Mutation induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis.

Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores, however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.     

Genetic effects of cosmic radiation in Drosophila melanogaster]

To examine possible effects of space radiation on living organism, we have analyzedtwo types of mutations, sex-linked recessive lethal mutations and somatic mutations, in fruit fly of the species Drosophila melanogaster. Drosophila strains used were wild type strains and a radiation-sensitive strain mei-41. Two different developmental stages of samples were sent into space; young adult males to analyze sex-linked recessive lethal mutations and about 30hr-old larvae to detect somatic mutations in wing epidermal cells. For wild type and mei-41 strains each, about 200 adult male flies and about 6,000 larvae were loaded on space shuttle Endeavour. The male flies returned from space were mated to virgin female flies of a tester strain, and the presence of the lethal mutations was analyzed at F2 generation. The frequencies of sex-linked recessive lethal mutations in flight groups were 2 and 3 times higher for wild type Canton-S and mei-4 1, respectively, than those in ground control groups. Most larvae sent to space emerged as adult flies within about 10 days after the landing. The presence of wing-hair somatic mutations, which give morphological change in hairs growing on the surface of wing epidermal cells, was analyzed under microscope. In wild type strain Muller-5, the frequency of wing hair mutant spots in flight group was about 1.5-fold higher than that in ground control, and in Canton-S-derived wild type strain the frequencies were similar between the two groups. By contrast, for mei-41 strain the mutation frequency was lower in flight group than in control group. The observed higher frequency of lethal mutations in the flight group might be due to a possibility that radiation effects on reproductive cells could be greatly enhanced under micro gravity. However, if this would be the case, we do not have appropriate explanation for the apparent absence of such synergistic effects on somatic wing-hair mutation system.     

Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Studies on an unstable phenotype induced by UV irradiation

Unstable clones excreting L-lysine into their growth medium are obtained at a very high frequency following UV irradiation in both haploid and diploid strains of Saccharomycopsis lipolytica, provided they carry a mutation affecting the first enzyme of the lysine pathway and confering resistance to end product inhibition. The phenotype can be stabilized in some sublines; it appears as dominant and coupled with a decrease in spore viability. Excretion in batch cultures is confined to the end of the exponential phase, and seems not to consist in a simple release of the lysine pool content.     

Effects of reduced natural background radiation on Drosophila melanogaster growth and development as revealed by the FLYINGLOW program

Natural background radiation of Earth and cosmic rays played a relevant role during the evolution of living organisms. However, how chronic low doses of radiation can affect biological processes is still unclear. Previous data have indicated that cells grown at the Gran Sasso Underground Laboratory (LNGS, L'Aquila) of National Institute of Nuclear Physics (INFN) of Italy, where the dose rate of cosmic rays and neutrons is significantly reduced with respect to the external environment, elicited an impaired response against endogenous damage as compared to cells grown outside LNGS. This suggests that environmental radiation contributes to the development of defense mechanisms at cellular level. To further understand how environmental radiation affects metabolism of living organisms, we have recently launched the FLYINGLOW program that aims at exploiting Drosophila melanogaster as a model for evaluating the effects of low doses/dose rates of radiation at the organismal level. Here, we will present a comparative data set on lifespan, motility and fertility from different Drosophila strains grown in parallel at LNGS and in a reference laboratory at the University of L'Aquila. Our data suggest the reduced radiation environment can influence Drosophila development and, depending on the genetic background, may affect viability for several generations even when flies are moved back to normal background radiation. As flies are considered a valuable model for human biology, our results might shed some light on understanding the effect of low dose radiation also in humans.     

Isolation and properties of genetically defined strains of the methylotrophic yeast Hansenula polymorpha CBS4732

Genetically defined strains of the yeast Hansenula polymorpha were constructed from a clone of H. polymorpha CBS4732 with very low mating and sporulation abilities. Mating, spore viability, and the percentage of four-spore-containing asci were increased to a level at which tetrad analysis was possible. Auxotrophic mutations in 30 genes were isolated and used to construct strains with multiple markers for mapping studies, transformation with plasmid DNA, and mutant screening. Various other types of mutants were isolated and characterized, among them mutants that displayed an altered morphology, methanol-utilization deficient mutants and strains impaired in the biosynthesis of alcohol oxidase and catalase. Also, the mutability of H. polymorpha CBS4732 vs H. polymorpha NCYC495 was compared. The data revealed clear differences in frequencies of appearance and mutational spectra of some mutants isolated. Many of the mutants isolated had good mating abilities, and diploids resulting from their crossing displayed high sporulation frequencies and high spore viability. Most of the markers used revealed normal Mendelian segregation during meiosis.The frequency of tetratype spore formation was lower than in Saccharomyces cerevisiae suggesting a lower frequency of recombination during the second meiotic division. The properties of genetically defined strains of H. polymorpha CBS4732 as well as their advantages for genetics and molecular studies are discussed.     

Use of a genome-wide approach to identify new genes that control resistance of Saccharomyces cerevisiae to ionizing radiation

We have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae, to screen for new mutants conferring sensitivity to ionizing radiation. In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains after irradiation. Of the top 33 strains most sensitive to radiation in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied. Only one, which deleted for the ORF YDR014W (which we name RAD61), conferred reproducible radiation sensitivity in both the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to ionizing radiation.     

Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H₂O₂) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H₂O₂, as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Efficacy of native and recombinant Cry1B protein against experimentally induced and naturally acquired ovine myiasis (fly strike) in sheep

Several hundred strains of Bacillus thuringiensis (Bt), isolated in New Zealand from samples of soil and sheep fleece, were tested for toxicity to larvae of the blowfly Lucilia cuprina Wiedemann. Characterization of the Bt strains revealed that three of the more active strains produced Cry1Ba (an insecticidal protein present in Bt mother cell crystal inclusion) that was toxic to blowflies. These strains were evaluated for the ability to prevent experimentally induced fly strike in a bioassay by using first instars. Results with undiluted spore/crystal preparations were variable, but they generally prevented fly strike on sheep maintained on pasture for 3-6 wk. Spore viability was satisfactory throughout the trials and environmental factors (e.g., precipitation and UV radiation) seemed to have minimal effect on persistence. The loss of fly strike protection in these experiments correlated with the movement of spore/crystal toxicity away from the skin as a result of wool growth. Solubilized protein preparations were not as potent as spore/crystal preparations and fly strike protection lasted only from 1 to 3 wk. Vegetative forms of the Cry1Ba-producing strains of Bt did not establish on the fleece of sheep, did not produce significant sporulation, and no protection against fly strike was achieved. Escherichia coli expressing recombinant Cry1Ba protein was toxic to larvae in vitro but did not effectively protect sheep from fly strike because blowfly larvae were able to establish readily 8 d posttreatment. In a single field experiment involving 80 sheep per group, a spore/crystal preparation from a Bt strain expressing Cry1Ba provided less protection from naturally acquired fly strike than afforded by a commercially available dip.     

Sustained and rapid chromosome movements are critical for chromosome pairing and meiotic progression in budding yeast

Telomere-led chromosome movements are a conserved feature of meiosis I (MI) prophase. Several roles have been proposed for such chromosome motion, including promoting homolog pairing and removing inappropriate chromosomal interactions. Here, we provide evidence in budding yeast that rapid chromosome movements affect homolog pairing and recombination. We found that csm4Δ strains, which are defective for telomere-led chromosome movements, show defects in homolog pairing as measured in a "one-dot/two-dot tetR-GFP" assay; however, pairing in csm4Δ eventually reaches near wild-type (WT) levels. Charged-to-alanine scanning mutagenesis of CSM4 yielded one allele, csm4-3, that confers a csm4Δ-like delay in meiotic prophase but promotes high spore viability. The meiotic delay in csm4-3 strains is essential for spore viability because a null mutation (rad17Δ) in the Rad17 checkpoint protein suppresses the delay but confers a severe spore viability defect. csm4-3 mutants show a general defect in chromosome motion but an intermediate defect in chromosome pairing. Chromosome velocity analysis in live cells showed that while average chromosome velocity was strongly reduced in csm4-3, chromosomes in this mutant displayed occasional rapid movements. Lastly, we observed that spo11 mutants displaying lower levels of meiosis-induced double-strand breaks showed higher spore viability in the presence of the csm4-3 mutation compared to csm4Δ. On the basis of these observations, we propose that during meiotic prophase the presence of occasional fast moving chromosomes over an extended period of time is sufficient to promote WT levels of recombination and high spore viability; however, sustained and rapid chromosome movements are required to prevent a checkpoint response and promote efficient meiotic progression.     

An estimation of Canadian population exposure to cosmic rays

The worldwide average exposure to cosmic rays contributes to about 16% of the annual effective dose from natural radiation sources. At ground level, doses from cosmic ray exposure depend strongly on altitude, and weakly on geographical location and solar activity. With the analytical model PARMA developed by the Japan Atomic Energy Agency, annual effective doses due to cosmic ray exposure at ground level were calculated for more than 1,500 communities across Canada which cover more than 85% of the Canadian population. The annual effective doses from cosmic ray exposure in the year 2000 during solar maximum ranged from 0.27 to 0.72 mSv with the population-weighted national average of 0.30 mSv. For the year 2006 during solar minimum, the doses varied between 0.30 and 0.84 mSv, and the population-weighted national average was 0.33 mSv. Averaged over solar activity, the Canadian population-weighted average annual effective dose due to cosmic ray exposure at ground level is estimated to be 0.31 mSv.     

Cell growth and morphology of Dictyostelium discoideum in space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiation-sensitive mutant and the parental wild-type strain, were used to investigate the effects of microgravity and/or cosmic radiation on their morphology through the whole life span from spores to fruiting bodies for about 7 days in space shuttle of NASA. We found almost no effect of space environment on amoeba cell growth in both strains. It was also observed that almost the same number and shape of fruiting bodies in space compared to the control experiments on earth. These results suggest that there is little effect of microgravity and space radiation on germination, cell aggregation, cell differentiation and cell morphology in the cellular slime mold.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. I. Development of isogenic strains and spontaneous mutability

7 different mutations that confer sensitivity to inactivation by ultraviolet light have been investigated for their effect on spontaneous mutation at the ad-3A and ad-3B loci in haploid strains of Neurospora crassa. The collection and development of strains isogenic to wild-type 74A is described as well as experiments to determine the effects of each mutation on the spontaneous ad-3 mutation frequency. 6 of the strains showed spontaneous ad-3 mutant frequencies not significantly different from the wild-type strain. Strain uvs-3 is highly mutable spontaneously with marked variation from experiment to experiment; the mean mutation frequency in this strain in about 40-fold higher than that found in the wild-type strain.     

Recovery of Deinococcus radiodurans from radiation damage was enhanced under microgravity.

Effect of microgravity on recovery of bacterial cells from radiation damage was examined on the IML-2 mission in 1994 using extremely radioresistant bacterium Deinococcus radiodurans. The cells were lyophilized and exposed to 60Co gamma-rays with doses 2 to 12 kGy before the space flight. At the end of the mission, the cells were mixed on board with liquid nutrient medium to allow the cells to start recovery process from the radiation damage. Afterwards the cells were stored at 4 degrees C until landing. The influence of cosmic radiation was negligible, because total absorbed dose of space radiation measured during the mission was less than 2 mGy and this bacterium does not decrease its viability after both gamma-rays and high-LET heavy charged particles irradiation with doses up to 5 kGy. The survival of the cells incubated in space increased significantly compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under microgravity.     

Phenotypic Mutations Induced During Storage in Barley and Pea Seeds

Previous work on chlorophyll-deficiency mutations in pea andbarley has shown that a significant increase in mutations isinduced by storing seeds under various conditions which leadto losses of viability to about 50 per cent. The work here showsthat a detectable increase in mutation frequency is also associatedwith much smaller losses of viability. Pea seeds were storedat 35 °C and 16.5 per cent moisture content for 40 and 57d when viability fell from 99 to 93 and 82 per cent, respectively.At the same time mutation frequency (percentage of seeds containingrecessive point mutations) increased from 1.62 per cent in thecontrol treatment to about 3 to 4 per cent. Barley seeds at15.5 per cent moisture content were stored at 50 °C for42 and 54 h, and at 35 °C for 28 and 39 d. During theseageing treatments viability fell from 98 to 75, 26, 93 and 48per cent respectively and the mutation frequency increased fromzero to between about 0.3 to 0.9 per cent. In both species theinduction of mutation by ageing treatments was significant butthe differences between the various ageing treatments were not.It is concluded that there is probably no safe threshold lossof viability which completely avoids mutation, and these resultssupport the view that for genetic conservation seeds shouldbe stored under conditions which minimise loss of viability. Pisum sativum L., pea, Hordeum distichum L., barley, mutation frequency, seed storage, seed viability     

Analysis of Mitotic and Meiotic Defects in Saccharomyces Cerevisiae Srs2 DNA Helicase Mutants

The hyper-gene conversion srs2-101 mutation of the SRS2 DNA helicase gene of Saccharomyces cerevisiae has been reported to suppress the UV sensitivity of rad18 mutants. New alleles of SRS2 were recovered using this suppressor phenotype. The alleles have been characterized with respect to suppression of rad18 UV sensitivity, hyperrecombination, reduction of meiotic viability, and definition of the mutational change within the SRS2 gene. Variability in the degree of rad18 suppression and hyperrecombination were found. The alleles that showed the severest effects were found to be missense mutations within the consensus domains of the DNA helicase family of proteins. The effect of mutations in domains I (ATP-binding) and V (proposed DNA binding) are reported. Some alleles of SRS2 reduce spore viability to 50% of wild-type levels. This phenotype is not bypassed by spo13 mutation. Although the srs2 homozygous diploids strains undergo normal commitment to meiotic recombination, this event is delayed by several hours in the mutant strains and the strains appear to stall in the progression from meiosis I to meiosis II.     

Meiotic Chromosome Pairing in Triploid and Tetraploid Saccharomyces Cerevisiae

Meiotic chromosome pairing in isogenic triploid and tetraploid strains of yeast and the consequences of polyploidy on meiotic chromosome segregation are studied. Synaptonemal complex formation at pachytene was found to be different in the triploid and in the tetraploid. In the triploid, triple-synapsis, that is, the connection of three homologues at a given site, is common. It can even extend all the way along the chromosomes. In the tetraploid, homologous chromosomes mostly come in pairs of synapsed bivalents. Multiple synapsis, that is, synapsis of more than two homologues in one and the same region, was virtually absent in the tetraploid. About five quadrivalents per cell occurred due to the switching of pairing partners. From the frequency of pairing partner switches it can be deduced that in most chromosomes synapsis is initiated primarily at one end, occasionally at both ends and rarely at an additional intercalary position. In contrast to a considerably reduced spore viability (~40%) in the triploid, spore viability is only mildly affected in the tetraploid. The good spore viability is presumably due to the low frequency of quadrivalents and to the highly regular 2:2 segregation of the few quadrivalents that do occur. Occasionally, however, quadrivalents appear to be subject to 3:1 nondisjunction that leads to spore death in the second generation.     

Lack of Chemically Induced Mutation in Repair-Deficient Mutants of Yeast

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.     

~(60)Coγ射线对不同细胞形态出芽短梗霉的辐射诱变效应

使用~(60)Coγ射线辐照诱变的方法处理出芽短梗霉AureobasidiumPullulansAP92菌株的原生质体、菌丝体片段、分生孢子悬液,经初筛、复筛与对突变株的遗传稳定性研究,发现采用原生质体进行诱变,所获突变株的正突变率、单株产多糖的提高幅度、正突变株的产多糖遗传稳定性均明显高于菌丝体与分生孢子。比较出发菌株AP92与经原生质体诱变获得的正突变株A81的性能,有如下明显改善：产多糖能力从16．35g／L提高到29．69g／L,糖转化率从32．7％升到61．4％,残糖从13．269／L降到3．06g／L,而发酵周期则可缩短约24小时。结果证明,对原生质体进行~(60)Coγ射线诱变,是优化出芽短梗霉菌种的有效途径。     

The type of mutations induced by carbon-ion-beam irradiation of the filamentous fungus Neurospora crassa

Heavy-ion beams are known to cause great damage to cellular components and are particularly renowned for their ability to generate DNA double-strand breaks (DSBs). To gain insight into the mutagenic effect of carbon-ion beams and how such damage is repaired by the cell, Neurospora crassa mutants deficient in one of three components involved in the repair of DSBs, nonhomologous end-joining (NHEJ), homologous recombination repair (HR), and the Mre11-Rad50-Xrs2 (MRX) complex, were irradiated with a carbon-ion beam and killing effect, mutation frequencies, and the type of mutation incurred by survivors were analysed. The sensitivity of the NHEJ-deficient strain (mus-52) was higher than that of the wild-type and the HR-deficient (mei-3) strains at low doses of radiation, but was little changed as the level increased. As a result both the wild-type and HR-deficient strains were more sensitive than the NHEJ-deficient strain at high radiation levels. In addition, the frequency of forward mutation at the adenine-3 (ad-3) loci of the NHEJ-deficient mutant was lower than that of the wild-type strain at all levels, while the mutation frequency of the HR-deficient strain was consistently ∼3-fold higher than the wild-type. From the comparison of mutation type of each strain, deletions were frequently observed in wild-type strain, whilst base substitution and deletion in the mus-52 and mei-3 strains. These mutations resulting from carbon-ion-beam irradiation depend on the mechanism invoked to cope with DSBs. Furthermore, in wild-type cells, these mechanisms likely compete to repair DSBs.