Inactivation of transforming DNA by ultraviolet light. 3. Further observations on the effects of 365 nm radiation

In the doubly auxotrophic strain ad 3A 38701 inos 37401, nitrosoethylurethane (NEU) produces a storage effect for adenine reversions but not for inositol reversions. Shaking treated spores in water for several hours destroys their response to storage. Short heat-treatment during or before plating increases the frequency of adenine reversions but not that of inositol reversions. Storage and heat-treatment decrease the inositol-specificity of NEU and may reverse it into adenine-specificity. Comparison with similar results obtained for diepoxybutane (DEB) shows that the cellular effects of NEU are more complex than those of DEB.     

The stabilisation of a transient mutagen-sensitive state in Neurospora by the protein synthesis inhibitor actidione

Summary Experimental evidence has been obtained to show that a transient mutagen sensitive state, believed to be induced in Neurospora by DEB, can be stabilised by the protein synthesis inhibitor actidione. Sensitisation can thus be separated from the complicating effects of traces of the DEB retained by the cells following washing. The bearing of these results on the interpretation of the DEB after-effect and DEB mutation induction curves is briefly discussed.Research supported by the Medical Research Council.     

The influence of temperature on the ultraviolet induced revertant frequencies of two auxotrophs ofNeurospora crassa

Summary The frequency of ultraviolet-induced adenine reversions relative to inositol reversions in the strainK3/17 ad-3A 38701: inos 37401 is influenced by temperature. Fewer adenine revertants are obtained from a given ultraviolet dose adminstered at 30°C than from the same dose administered at 2°C. The inositol reversion frequency and the percentage survival are not greatly affected by these differences in temperature. Temperature differences are only effective if a) applied during irradiation b) higher temperatures above 15°C are used. Possible explanations for these results are considered.With 2 Figures in the Text     

A case of mutagen specificity attributable to a plating medium effect

Summary In anadn ^– met ^– di-auxotrophic strain ofSchizosaccharomyces pombe met ⁺ reversions are several hundred times more frequent thanadn ⁺ reversions after treatment with ultra-violet light. They are only slightly more frequent thanadn ⁺ reversions when HNO₂ is used as a mutagen (mutagen specificity). The poor response of theadn-1,199 allele to the mutagenic action of U.V. can be largely overcome by replacing themet-4,D19 allele with its normalmet ⁺ allele (influence of the genetic background). It was shown that both the mutagen specificity and its dependence on the genetic background are due, largely at least, to the inhibition ofadn ⁺ reversions on a plating medium containingl-methionine. This inhibition is very strong for U.V.-induced reversions but only weak for HNO₂-induced ones. It would be wrong to assume that other mutants at theadenine-1 locus behave in the same manner.With 1 Figure in the Text     

Spontaneous mutability in yeast. I. Stability of lysine reversion rates to variation of adenine concentration

Novick and Szilard demonstrated that increasing the concentrations of adenine enhance the mutation rate of E. coli. We have found that the spontaneous mutation rate of the yeast Saccharomyces cerevisiae remains constant over a 200-fold range of adenine concentration.The system that we commonly use for measuring spontaneous mutation rate is reversion of the super-suppressible mutant lys1-1. In this system, growth of the yeast is limited by limiting the amount of lysine in the medium. A reversion to lysine independence will continue to grow. One of the other super-suppressible mutants in the test system is ade2-1, a mutant that causes accumulation of red pigment. By adjusting the concentration of adenine slightly above that of lysine, reversions of super-suppressors produce white colonies and reversions of the lys1-1 locus itself produce red colonies.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

Recovery of Clostridia on Catalase-Treated Plating Media

Four plating media commonly used for culturing clostridia were tested for their ability to support growth of several Clostridium species after storage of the plates for 1 to 10 days at 4 and 25°C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar rapidly became incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens agar base and Brewer anaerobic agar were less affected. Plate counts of vegetative cells of nine of the less fastidious Clostridium species on untreated LV and BHI agars, stored for 3 days at 4°C, were 60 to 90% lower than counts on catalase-treated media. Counts on Shahidi Ferguson perfringens agar base were only 1 to 24% lower on untreated medium with the same species. Addition of 500 U of purified beef liver catalase to the surface of the 3-day-old agars before inoculation resulted in substantial restoration of the ability of the media to support colony formation from vegetative cells except with the most strictly anaerobic species (nonproteolytic C. botulinum types B, E, and F, and C. novyii types A and B). A similar response was obtained with spores of the less fastidious species on catalase-treated media. Our results suggest that inhibition of most Clostridium species on LV and BHI agars may be due to accumulation of peroxide during preparation, storage, and incubation of the media, and also suggest that the presence of glucose in these media is a major factor contributing to their inability to support growth. It is believed that the addition of exogenous catalase prevents the accumulation of peroxide(s), thus allowing colony formation from vegetative cells of the clostridia under what would otherwise be unsuitable cultural conditions.     

Physiological modification of alkylating agent induced mutagenesis. II. Influence of the numbers of chromosome replicating forks and gene copies on the frequency of mutations induced in Escherichia coli.

The frequency of reversions induced in Escherichia coli K-12 trpA58 by any of five different monofunctional alkylating agents increased as the growth rate of the organism was raised prior to mutagen treatment. The increase in mutation frequency did not correlate with growth rate-dependent changes in cell area or total cellular protein and DNA. After treatment of cells with N-methyl-N-nitrosourea (MNUA), no growth rate-dependent change was observed in the total DNA alkylation or percentage of O6-methylguanine present in the DNA extracted. The frequency of reversions induced by one mutagen, methyl methanesulphonate (MMS), increased in proportion to the average number of trpA gene copies per cell, whereas the frequency of reversions induced by the other compounds was dependent on the average number of chromosome replicating forks per cell. This difference was attributed to the different ratios of DNA base alkylation products observed, formed after treatment with MMS, an SN2-type reagent, or after treatment with the SN1-type reagents ethyl methanesulphonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNUA and N-ethyl-N-nitrosourea (ENUA). Possible reasons for the dependence of mutation frequency on the number of replicating forks per cell are discussed.     

Genetics of somatic mammalian cells. XIV. Genetic analysis in vitro of auxotrophic mutants

Genetic analysis has been carried out on auxotrophic mutants produced by treatment of Chinese hamster ovary and the Chinese hamster lung cells with mutagenic agents in vitro. Thirty-six different mutants were subjected to complementation analysis and biochemical tests. The different mutations studied result in growth requirements for proline; glycine; glycine or folinic acid; adenine or several of its precursors; inositol; adenine plus thymidine; and glycine plus adenine plus thymidine. The mutants which require glycine fall into four different complementation classes while those requiring adenine or hypoxanthine form two different complementation classes. The biochemical blocks of the latter two classes both occur somewhere in the steps involved in conversion of 5-phosphoribosyl-1-pyrophosphate to 5-aminoimidazole 4-carboxylic acid ribonucleotide. The auxotrophic mutants described exhibit all-or-none responses to their specific nutrilite supplements and are stable with respect to reversion. They involve alterations in ten different genes, and hence form a useful set of mutants for a variety of genetic studies. All the auxotrophies produced in a single exposure to a mutagen are due to single gene mutations, even when multiple nutritional requirements were produced. All the mutations studied are recessive.     

Quantitative Measurement of the Ability of Different Mutagens to Induce an Inherited Change in Phenotype to Allow Maltose Utilization in Suspension Cultures of the Soybean, GLYCINE MAX (L.) Merr

Using a newly developed plating system, we have measured cell survival and the frequencies of variation in an inherited trait after treatment of soybean cell suspensions with different mutagens: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG), hycanthone (1-{[2-(diethylamino) ethyl] amino}-4-(hydroxymethyl)-9H-thioxanthen-9-one and ultraviolet light (UV).—The heritable variation selected for displays a phenotype of rapid growth on maltose as carbon source. The marker is stable in the absence of maltose, and prolonged growth of variant cells on sucrose has not shown reversions to slow growth. Doubling time in suspension cultures is decreased from 100 hr to ca. 30 hr by the mutation. Both wild-type and variant cells grow on sucrose with a 24-hr doubling time. Thus, lethality after mutagen treatment can be estimated rapidly by growth on sucrose, whereas variants are scored on maltose medium. The spontaneous frequency of variants was 1.2 x 10^-7; induced frequencies ranged from a low of 3.6 x 10^-5 for EMS to a high of 10^-3 for hycanthone. The high frequency of variants induced by hycanthone, a frame-shift mutagen, and the observation that UV induces variants in haploid cells with much higher frequency than in diploid cells suggests a recessive mutation.     

An investigation into the mutagenic after-effect of butadiene diepoxide usingNeurospora crassa

Summary Butadiene diepoxide (DEB) was used to induce reverse mutations in anad-3A mutant ofNeurospora crassa. It was found that the mutagenic action of DEB continued when the twice washed and resuspended conidia were left standing in water at room temperature. The after-effect can be prevented if the conidia are kept stirred by shaking or gaseous bubbling for 4–5 hours. The after-effect does not develop if the conidia are plated immediately after washing, or if they are kept at 0–4°C until plating. The experimental data indicate that the after-effect is caused by traces of DEB, or by mutagenic reaction products of DEB, which are not readily removed by the ordinary washing procedure, but which may be removed by diffusion from the cells when these are kept suspended for a prolonged period.With 3 Figures in the Text     

An integrated culture and real-time PCR method to assess viability of disinfectant treated Bacillus spores using robotics and the MPN quantification method

Using robotics and the MPN technique, a 96-microwell method was developed to compare two procedures for enumeration of viable chlorine-treated B. atrophaeus spores: broth-culture enrichment followed by real-time polymerase chain reaction analysis; and filter plating on agar. Recoveries of chlorine-treated spores were improved by broth enrichment over filter plating, whereas recoveries of non-treated spores were not different in the two procedures.     

Recovery of Viability and Radiation Resistance by Heat-Injured Conidia of Penicillium expansum Lk. ex Thom

Spores heated in water at 54 C for up to 1 hr were plated on nutrient agar immediately or held for 3 days in aerated water at 23 C and then plated. Under these conditions, holding was optimal for recovery, increasing survival percentage up to 20-fold over values for immediate plating. Recovery was prevented partially or completely, however, when spores were held in any of the following solutions: glucose, potassium phosphate, ammonium or sodium acetate, sodium azide, or 2,4-dinitrophenol, or in the sodium or potassium salts of pyruvate, and tricarboxylic acid cycle acids. Both anaerobiosis and incubation at 0 C prevented recovery. Survivors of a heat treatment were more sensitive to gamma radiation than were unheated spores. Conditions which affected the recovery of viability had the same effect on restoration of radiation resistance. Thus, many of the processes for restoration of radiation resistance seem involved also in recovery of viability after heating. After a 99% inactivating treatment (about 30 min at 54 C), heated spores respired as fast as unheated spores, or faster. Malate, citrate, succinate, and acetate stimulated respiration in unheated spores and inhibited it in heated spores.     

Analysis of a case of mutagen specificity in Neurospora crassa. IV. Interactions between UV and three chemical mutagens

Summary In the strain ad 3A 38701 inos 37401, UV usually produces about twice as many inositol-as adenine-reversions. We have shown previously that this inositol-specificity of UV can be reversed into adenine-specificity by pre- or post-treatment with low doses of the adenine-specific DEB. We now have tested two more adenine-specific mutagens, nitrous acid (NA) and a mixture of hydrogen peroxide and formaldehyde (HF) and one inositolspecific mutagen nitrosoethylurethane (NEU) for interaction with UV. The former two substances behaved like DEB in reversing the inositol-specificity of UV when given as pre-or post-treatment. Pre-treatment with NEU always enhanced the frequency of inositol-reversions beyond additivity. At low doses, it also enhanced the frequency of adenine-reversions. The results are discussed in relation to mutagen specificity. They indicate that specificity may arise from the effects of treatment on cellular events in any one of the three major parts of the mutational pathway: repair, expression, growth of completed mutants into clones.     

Use of acridine yperite in the selection of Act. nodosus, a producer of amphotericin B]

The effect of acridine-yperite (AY) on Act. nodosus LIA 0861 at various physiological stages of development was studied. It was shown that both the spores and the germs were sensitive to AY. The level of the mutagen effect depended on the exposure time and the physiological state of the Act. nodosus cells during the treatment. The treatment of the spore suspension with AY had an insignificant effect on the strain morphological variation, while the number of the morphologically changed colonies markedly decreased when 5-hour germs were subjected to the mutagen effect. A decrease in the average antibiotic activity of Act. nodosus was observed, when its spores were subjected to the treatment with AY, which was associated with a decrease in the number of the plus variants. The treatment of 5-hour germs increased the average antibiotic activity of the culture which correlated with an increase in the number of the plus variants.     

Rates of forward and reverse mutation inDrosophila after exposure to mustard gas and X-rays

After treatment with mustard gas, reversions of the mutantforked ³ⁿ were observed with a frequency of 1 in 7,500. In the absence of indications for either suppressors or chromosome-rearrangements, these data provide evidence that a chemical mutagen can produce back mutations inDrosophila. Half the number of reversions was characterized by mosaic manifestation. This shows that delayed appearance after chemical treatment also holds for true gene mutations. One partial reversion to nearly normal type was not due to back mutation, but to a rearrangement, presumably involving a duplication of theforked containing region.The study on reversion off ³ⁿ was combined with tests for recessive visibles at 15 selected loci of the X-chromosome. Mutations at theruby locus were most frequently induced by mustard gas (1 in 1,700). About one quarter of the forward mutations were fractionals. After exposure to 5,000 r X-irradiation both reversions off ³ⁿ and forward mutations at the loci under study were observed with frequencies comparable to those induced by mustard gas. Thus, no indication for mutagenspecific differences in mutational response have been obtained. After treatment with mustard gas a higher ratio of visibles to lethals was observed than after exposure to X-irradiation. It is pointed out that comparisons of the mutagenic effect of a chemical mutagen with that of X-radiation, even if restricted to visible mutations, inevitably involve an underestimate for the chemical, due to delayed effects of the latter.The experiments were carried out mainly at the Department of Genetics, State University of Utrecht (Director: Prof. Dr.C. L. Rümke). A. J. M. van Hedel, G. J. O. Jansen, V. Labordus andS. C. M. Schouten collaborated on parts of this project.     

Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coli. Among other nitroso compounds tested only N-methyl-N′-nitro-N-nitrosoguanidine (MNG) was an active mutagen in the system used.     

Sodium azide-induced mutagenesis in Saccharomyces cerevisiae.

Sodium azide (0.5--2.0 X 10(-5) M), applied for 24 h on cells growing in complete medium, increased up to 26 times the frequency of reversions and locus-specific suppressor mutations of allele ilv1-92 in diploid strain D7 of Saccharomyces cerevisiae. Similarly, it enhanced the frequency of reversions and/or mitotic gene conversions of alleles trp5-12/trp5-27 up to 19 times. Reconstruction experiments showed that the increase of mutations in complete medium was not due to a selection of prototrophic types under growth conditions and, therefore, that sodium azide acts as a weak mutagen in S. cerevisiae under growth conditions at a low pH. No mutagenic or convertogenic effect was observed when azide was applied to resting cells in buffer at pH 4.2.     

Superoxide and hydrogen peroxide in oxygen damage.

Escherichia coli were damaged and killed by exposure to hyperbaric oxygen. Lethality was measured as the decrease in the number of colonies formed upon plating the exposed cells onto rich agar. Damage was assessed by plating onto both rich and minimal agar. Cells which gave rise to visible colonies on rich but not on minimal agar were considered to be damaged. That this differential colony count was largely due to reparable damage rather than to stable mutagenesis was shown by replica plating from the rich onto the minimal agar. Most of the cells which had been unable to grow when directly plated onto minimal agar regained this ability after growth upon rich agar. Repair of the damage imposed by exposure to oxygen was thus more readily accomplished on a nutritionally rich medium. The enzymes superoxide dismutase, catalase, and peroxidase appeared to protect against oxygen damage. It is thus likely that both O₂^? and H₂O₂ are important agents of oxygen toxicity. In accord with this conclusion were the observations that augmented intracellular levels of these enzymes correlated with increased resistance towards oxygen damage, whereas increased respiratory capacity correlated with increased sensitivity towards hyperbaric oxygen.     

Production,long term preservation and synchronous germination of aerial spores of Streptomyces

Vigorous vegetative growth of various Streptomyces species (S. auroefaciens, S. collinus and S. granaticolor) was achieved in a new semisynthetic liquid medium. Unlike the media commonly used for the cultivation of the submerged mycelia of different streptomycetes, this one does not contain insoluble material which enables direct and reliable measurement of net production of biomass. The medium was formulated to meet the nutritional requirements of all the three species. Is also supported production of antibiotic in each of the strains. A method for bulk preparation of Streptomyces aerial spores, involving cultivation on agar plates covered with cellophane, was developed. Advantage of this method lies in higher yields of spores, their higher purity and easier harvesting. The spores were activated by amild treatment with an Ultra-Turrax homogenizer resulting in the breakage of fibrous sheath, suspended in 20% glycerol, and stored at ?60°C. Thus, treated spores germinated synchronously even after several months of the storage. Hence, such spore material may be used for precise inoculation in a large series of experiments implying synchronous germination, and the inoculations can be carried out from the same batch over a long period.