic regulation could broaden cancer therapeutic strategies to include epigenetic modifiers combined with BER inhibitors.

Single-strand DNA breaks in human hair root cells exposed to mobile phone radiation

Purpose: To analyze the short-term effects of radiofrequency radiation (RFR) exposure on genomic deoxyribonucleic acid (DNA) of human hair root cells.Subjects and methods: Hair samples were collected from eight healthy human subjects immediately before and after using a 900-MHz GSM (Global System for Mobile Communications) mobile phone for 15 and 30 min. Single-strand DNA breaks of hair root cells from the samples were determined using the ‘comet assay’.Results: The data showed that talking on a mobile phone for 15 or 30 min significantly increased (p < 0.05) single-strand DNA breaks in cells of hair roots close to the phone. Comparing the 15-min and 30-min data using the paired t-test also showed that significantly more damages resulted after 30 min than after 15 min of phone use.Conclusions: A short-term exposure (15 and 30 min) to RFR (900-MHz) from a mobile phone caused a significant increase in DNA single-strand breaks in human hair root cells located around the ear which is used for the phone calls.

Genotoxic effects of low 2.45 GHz microwave radiation exposures on Sprague Dawley rats

This paper investigates the genotoxic effects of 2.45 GHz microwave (MW) radiation exposure at low specific absorption rates (SAR). 200 Sprague Dawley rats were exposed to SAR values between 0.48 and 4.30 W.kg-1 and the DNA of different tissues extracted, precipitated and quantified. Induced deoxyribonucleic acid (DNA) damages were assessed using the methods of DNA Direct Amplification of Length Polymorphisms (DALP) and the Single Cell Gel Electrophoresis (SCGE). Densitometric gel analysis demonstrated distinctly altered band patterns within the range of 40 and 120 bp in exposed samples and in the tail DNA of the same animals before exposure compared with control. Results were re-affirmed with SCGE (comet assay) for the same cells. Different tissues had different sensitivities to exposures with the brains having the highest. DNA damages were sex-independent. There was statistically significant difference in the Olive moment and % DNA in the tail of the exposed tissues compared with control (p < 0.05). Observed effects were attributed to magnetic field interactions and production of reactive oxygen species. We conclude that low SAR 2.45 GHz MW radiation exposures can induce DNA single strand breaks and the direct genome analysis of DNA of various tissues demonstrated potential for genotoxicity. Key words: Microwave radiation, single strand break, genotoxicity.

Monitoring the (photo)genotoxicity of photosensitizer drugs: Direct quantitation of single-strand breaks in deoxyribonucleic acid using an oligonucleotide chip

Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (⩾93.6 kJ/m 2) chlorpromazine (⩾0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04–0.20 mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.

Calculation of DNA strand breakage by neutralisation effect after 125I decays in a synthetic oligodeoxynucleotide using charge transfer theory

The decay of the radioisotope (125)I into (125)Te is typically followed by the emission of two groups of approximately 10 electrons each via Auger processes. In deoxyribonucleic acid (DNA) with (125)I incorporated, these electrons produce various types of damage to DNA, e.g. single strand breaks (SSBs) and double strand breaks (DSBs) through direct actions of physical tracks, or indirect actions of radicals produced in water. Among the direct actions one should consider not only the excitation and ionisation of DNA by Auger electrons, but also the neutralisation of highly charged (125 m)Te ions with electrons from neighbouring molecules. Comparison between experiment and simulation done recently revealed that without including neutralisation effect the simulated yield of SSBs was 50% less than the measured result. In the present work a calculation of DNA strand breakage by the neutralization effect in a 41-mer synthetic oligodeoxynucleotide (oligoDNA) model was done using the charge transfer theory. Calculation based on transfer rate using the newly evaluated electronic coupling of DNA bases showed that the positive charge (hole) transfer rate is of the order of magnitudes of several 10(13) s(-1), implying that a charge higher than 10 units might not build on a (125 m)Te atom. The potential energy accumulated on the decay base is transferred to bases along the DNA chain nearby and destroys those bases and ionises the sugar-phosphate group, leading a DNA SSB with a frequency of 0.2% per eV in average.

Basal colon crypt cells are more sensitive than surface cells toward hydrogen peroxide, a factor of oxidative stress

Products of oxidative stress are possibly important risk factors for colon cancer. It is necessary to assess their toxicity in tumour target cells, which include the stem cells and dividing daughter cells located in the bottom of the colon crypts. Here, we investigated the sensitivity of crypt cells towards hydrogen peroxide (H 2O 2), a key genotoxin associated with oxidative stress. Primary rat colonocytes, were isolated from different regions of the crypts by fractionated digestion. Differentiation was determined by measuring the alkaline phosphatase activity. Deoxyribonucleic acid (DNA) damage and oxidised DNA bases were determined using the modified version Comet assay with endonuclease III. Major findings were that rat colonocytes had high levels of endogenous DNA single strand breaks, with no significant difference from basal crypt cells to surface cells. However, cells of the basal crypt had more oxidised DNA pyrimidines, which were probably a reflection of preceding in vivo exposure. An in vitro treatment with H 2O 2 significantly increased DNA strand breaks in all fractions of rat colonocytes, but again cells of the basal crypt were more sensitive than cells of the surface epithelium. We conclude that cells from lower crypt sections are more sensitive towards H 2O 2 than differentiated cells at the surface of the crypt.

Deoxyribonucleic acid damage in human lymphocytes after percutaneous transluminal coronary angioplasty

ObjectivesWe investigated the presence of oxidative deoxyribonucleic acid (DNA) damage in the peripheral lymphocytes of patients undergoing percutaneous transluminal coronary angioplasty (PTCA) by using the micronucleus test and comet assay, which are sensitive biomarkers of DNA damage. BackgroundAlthough it has recognized that ischemia-reperfusion can induce oxidative DNA damage, its occurrence in patients undergoing PTCA has not yet been demonstrated. MethodsThree groups of patients were enrolled: 30 patients with documented coronary heart disease who underwent elective PTCA (group I); 25 patients who underwent elective coronary angiography for diagnostic purpose (group II); and 27 healthy, age- and gender-matched subjects (group III). For each subject, the frequency of micronucleated binucleated (MNBN) cells, DNA single-strand breaks (SSBs), endonuclease III-sensitive sites, and sites sensitive to formamidopyrimidine glycosylase (FPG) were analyzed before and after diagnostic procedures. ResultsThe mean basal values of MNBN cells (p = 0.04), DNA-SSBs (p = 0.001), endonuclease III-sensitive sites (p = 0.002), and FPG sites (p < 0.0001) were significantly higher in groups I and II than in group III. A high significant increase of MNBN cell frequency was observed in group I after the PTCA procedure (11.0 ± 1.3 vs. 19.8 ± 1.6, p < 0.0001), whereas no significant difference was observed in group II (10.2 ± 1.3 vs. 12.9 ± 1.4, p = 0.18). A significant positive correlation was observed between the increase in the MNBN cell rate and total inflation time during PTCA (R = 0.549, p = 0.0017). The levels of DNA-SSBs (11.7 ± 1.4 vs. 26.5 ± 3.0, p = 0.0003) and FPG sites (13.8 ± 1.8 vs. 22.5 ± 2.4, p = 0.01) were also higher after PTCA. ConclusionsOur results provide evidence for oxidative DNA damage after PTCA, likely related to ischemia-reperfusion injury.

Influence of genetic polymorphisms of biotransformation enzymes on gene mutations, strand breaks of deoxyribonucleic acid, and micronuclei in mononuclear blood cells and urinary 8-hydroxydeoxyguanosine in potroom workers exposed to polyaromatic hydrocarbo

Airborne exposure to polycyclic aromatic hydrocarbons (PAH) in the potroom of an aluminum reduction plant was studied in relation to genotoxic or mutagenic effects, and the possibility of host genotypes of different metabolizing enzymes modifying associations between PAH exposure and genotoxic or mutagenic response was assessed. Ninety-eight male potroom workers and 55 male unexposed blue-collar workers constituted the study population. Micronuclei in CD4+ and CD8+ lymphocytes, DNA (deoxyribonucleic acid) single-strand breaks, hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation frequency, and genotype for cytochrome P-4501A1, glutathione transferases M1, T1 and P1, and microsomal epoxide hydrolase were analyzed using peripheral mononuclear cells. Urine samples were collected for the analysis of 8-hydroxydeoxyguanosine. Micronuclei in peripheral CD4+ and CD8+ lymphocytes, DNA single-strand breaks, HPRT mutation frequency, and 8-hydroxydeoxyguanosine in urine did not differ between the potroom workers and the unexposed referents. With the exception of an observed exposure-response relationship for potroom workers with Tyr/Tyr genotype for microsomal epoxide hydrolase, between airborne PAH and CD8+ micronuclei, no correlations were found between any of the genotoxicity biomarkers and any of the exposure measures (airborne particulate PAH, airborne gas phase PAH, length of employment in the potroom, 1-hydroxypyrene in urine, or PAH-DNA adducts in peripheral lymphocytes), also when genotypes for biotransforamtion enzymes were considered. The results indicate that the employed biomarkers of mutagenic or genotoxic effects are not appropriate for surveillance studies of potroom workers exposed to current airborne levels of PAH. The significance of the correlation between airborne PAH and CD8+ micronuclei in Tyr/Tyr genotype subjects should be evaluated.

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.

Single-strand breaks in deoxyribonucleic acid in fire fighters accidentally exposed to o-nitroanisole and other chemicals.

The aim of the study was to detect single-strand breaks in deoxyribonucleic acid (DNA) in mononuclear blood cells of fire fighters exposed to o-nitroanisole and other substances released into the environment during an accident in a chemical plant. The level of DNA single-strand breaks in mononuclear blood cells was detected by alkaline elution. The results were compared for 16 fire fighters who worked in a contaminated area for about 8 h and two reference groups (one of fire fighters who had not worked in the contaminated area, group I, and one of persons without any apparent occupational exposure to genotoxic substances, group II). The mean normalized elution rate (nER) 19 d after the accident was slightly but statistically significantly (P < 0.05) higher for the exposed fire fighters [mean 1.48 +/- 95% confidence interval (95% CI) 0.21] than for reference group I (mean 1.21 +/- 95% CI 0.21) or reference group II (mean 1.17 +/- 95% CI 0.18). No statistically significant difference was found between reference groups I and II. Another analysis was performed three months after the first. The level of DNA single-strand breaks (mean nER 1.12 +/- 95% CI 0.11) was no longer increased in comparison with the levels of the reference groups. DNA single-strand breaks were increased in fire fighters exposed to o-nitroanisole and other substances. In comparison with the extent of DNA strand breaks found in other occupational groups the increase was only moderate. The observed decrease in DNA single-strand breaks to the reference level in exposed fire fighters three months later suggests a DNA repair mechanism for DNA single-strand breaks caused by o-nitroanisole.

Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: incision of ultraviolet-irradiated deoxyribonucleic acid in vivo.

A group of genetically related ultraviolet (UV)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD(+) strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.

Influence of chromosome integrity on Escherichia coli cell division.

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.

Repair of x-ray-induced deoxyribonucleic acid single-strand breaks in xth mutants of Escherichia coli.

An exonuclease III-deficient strain of Escherichia coli K-12, BW2001 (xthA11), was unable to perform rapid repair of X-ray-induced deoxyribonucleic acid single-strand breaks and appeared to have a defect in the priming of the 3'-termini necessary for initiation of repair synthesis at the breaks. This defect cannot be explained solely by the lack of exonuclease III activity, because other xth mutants tested, including a deletion mutant, repaired radiation-induced strand breaks at close to the normal rate.

Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.

Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli

Deoxyribonucleic acid (DNA) from bacteriophage T7 has been used to monitor the capacity of gently lysed extracts of Escherichia coli to perform repair resynthesis after ultraviolet (UV) irradiation. Purified DNA damaged by up to 100 J of UV radiation per m2 was treated with an endonuclease from Micrococcus luteus that introduces single-strand breaks in irradiated DNA. This DNA was then used as a substrate to study repair resynthesis by extracts of E. coli. It was found that incubation with the extract and exogenous nucleoside triphosphates under suitable assay conditions resulted in removal of all pyrimidine dimers and restoration of the substrate DNA to its original molecular weight. Repair resynthesis, detected as nonconservative, UV-stimulated DNA synthesis, was directly proportional tothe number of pyrimidine dimers introduced by radiation. The repair mode described here appears to require DNA polymerase I since it does no occur at the restrictive temperature in polA12 mutants, which contain a thermolabile polymerase. The addition of purified DNA polymerase I to extracts made from a polA mutant restores the ability to complete repair at the restrictive temperature.

Role of deoxyribonucleic acid polymerase III in the repair of single-strand breaks produced in Escherichia coli deoxyribonucleic acid by gamma radiation.

Cell survival, deoxyribonucleic acid (DNA) degradation, and the repair of DNA single-strand breaks were measured for Escherichia coli K-12 pol+, polA1, polC1026(ts), and polA1 polC1026(ts) cells after 137Cs gamma irradiation. The results indicate that DNA polymerase III is required for growth medium-dependent (type III) repair in polA+ or polA cells. In pol+ or polC cells, DNA polymerase I performs type II repair efficiently. The relative deficiencies of each of these strains in DNA repair generally correlate with their relative sensitivities to cell killing and with the extent of DNA degradation observed.

Mutation induction in bacteria by freeze-drying

The stability of the deoxyribonucleic acid (DNA) double helix to freezing and thawing both in vitro and in vivo has been well documented. Shikama (13), using thermal denaturation techniques, showed that purified DNA solutions were not affected by freeze-thaw procedures. Ashwood-Smith ( 1) presented evidence of the genetic stability of bacteria frozen and thawed under several different conditions. No mutation induction in Escherichiu coli was evident and no change in a wide spectrum of antibiotic resistance could be demonstrated after a long series of freeze-thaw selection procedures. Swartz (14), however, has published evidence that freezing and thawing ‘of E. coli produced single-strand breaks in DNA in vivo, but this has not been confirmed by other workers ( 1). The best evidence at this time, therefore, is that freezethaw procedures, per se, do not produce genetic damage, and this statement is substantiated by evidence presented in this paper. Genetic changes in bacteria induced by freeze-drying have not been reported, although Webb (16) has published data on the genetic effects of exposing E. co& in an aerosol to different degrees of humidity. The highest number of mutants was observed when the relative humidity of the aerosol was approximately 40%. Bridges (personal communication) has indicated that stocks of freeze-dried bacterial preparations have higher background reversion

Effects of chloramine on Bacillus subtilis deoxyribonucleic acid.

The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity.