J. Velemínský

Repair of single-strand breaks and fate of N-7-methylguanine in DNA during the recovery from genetical damage induced by N-methyl-N-nitrosourea in barley seeds

Abstract The recovery from various types of N -methyl- N -nitrosourea (MNU)-induced genetical damage, observed after two weeks storage of alkylated seeds with 30% water content at 25°, was found to be connected with the repair of single-strand breaks and/or apurinic sites in DNA of the seed embryo. The amount of N -7-methylguanine (7-MeG) in DNA was not influenced by this type of seed storage, showing that the excision of this base is not a part of the repair mechanism. The changes in the alkylation of proteins, lipids and RNA observed after storage of the seed are considered to be irrelevant to the repair process. Single-strand breaks and/or apurinic sites in barley DNA are presumed to be the main lesions responsible for the reparable types of genetical damage.

DNA repair in mutagen-injured higher plants

Abstract Knowledge of DNA repair in higher plants is rather poor as compared with that for bacteria, mammalian cells, etc. Some years ago, excision repair was believed to be absent from plants. In this review, data are summarized proving the occurrence of photoreactivation of UV-induced pyrimidine dimers in cells of Nicotiana tabacum, Gingko and carrot, the excision of dimers in protoplasts of carrot and in embryos of Lathyrus sativus, and the repair of DNA single-strand breaks induced in carrot protoplasts and barley embryonic cells by ionizing radiation. In irradiated barley embryos the unscheduled DNA synthesis and higher accessibility of induced primers to DNA polymerase I of E. coli were observed preferentially in G1 cells with diffused chromatin. These reactions were inhibited by caffeine and EDTA. Unscheduled DNA synthesis was also observed in synchronized irradiated root cuttings of Vicia faba and in barley embryos treated with 4-nitroquinoline oxide, the latter being inhibited by caffeine and hydroxyurea. Repair synthesis was also established in barley embryos treated with mutagenic N-methyl-N-nitrosourea under conditions that postponed the onset of germination after the treatment. The same conditions enhanced the repair of DNA single-strand breaks induced by this mutagen and several other monofunctional alkylating compounds. From tissues of barley and of Phaseolus multiflorus, endonucleases for apurinic sites were isolated and characterized. Some of them are located in chromatin, others in chloroplasts. The relation between DNA repair and genetic effects of mutagens in higher plants is also discussed.

Mutagenesis of Saccharomyces cerevisiae by sodium azide activated in Barley

Concentrated dialysate of the extract prepared from barley seeds treated with sodium azide increased up to 100--200 times the frequency of forward mutations to cycloheximide resistance in the excision-deficient UV-sensitive heploid strain rad2-5 of Saccharomyces cerevisiae, when applied to growing cells in complete medium at pH 4.2. Only a slight increase of mutation frequency (less than 4 times) was found in the haploid RAD+ strain treated in the same way as well as in haploid RAD+ and rad2-5 strains treated directly by sodium azide. In contrast with the barley-activated sodium azide, UV irradiation was more effective in the induction of cycloheximide resistance in the RAD+ strain than in the RAD2-5 mutant. The dialysate from azide-treated barley seeds, applied at both pH 4.2 and pH 9, also significantly increased the frequency of locus-specific suppressor mutations to isoleucine independence and -- to a lesser extent -- reversions and/or gene conversions in the trp5 locus in growing cells of the diploid strain D7. The dialysate was also mutagenic in resting cells of strains D7 and rad2-5 but with lower effectiveness.

Inhibitors of N-nitroso compounds-induced mutagenicity

N-Nitroso compounds are environmental mutagens that are present in the air, water, soil etc. or can be formed by nitrosation of various nitrosatable compounds. The present paper gives a survey of inhibitors of N-nitroso compounds-induced mutagenicity. Inhibitors covered include: thiols, metals, vitamins, phenolic acids, complex mixtures of plant, animal and human origin, organic solvents, inhibitors of mixed-function oxidases etc. Data on inhibitors that prevent the formation of N-nitroso compounds are not covered in this review.

Induction of DNA single-strand breaks in barley by sodium azide applied at pH 3

Sodium azide (1 to 50 mM), adjusted to pH 3 and applied for 2 h to presoaked barley seeds, induced a dose-dependent frequency of single-strand breaks in DNA of non-germinating embryos. This was demonstrated by sedimentation analyses of isolated DNA samples in alkaline sucrose gradients and in neutral sucrose gradients with 80% formamide. The doses applied also inhibited dose dependently the root length, seed germination and partially the seedling height. Only the sub-lethal doses (10 and 12.5 mM) induced a low frequency of chromatid breaks and translocations in the root tip metaphases. The sedimentation rate (in alkaline sucrose gradients) of calf thymus DNA treated with sodium azide at pH 3, was similar to that of the control DNA treated with buffer (pH 3) alone.

Recovery or increase of induced genetical effects dependent upon the moisture content of stored N-methyl-N-nitrosourea-treated barley seeds

Abstract ( 1 ) Barley seeds were treated with N -methyl- N -nitrosourea (MNU) and stored at 25° with 30% moisture. A strong recovery was observed of the M 1 seedling height and chromosome aberrations in root tips and of the M 2 chlorophyll mutant frequency. ( 2 ) In contrast, storage of MNU-treated seeds with 20% moisture led to a pronounced increase in biological injury. ( 3 ) After storage of MNU-treated seeds with 15% moisture, a small recovery in seedling height and in chromosome aberrations took place, but there was no significant change in frequency of the M 2 chlorophyll mutants. ( 4 ) Storage of MNU-treated seeds with 5% seed moisture had no significant effect on either type of biological injury in the M 1 generation or the M 2 chlorophyll mutant frequency. ( 5 ) Storage effects of MNU and ethyl methanesulphonate treatments of barley seeds are compared.

Storage effect in barley changes in the amount of DNA lesions induced by methyl and ethyl methanesulphonates

Abstract Barley seeds were treated with methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), stored at 15% water content and washed for 16–24 h. These treatments resulted in an increase of toxic and genetic effects. In teh DNA of embryos of such stored MMS- and EMS-treated seeds, a strong enhancement of the amount of single-strand breaks and/or alkali-labile sites took place. In contrast, the amount of alkylated sites, particularly of 7-methylguanine, was somewhat lower. It can be that the depurination and/or backbone breakage, which proceeds during the storage period, is responsible for the enhancement of toxic and genetic effects, whereas the influence of the alkylation of DNA during the storage period by the unreacted residual mutagen is negligible.

Effects of humic acids, para-aminobenzoic acid and ascorbic acid on the N-nitrosation of the carbamate insecticide propoxur and on the mutagenicity of nitrosopropoxur

Nitrosation of the carbamate insecticide propoxur at pH 3 and 37 degrees C was determined colorimetrically and found to be time- and sodium nitrite concentration-dependent. Nitrosated propoxur was mutagenic when exposed to the seeds of the higher plant Arabidopsis thaliana but the formation of nitrosopropoxur, the presumed mutagen, was inhibited by humic acids, para-aminobenzoic acid and ascorbic acid. These agents also reduced the mutagenicity of preformed nitrosopropoxur.

Reduction in the frequency of N-methyl-N-nitrosourea-induced somatic mutations in Tradescantia by pretreatment with low doses of alkylating agents

Treatment of Tradescantia cuttings with sub-mutagenic doses of N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea and methyl methanesulphonate before challenging doses of MNU reduced the frequency of somatic mutations in stamen hairs as compared with the effect of challenging dose alone. The highest response was about a 50% reduction in the mutagenic effect of the challenge dose.

Antimutagenic effects of diethyldithiocarbamate towards maleic hydrazide- and N-nitrosodiethylamine-induced mutagenicity in theTradescantia mutagenicity assay

InTradescantia, clone 4430, diethyldithiocarbamate (DEDTC) markedly decreased the frequency of somatic mutations induced by maleic hydrazide (MH) and N-nitrosodiethylamine (NDEA). In contrast, DEDTC had no such effect on N-methyl-N-nitrosourea-induced mutagenesis. The putative degradation and conversion products of MH (maleic acid diamide, succinic acid, maleic acid, lactic acid and hydrazine) exhibited no mutagenic activity in theTradescantia mutagenicity assay.