An efficient mutational method for photosynthetic bacteria

The pigment and auxotrophic mutants of Rhodobacter sphaeroides Y6 were obtained by treatment with ethyl methanesulfonate (EMS) followed by lithium chloride (LiCl). Treatment with 0.081 MEMS and subsequent treatment with 0.071 M LiCl resulted in 12% higher frequency og than that by 0.081 mol/L EMS alone, and the same frequency of pigment mutations than application of 0.081 M EMS alone; the frequency of auxotrophic mutations increased 2.5-fold when treatment with lithium chloride was applied. A blue shift by 10 nm was recorded in the absorption spectrum of carotenoids form YM5-3 green mutant; considerable accumulation of neurosporine was revealed by HPLC and mass spectrometry. The method is efficient for isolating the mutants of photosynthetic bacteria. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 6, pp. 758–764. The text was submitted by the authors in English.     

Nuclear and extranuclear mutations in yeast induced by ethyl methanesulfonate

When zero-point mutations were induced in the yeastSaccharomyces cerevisiae using ethyl methanesulfonate (EMS) no differences were found in the frequency of auxotrophic mutants formed by a short and a prolonged treatment of the agent at equal survival level. The expression of a part of the mutations induced by a prolonged EMS treatment was delayed by one or two division cycles. The total frequency of auxotrophs due to both the zero point and delayed mutations, however, is still considerably lower than the frequency of auxotrophs induced by a prolonged treatment of EMS in some bacterial species. Both the prolonged and short EMS treatment induces in yeast also extranuclear respiration-deficient (RD) mutants at a relatively high frequency; in wild strains at equal survival level the prolonged treatment produces a higher number of RD mutants than the short one. In strain which is more susceptible to the lethal EMS effect than wild strain the number of RD mutants produced by the agent is much higher than in the wild strain. The results support the assumption of the different DNA arrangement in yeast nuclei and mitochondria and indicate the possible effect of repair mechanisms during the induction of mutations causing the respiration deficiency.     

Isolation of auxotrophic mutants ofAspergillus niger by ethylmethane sulphonate treatment

The alkylating agent EMS can be employed for the isolation of auxotrophic mutants of theAspergillus niger strain K 10. The maximum number of auxotrophic mutants (2.9–5.1%) corresponded approximately to the number of mutants obtained by EMS treatment of yeasts, but it was by order lower than the number of mutants generally obtained by EMS treatment in bacteria. The majority of isolated mutants grew worse than the parent strain in the liquid medium and also formed lower amount of organic acids. The organic acid which was most frequently accumulated by mutants was citric acid.     

Effects of sodium acetate and sodium chloride on EMS-induced chlorophyll mutations in barley

Sodium acetate solutions to which sodium chloride was added, and acetate or chloride alone have been used as pre-, simultaneous, and post-treatment of dry and pre-soaked seeds of barley to study their effect on the types and frequencies of ethyl methanesulphonate (EMS)-induced chlorophyll mutations in spring barley, variety Elsa, and winter barley, varieties $\text{436}\text{35}$ and Ager. Application of acetate/chloride on dry seeds before or simultaneously with EMS both resulted in the frequency of chimeral plants with chlorophyll-deficient sectors in M₁ and chlorophyll mutants in M₂ approximately being halved as compared with the controls (EMS treatment alone).An opposite effect was observed after simultaneous treatment with acetate/ chloride and EMS (pH 4.5 and pH 7.0) and application of acetate/chloride after EMS treatment of pre-soaked seeds. In this case the mutagen sensitivity, i.e. the frequency of chimeral plants with induced chlorophyll-deficient sectors in M₁ and of chlorophyll mutants in M₂, was approximately doubled as compared with the control.Separate application of both acetate or chloride as a simultaneous treatment with EMS resulted also in an increase in the chlorophyll mutation frequency as compared with EMS treatment alone.Based on these results some aspects of the acetate/chloride effect are briefly discussed.     

富马酸生产菌少根根霉的诱变筛选

以实验室原少根根霉为出发菌种,通过紫外线和LiCl诱变处理,发现当紫外线照射时间为3min,并在质量分数4％LiCl的平板中培养,可诱变出富马酸高产菌。利用溴甲酚绿加塑料小管的平板进行初筛,其生成的富马酸通过塑料小管底部渗透到指示培养基中,产生变色圈,根据变色圈大小可初步判断诱变株的产酸能力,大大缩短了筛选时间。在葡萄糖质量浓度为80g/L时,诱变后的3#菌种在发酵72h后能产生55．02g/L的富马酸,比原菌种的富马酸产量提高了2．49倍。     

Effect of ethyl methane sulphonate on biomass and protein production by Candida tropicalis

Large doses of ethyl methanesulphonate (EMS) greatly increased the induction of auxotrophic mutants in Candida tropicalis. The maximum yield of biomass and protein was recorded in some mutants isolated after treatment with 60, 80 and 100 ppm EMS. The electrophoretic protein profile revealed typical banding patterns for both C. tropicalis wild type and mutants.     

Optimization on the selection of auxotrophic mutants in Candida utilis by snail enzyme treatment

The determination of the best conditions for the application of the snaill enzyme digestion method in the enrichment of auxotrophic mutants in Candida utilis was carried out following Box and Wilson's mathematical method. The selection procedure proposed was tested in the enrichment of auxotrophic mutants from a mutagenized culture of a wild-type strain. Mutant frequency was increased 46-fold by treatment with snail enzyme. The method also proved useful in the selection of additional auxotrophic mutations from single auxotrophs.     

Spontaneous Auxotrophic and Pigmented Mutants Occurring at High Frequency in Bacillus pumilus NRRL B-3275

Broth cultures of Bacillus pumilus NRRL B-3275 (BpB1) grown at 25, 30, or 37 C contain 1 to 2% spontaneous auxotrophic mutants in both the exponential and stationary phases of growth. Of 70 such mutants isolated from cultures grown at 37 C, approximately two-thirds reverted at such a high frequency as to preclude their study. Of the remaining 22 mutants, 18 required a single amino acid, 1 required adenine, and 1 required uracil. Two of the auxotrophs each required two unrelated amino acids resulting from two independent mutations. All of the mutations reverted spontaneously. Enhanced reversion of approximately one-third of the mutations was obtained with nitrosoguanidine, ethyl methane sulfonate, or diethyl sulfate, or with more than one of these mutagens. The reversion of one mutation was enhanced by 2-aminopurine. The reversion of the remaining mutations was not enhanced by the above mutagens, nor by mutagens known to induce (and revert) frameshift mutations in other bacterial systems. Nine of 10 mutants examined did not show a selective growth advantage over the parents. All but three of the mutations could be linked by PBS1 transduction to one of the previously described auxotrophic markers in strain BpB1. No evidence was obtained for clustering of the mutations on the BpB1 genome. Six of the mutations conferred a requirement for serine. One linked by transduction to trp-2, three linked to argA1, and two (ser-2, -3) linked to argO1. Pigmented mutants (containing a carotenoid-like pigment), which occur spontaneously in BpB1 cultures at a frequency on the order of 1 to 5 mutants per 10(4) cells, link by transduction to ser-2, -3. Spontaneous mutants of strain BpB1 resistant to rifampin, streptomycin, erythromycin, 5-fluorouracil, or 5-methyltryptophan occur at a frequency similar to that of strains of B. pumilus which do not exhibit a high rate of spontaneous mutation to auxotrophy. It is suggested that certain sites or regions of the BpB1 genome exhibit a high rate of spontaneous mutation.     

The replication map of the chromosome ofMycobacterium phlei

It was the aim of the present work to construct the replication map of the chromosome ofMycobacterium phlei. The method of mutagenesis of the replication point by N-methyl-N-nitroso-N’-nitroguanidine in synchronously dividing populations and the method of analysis of gene frequency were applied. The order of replication of 19 genes on the chromosome was determined by means of induction of back mutations and forward mutations in auxotrophic mutants PAleu and PAmet and in double auxotrophic mutants with methionine as a reference marker.     

Mutation frequency decline in Escherichia coli B/r after mutagenesis with ethyl methanesulfonate

Nonsense-defective auxotrophic strains of Escherichia coli B/r were used to study mutation frequency decline (MFD) after mutagenesis with ethyl methanesulfonate (EMS). The mutation frequencies for prototrophic revertants that were either converted or de novo glutamine tRNA suppressor mutations declined as treated auxotrophic parental cells were incubated with glucose but without required amino acids (a condition typically producing MFD). The decline for converted suppressor mutations was more rapid than the decline for de novo suppressor mutations after low or moderate EMS treatment, but both suppressor mutation types showed the same slow decline after extensive treatment. The declines for both types of suppressor mutation were eliminated in uvrA-defective cells, and the rapid decline seen for converted suppressor mutations appeared as a slow decline in mfd-defective cells. The results are interpreted that true MFD (the rapid process) affects only the EMS-induced converted glutamine tRNA suppressor mutations. This would account for the rapid decline that is blocked in cells with an mfd defect and in cells with deficient excision repair activity (uvrA or excessive DNA damage). In addition, a second non-specific antimutation mechanism is proposed that is dependent on excision repair only and accounts for the slow decline seen with converted suppressor mutations in some instances and with de novo suppressor mutations at all times. The true MFD mechanism may consist of a physiologically dependent facilitated excision repair specifically for premutational residues located in the transcribed strand of the target DNA sequence (for O6-ethylguanine in cells treated with ethyl methanesulfonate or pyrimidine-pyrimidine photoproducts after UV irradiation).     

Effects of caffeine or EDTA post-treatment on EMS mutagenesis in soybean

Seeds of soybean cultivar LD4 were mutagenically treated with EMS (0.3, 0.5, 0.6, 0.9, 0.5 and 1.8%) for 3 h only or plus caffeine (50 mM) or EDTA (1 mM) post-treatment for 5 h. The experimental results indicated that: (1) of the different concentrations of EMS treatment, the M2 mutation frequency induced with 0.6% EMS was the highest (9.7%). When the EMS concentration was over 0.9%, the mutation frequency decreased rapidly. (2) Of the EMS treatments plus caffeine or EDTA post-treatment, the mutagenic effect of 0.6% EMS was the best for inducing morphological variations. Caffeine post-treatment decreased notably the mutation frequency of EMS treatment; when concentrations of EMS were very high (1.5% and 1.8%), mutation frequencies of EDTA post-treatment were still 5.0% and 4.88%, but no mutants were found in EMS treatment or plus caffeine post-treatment. (3) In the M2 mutation spectrum, 11 kinds of mutant types were observed in EMS treatment or plus caffeine or EDTA post-treatment. Relative frequencies of some mutant types (growth period, plant height, grain size, leaf shape and sterility, etc.) were similat among the three treatments, but EDTA post-treatment could change the relative frequencies of yield characteristics (number of pods and grains, grain weight/plant) induced by EMS treatment only.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Ethyl methanesulfonate mutagenesis in extremely halophilic archaebacteria: Isolation of auxotrophic mutants ofHaloferax mediterranei andHaloferax gibbonsii

The lethality and mutagenicity in ethyl methanesulfonate (EMS)-treated cells of five archaebacterial strains belonging to each of the three described genera of non-alkaliphilic halobacteria were investigated. In order to test the efficiency of the mutagenesis under a variety of experimental conditions, we chose the fast-growing halobacteriumHaloferax mediterranei as a model strain. A strong induced mutagenicity was found, since the spontaneous mutation rate (expressed as the rate of resistance to the antibiotic josamycin) increased up to 500-fold after mutagen exposure. The mutagenesis was also successfully used in obtaining auxotrophic mutants. Although a heterogeneous response to the induced effects caused after EMS exposure was detected for the other halophilic archaebacteria tested, a clear, efficient mutagenicity ofHalobacterium halobium andHaloferax gibbonsii was observed; auxotrophic mutants of this halobacterium were also produced. Optimal experimental conditions for EMS mutagenesis of some halobacteria were determined.     

The mutation range ofBrevibacterium sp. M27

The mutation range was studied inBrevibacterium sp. M27 after UV irradiation and after treatment with N-methyl-N’-nitro-N-nitrosoguanidine. The induction of auxotrophic mutants and mutants resistant to streptomycin and tetracycline was investigated. A collection of auxotrophic mutants for the studies of genetic transfer in this model was prepared.     

Selection of stable mutants from cultured rice anthers treated with ethyl methane sulfonic acid

To increase the frequency of stable mutants from cultured anthers of rice, the effects of EMS treatment on callus induction, plant regeneration and mutant induction were investigated according to the timing of treatment after anther inoculation on the medium. The frequency of callus induction was highest in anthers treated with 0.5% EMS 10 days after culture. Anthers treated directly at the initiation of culture exhibited a very low callus induction level, and the such calluses exhibited a poor plant regeneration capacity. The frequency of regeneration of green plants was significantly decreased by EMS treatments immediately after anther inoculation as compared with control. The frequencies of stable mutants were 20.7% and 12.0% in EMS treatments at 10 and 20 days, but unstable mutants were 43.1% and 52.6%, respectively. A total of 14 stable mutants, semidwarf mutants (4 lines), grain-shape mutants (2 lines) and glabrous mutants (8 lines) were selected from doubled haploid lines of the A₂ generation. The frequencies of callus induction, green plant regeneration and stable mutants were maximal in anthers treated with 0.5% EMS 10 days after culture.     

Variability of cultures of Pullularia pullulans with different ploidy levels

As the level of ploidy rises in Pullularia pullulans, this causes an increase in the frequency of spontaneous and UV-induced auxotrophic mutants as well as mutants with a modified respiration activity while the frequency of morphological mutants decreases. The latter can arise as a result of recessive and dominant mutations. A higher frequency of morphological mutants in the haploid may be result of recessive mutations. It is likely that the frequency of dominant mutations increases in cultures with a higher level of ploidy since, as the difference between the frequency of UV-induced mutants and the frequency of spontaneous morphological mutants increases.     

The multiple-cluster mutation complex in mutagenesis with higher plants

Summary After treatment of dry and pre-soaked seeds of barley with gamma-rays, EMS, NEU and EI, the frequency of multiple mutations (multimutations) was higher with EMS and NEU treatment, while cluster mutations appeared in greater numbers following treatment with gamma rays and NEU. Pre-soaking the seeds led to a reduction in the frequency of total mutations, cluster mutations and multimutations. This has been explained as a result of the application of lower doses and the induction of mutations at a relatively later stage in ontogenetic development in the case of pre-soaked seeds.Some new mutation types in barley have been described and some of the old types have been given names representing the mutation characters more precisely.The compound mutation frequency of different seedling mutation types, when taken separately, was found to be independent of the mutagen employed and the stage of treatment. The size of mutated chimeras in M ₁ plants, as indicated by the segregation ratio of mutants in M ₂, was largest in albina, xantha, chlorina, albina-tigrina, chl-terminalis and eceriferum, and lowest in viridis, viridoalbina etc. This could be expected if the unstable premutations induced by mutagenic treatment are resolved into mutations at different intervals after their initiation, or it can be explained by the induction of dominant mutations, or lethal changes together with visible mutations.     

Mutations Affecting the Chemosensory Neurons of Caenorhabditis Elegans

We have identified and characterized 95 mutations that reduce or abolish dye filling of amphid and phasmid neurons and that have little effect on viability, fertility or movement. Twenty-seven mutations occurred spontaneously in strains with a high frequency of transposon insertion. Sixty-eight were isolated after treatment with EMS. All of the mutations result in defects in one or more chemosensory responses, such as chemotaxis to ammonium chloride or formation of dauer larvae under conditions of starvation and overcrowding. Seventy-five of the mutations are alleles of 12 previously defined genes, mutations which were previously shown to lead to defects in amphid ultrastructure. We have assigned 20 mutations to 13 new genes, called dyf-1 through dyf-13. We expect that the genes represented by dye-filling defective mutants are important for the differentiation of amphid and phasmid chemosensilla.     

The induction of mutants of Acinetobacter calcoaceticus NCIB8250 and their selection by Vancomycin.

The mutagenic and lethal effects of N-methyl-N-nitro-N-nitrosoguanidine (NTG), ethyl methanesulphonate (EMS), ultraviolet light iffadiation and near-ultraviolet light irradiation with 8-methoxypsoralen on the bacterium Acinetobacter calcoaceticus NCIB8250 were examined. The production of auxotrophic mutants was used as a measure of mutagenic efficiency. Under appropriate conditions all four agents were mutagenic. EMS and NTG although more effective than irradiation, did not cause such a high frequency of mutation as has been observed with other bacteria. A combination of vancomycin and penicillin V gave enrichment of non-metabolizing bacteria and optimum conditions were found for the use of these compounds in a selection technique.     

The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.