Mutagenic action of N-nitrosodimethylurea on Actinomyces rimosus and Penicillium chrysogenum]

High mutagenic activity of N-nitrozodimethylurea (NDMU), an agent of the group of the nitrozo compounds not studied in detail was shown with respect to prototrophic and auxotrophic strains of Actinomyces rimosus, an organism producing oxytetracycline and Penicillium chrysogenum, an organism producing penicillin. The rate of direct and back mutations in the auxotrophic strain of Act. rimosus under the effect of NDMU was many times higher than that of spontaneous mutations. NDMU was used at one of the selection stages at which a more active variant of Act. rimosus was obtained. This is evident of a possible use of the mutagen for induction of variation with respect to the quantitative feature of oxytetracycline production. A great number of morphologically changed forms and biochemical mutants of Pen. chrysogenum formed under the effect of this substance. NDMU induced a mutant of Pen. chrysogenum capable of selective synthesis of 6-aminopenicillinic acid without addition of the precursor.     

Ratio of the mutation spectrum to the physiological state of the Penicillium chrysogenum cell. Auxotrophic mutations induced by N-nitroso-N-methylbiuret in exposure at different stages of DNA replication

The effect of UV light on activated conidia of Penicillium chrysogenum, strain 39 was studied. It was found that the spectrum of the auxotrophic mutations induced by UV light during replication of DNA changed with the dose of the mutagen and was specific to every dose. The schemes of predominating mutation induction during DNA replication under the effect of 2 doses of UV light were developed.     

Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.     

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.     

Mutagenesis in multinucleate cells: the effects of N-methyl-N'-nitro-N-nitrosoguanidine on Phycomyces spores

Multinucleate cells, such as the spores of the fungus Phycomyces, are unsuitable for the isolation of recessive mutants. Nuclear killing by N-methyl-N'-nitro-N-nitrosoguanidine (henceforth nitrosoguanidine) eliminates all but one of the nuclei in some of the cells and allows the expression of recessive mutations. Even in the best conditions, only about 35% of the survivors have a single functional nucleus. Functionally uninucleate cells can be positively selected. This involves the exposure to nitrosoguanidine of the spores of a heterokaryon and selection for a recessive marker present in a small fraction of its nuclei. The optimal conditions for nitrosoguanidine mutagenesis in Phycomyces differ from those for bacteria and yeast. Buffer composition and pH are less important than in other organisms. Survival is an exponential function and mutation induction a linear function of the dose of the mutagen (concentration X time). Spore germination leads to an immediate increase in the number of gene copies per cell, thus further hindering the expression of recessive mutations; dominant mutations are then nearly always isolated in heterokaryotic form.     

Dispersal of fungal spores from three types of air handling system duct material

Exposure to airborne microorganisms in indoor environments may result in infectious disease or elicit an allergic or irritant response. Air handling system components contaminated by fungi have been implicated in the dispersal of spores into the indoor environment, thereby serving as a route of exposure to occupants. This study was conducted to provide quantitative data on the dispersal of spores from fungal colonies growing on three types of duct material. Galvanized metal, rigid fibrous glass ductboard, and fiberglass duct liner were soiled and contaminated with a known concentration of Penicillium chrysogenum spores. The duct materials were incubated in humidity chambers to provide a matrix of growing, sporulating fungal colonies at a contamination level of 109 colony forming units (CFU) per duct section, consistent for all materials. For each experiment a contaminated duct section was inserted into the air handling system of an experimental room, and the air handling system was operated for three 5-minute cycles with an air flow of 4.2 m3 min–1. The duct air velocity was approximately 2.8 m sec–1. The airborne concentration of culturable P. chrysogenum spores (CFU m–3), total P. chrysogenum spores (spores m–3), and total P. chrysogenum-sized particles (particles m–3) were measured in the room using Andersen single-stage impactor samplers, Burkard slide impactor samplers, and an aerodynamic particle sizer, respectively. The highest airborne concentrations (104 CFU m–3; 105 spores m–3; 104 particles m–3) were measured during the first operating cycle of the air handling system for all duct materials with decreasing airborne concentrations measured during the second and third cycles. There was no significant difference in spore dispersal from the three contaminated duct materials. These data demonstrate the potential exposure for building occupants to high concentrations of spores dispersed from fungal colonies on air handling system duct materials during normal operation of the system.     

Differential mutation of transgenic and endogenous loci in vivo

Although chemicals usually induce very similar frequencies of mutations in transgenes and endogenous genes in vivo when given acutely, chronic exposure to N-ethyl-N-nitrosourea (ENU) produced a more complex pattern in which the endogenous locus was spared many mutations. Here, we demonstrate that the effect is neither ENU-specific nor locus-specific, and thus, may be important in the extrapolations of risk assessment and in understanding mutational mechanisms. During chronic mutagen exposure, mutations at the transgene accumulate linearly with time, i.e. in direct proportion to the dose received. In contrast, mutations at the endogenous gene are much less frequent than those of the transgene early in the exposure period and the accumulation is not linear with time, but rather accelerates as the exposure continues. Previous comparisons involved the endogenous Dlb-1 locus and the lacI transgene from the Big BlueMouse in the small intestine. These experiments involved the Dlb-1 locus and the lacZ transgene from the MutaMouse in the small intestine and the hprt locus and the lacZ transgene in splenocytes. Comparisons were made in both tissues after acute and chronic exposures to ENU, the original mutagen, and in the small intestine after exposures to benzo(a)pyrene. All comparisons showed that during chronic exposures mutations at the transgene accumulate linearly with the increasing duration of exposure, whereas induced mutations of the endogenous gene initially accumulate at a slower rate. Thus, the difference in mutational response observed during low chronic treatment is not unique to a particular transgene, endogenous gene, tissue, or mutagen used, but may be a general phenomenon of such genes.     

Solid-state and submerged fermentations show different gene expression profiles in cephalosporin C production by Acremonium chrysogenum

Despite the importance of Acremonium chrysogenum as the only cephalosporin C (CPC) producer, there is still a limited understanding about the molecular mechanisms regulating antibiotic biosynthesis in this fungus. Based on the previously described relationship between environmental pH and antibiotic production in numerous filamentous fungi, we studied the expression of genes related to CPC production in A. chrysogenum. We report for the first time similarities and differences, characterizing CPC production by A. chrysogenum under a variable pH environment, in submerged and solid-state fermentation. This characterization is supported by measurements of parameters, like CPC production, pH, growth, and expression levels of several genes involved, directly or indirectly, in CPC production. Interesting differences in intermediate (Pen N) and certain biosynthetic gene expression levels were observed. Our results point out some relationships between physiological features and gene expression that open important improvement perspectives for both culture systems.     

Segregation of Induced Frameshift Mutations and the Sequence of Gene Replication in ESCHERICHIA COLI K12

We have constructed a strain of E. coli K12 carrying six mutations induced by the acridine half-mustard ICR-191. The mutations are widely spaced on the E. coli linkage map and are all easily reverted by ICR-191. Mapping of ten independent revertants for each of five markers indicated that the reversions induced by ICR-191 occurred near the original mutations. Exponentially and nonsynchronously growing cultures of this strain were exposed to ICR-191 for 0.85 generation, quickly washed free of mutagen, and resuspended in the original medium minus mutagen. Total viable cell number maintained its exponential increase both during and immediately after exposure to mutagen, whereas the number of revertants of any particular type remained constant for a characteristic period after removal of mutagen before finally assuming an exponential increase. Theoretically, the length of such a segregation lag should depend on the position of the particular reverted gene in the sequence of gene replication: the earlier a gene is replicated in the chromosome replication cycle, the longer its segregation lag should be. Our results are consistent with this prediction and fit a unidirectional, clockwise replication scheme with an origin between 55 and 74 min on the E. coli linkage map. The results also fit a very asymmetric bidirectional replication scheme.     

Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.     

The characterization of color mutations in Bangiaceae (Bangiales, Rhodophyta)

The color mutations in Bangiaceae were investigated by treating the blades, conchocelis and conchospores phase of Bangia sp., Porphyra yezoensis, and P. haitanensis sampled in China with mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). A high percentage of mutation in different expression characteristics in all three phases were shown within optimum mutagen concentrations. Among mutagenized blades, mutations occurred on single cells, which is a direct outcome of mutation of haploid cells. The mutation of mutagenized conchocelis resulted in a two-step process: low-level expression in conchocelis phase, and high-level expression in progeny, explaining that mutation took place in diploid cells. The mutations of conchospores were expressed immediately at germination of spores, indicating a change in ploidy. This paper reports the process of meiosis and its effect on frond development, and the relation between color mutations and morphological characteristics expressed by mutations in Bangiaceae.     

Effect of nonmutagenic doses of 1,4-bis-diazoacetylbutane on Streptomyces griseus

Streptomyces griseus 15 was subjected to the action of 1,4-bis-diazoacetyl butane (DAB) taken at concentrations of 10 to 50,000 micrograms/ml. Small doses (10-100 micrograms/ml) of DAB had no mutagenic action and activated the cultural growth (the viability and the survival rate of spores increased on solid media, while the biomass yield rised in liquid media). Experiments were conducted using the method of orthogonal planning of a bifactorial experiment, and the role of the exposure time rised with a decrease in the mutagen concentration.     

Auxotrophic mutants of the producer of the antibiotic mycoheptin Streptoverticillium mycoheptinicum

The lethal and mutagenic effects of N-nitrozo-N-methyl biuret (NMB), N-nitrozo-N-methyl urea (NMU) and UV light on Streptoverticillium mycoheptinicum, strains O883 and 852, were studied. The concentrations of NMB were 0.005, 0.1 and 0.25 per cent, the exposure time was 2, 4 and 6 hours. The concentration of NMU was 1 per cent and the exposure time was 1, 2, 3, 4 and 5 hours. The dose of UV light was 2000 erg/mm2. When the spores of Streptoverticillium mycoheptinicum, strain O883, were treated with NMB, the frequency of auxotrophic mutants increased from 0.63 to 3.4 per cent with an increase of the mutagen concentration from 0.05 to 0.25 per cent and the exposure time from 2 to 6 hours. More than 80 auxotrophic mutants were selected. When Streptoverticillium mycoheptinicum, strain 852, was treated with NMU, the frequency of auxotrophic mutants ranged from 0.5 to 2.4 per cent. Fifty-seven auxotrophic mutants were selected. The majority of the auxotrophic mutants selected with the use of NMB and NMU was unstable. Exposure of Streptoverticillium mycoheptinicum, strains 852, 10/69 Met and 54/100 Lys to UV light resulted in formation of groups of polyauxotrophic mutants.     

Causes of cell death following exposure to different fungicides and heat

The lethal effects of several agricultural fungicides, the mutagen ICR-170, and heat have been studied in multinucleated spores from heterokaryotic mycelia ofPhycomyces blakesleeanus. The fungicides captafol, captan, Manzate (maneb), Benlate (benomyl), and ZZ-melprex (dodine) induce exclusively cytoplasmic lethality. The mutagen ICR-170 causes three different kinds of lethal events: mitotic, recessive, and cytoplasmic. Heat exposure of dormant spores results in only cytoplasmic lethal damage. Germination increases heat sensitivity, particularly that of nuclei; while most of the lethality in germinating spores is still cytoplasmic, there is a definite contribution of mitotic lethality, i.e., events blocking the division of the affected nuclei.     

Manganese mutagenesis in yeast. A practical application of manganese for the induction of mitochondrial antibiotic-resistant mutations.

When yeast cells were incubated for 4 to 8 h in yeast extract-peptone-glucose medium, pH 6, containing 8 mM-manganese, and then plated on selective media, there was a strong induction of antibiotic-resistant mutations. Indirect evidence suggests that practically all resistant mutants selected were of independent origin. The analysis of manganese-induced resistant mutants showed that most were extranuclear, while those tested showed recombination with known mitochondrial markers. Our results suggest that manganese can be considered as a mutagen which specifically induces mitochondrial mutations in Saccharomyces cerevisiae.     

Effects of Male Germ-Cell Stage on the Frequency,Nature, and Spectrum of Induced Specific-Locus Mutations in the Mouse

By means of the mouse specific-locus test (SLT) with visible markers, which is capable of detecting intragenic mutations as well as larger lesions, about 20 mutagens have been studied comparatively across arrays of male germ-cell stages. In addition, a very large historical control, accumulated over decades, provides data on spontaneous mutations in males. Each mutagen has a characteristic germ-cell-stage sensitivity pattern. Although most chemicals yield their maximum numbers of mutations following exposure of spermatozoa and late spermatids, mutagens have now been identified that peak in each of the major stages of spermatogenesis and spermiogenesis, including those in which effects on recombination can also be induced. Stem-cell spermatogonia have yielded positive results with only five of 15 mutagenic chemicals. In postspermatogonial stages, all chemicals, as well as radiations, induce primarily large lesions (LL). By contrast, in spermatogonia (either stem-cell or differentiating) all chemicals except one (bleomycin) produce very few such lesions. The spectrum of relative mutation frequencies at the seven loci of the SLT is characteristic for treated germ-cell stage and mutagen. Treatments that induce primarily LL are characterized by a great preponderance of s (Ednrb)-locus mutations (possibly due to a paucity of haplo-insufficient genes in the surrounding region); and those that induce very few, if any, LL by a great preponderance of p-locus mutations. Spontaneous locus-spectra differ from both types of treatment-induced spectra; moreover, there are two distinct types of spontaneous spectra, depending on whether mutations occurred in mitotic cells or during the perigametic interval.     

Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling.

Aerobiological monitoring was conducted in an experimental room to aid in the development of standardized sampling protocols for airborne microorganisms in the indoor environment. The objectives of this research were to evaluate the relative efficiencies of selected sampling methods for the retrieval of airborne fungal spores and to determine the effect of human activity on air sampling. Dry aerosols containing known concentrations of Penicillium chrysogenum spores were generated, and air samples were taken by using Andersen six-stage, Surface Air System, Burkard, and depositional samplers. The Andersen and Burkard samplers retrieved the highest numbers of spores compared with the measurement standard, an aerodynamic particle sizer located inside the room. Data from paired samplers demonstrated that the Andersen sampler had the highest levels of sensitivity and repeatability. With a carpet as the source of P. chrysogenum spores, the effects of human activity (walking or vacuuming near the sampling site) on air sampling were also examined. Air samples were taken under undisturbed conditions and after human activity in the room. Human activity resulted in retrieval of significantly higher concentrations of airborne spores. Surface sampling of the carpet revealed moderate to heavy contamination despite relatively low airborne counts. Therefore, in certain situations, air sampling without concomitant surface sampling may not adequately reflect the level of microbial contamination in indoor environments.     

The effects of pathogen infection and mutation on life-history characters in Arabidopsis thaliana

The nature of the interaction among deleterious mutations is important to models in many areas of evolutionary biology. In addition, interactions between genetic and environmental factors may affect the predictions of such models. Individuals of unknown genotypes of Arabidopsis thaliana, ecotype Marburg, were exposed to five levels of chemical (EMS) mutagenesis and three levels of Pseudomonas syringae infection. Survival, growth and flowering characteristics of each individual were measured. The logarithm of fitness is expected to be a linear function of mutation number if mutations act independently. Furthermore, the expected number of mutations should be approximately a linear function of time of exposure to mutagen. Therefore, nonlinear effects of mutagen exposure on the logarithm of fitness characters would suggest epistasis between mutations. Similarly, if pathogen infection and mutation act independently of each other, their effects should be additive on a log scale. Statistical interactions between these factors would suggest they do not act independently; particularly, if highly mutated individuals suffer more when infected than do less mutated individuals, this suggests that pathogens and mutations act synergistically. Pseudomonas-infected individuals were shown to have an increased probability of flowering under conditions of short day length, but to ultimately produce fewer flowers than uninfected individuals. This suggests a plastic response to stress and, despite that response, an ultimately deleterious effect of infection on fitness. Leaf rosette growth was negatively and linearly related to the expected number of mutations, and the effects of mutation on different life-cycle stages appeared to be uncorrelated. No significant interactions between pathogen and mutation main effects were found. These results suggest that mutations act multiplicatively with each other and with pathogen infection in determining individual fitness.     

Characterization of trimethylpsoralen as a mutagen for mouse embryonic stem cells

Given a large number of genes with unknown functions in model organisms, collections of mutants are valuable resources for studying gene function. For the mouse, embryonic stem cell technology offers the possibility to manipulate the genome and select for mutations in vitro. Mutant mice can then be generated from clones of interest to study the phenotype of these animals. We manipulate the genome of mouse embryonic stem (ES) cells chemically using the mutagen trimethylpsoralen (TMP). TMP predominantly causes deletions in the genome of Caenorhabditis elegans and Escherichia coli, but has not been established as a mutagen in mammalian systems yet. We have characterized TMP as a mutagen for mouse ES cells regarding death rates, mutation frequencies, and mutation spectrum. Allowing for 12.5% of cell survival, the mutation frequency at the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus was 3.5 x 10(-5) on average. The characterization of a non-redundant set of 17 Hprt-deficient ES clones revealed that only 12% of clones contained genomic deletions and almost 50% were point mutations. Base substitutions were mostly transversions and all affected AT base pairs. We conclude that the mutation spectrum of TMP in mouse ES cells is different from that observed in C. elegans and E. coli.     

Mutagenic fingerprint of ozone in human cells

Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.