V. Pokorný

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.

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.

Caffeine enhancement of alkylating agent-induced injury in barley: its connection to DNA single-strand breaks and their repair

Abstract Non-germinating barley seeds were treated with N -methyl- N -nitrosourea (MNU), methyl methanesulphonate (MMS) or ethyl methanesulphonate (EMS) and then exposed either to non-toxic caffeine doses ( i.e. doses having no biological effect and inducing no DNA single-strand breaks when applied alone) or to toxic doses ( i.e. doses reducing seed germination and seedling height but inducing no DNA single-strand breaks). Non-toxic doses of caffeine, applied to MNU, MMS and EMS treated seeds enhanced the mutagen-induced reduction of germination and seedling height but did not disturb the recovery from this damage, achieved by storage of the seeds at 30% water content. At the DNA level, these doses of caffeine did not increase the amount of mutagen-induced single-strand breaks in the treated (preformed) DNA and did not inhibit repair of mutagen-induced single-strand breaks during seed storage. However, non-toxic doses of caffeine affected the size of DNA strands, newly synthesized during 3 days of seed germination. Whereas the size of DNA strands after MMS and EMS treatment approached the size of the newly synthesized DNA in the controls, the DNA strands from treatment with MMS or EMS plus non-toxic caffeine doses were considerably smaller. Thus, it seems likely that caffeine, although applied before the DNA-synthesis, enhanced the biological damage by acting in the S-phase and inhibiting the repair of DNA damage in the newly synthesized DNA strands. Interaction of toxic caffeine doses with MNU led both to the potentiation of the mutagen-induced reduction of germination and seedling height, and to the inhibition of recovery from this damage during seed storage. On the DNA level. even toxic doses of caffeine did not increase the amount of MNU-induced single-strand breaks in the preformed DNA but inhibited completely the repair of these breaks during the seed storage.

The action of 1-alkyl-1-nitrosoureas and 1-alkyl-3-nitro-1-nitrosoguanidines on the M1 generation of barley andArabidopsis thaliana (L.) heynh.

The activity of 1-methyl-1-nitrosourea (MNH), 1-ethyl-1-nitrosourea (ENH), 1-methyl-3-nitro-1-nitrosoguanidine (MNG) and 1-ethyl-3-nitro-1-nitrosoguanidine (ENG) was tested on seeds of barley andArabidopsis. The activity of nitrosoamides tested was expressed by the germination and M1 seedling height reduction of barley and M1 root length reduction ofArabidopsis.1)After the action of both nitrosoureas (MNH and ENH) the germination of barley is at the same level as that of controls, even at concentrations, leading to a maximal reduction in the height of seedlings. After the action of both nitrosoguanidines (MNG and ENG) germination decreases in parallel with the decreasing seedling height. InArabidopsis no such differences in the relation germination to root length reduction were observed after nitrosoureas and nitrosoguanidines treatment. The differences in the M1 generation of barley andArabidopsis after nitrosoguanidines treatment may be the reason for the non-mutagenic action of MNG and ENG in barley.2)All 4 tested nitrosoamides are active between pH 3 and pH 6 and almost entirely ineffective at pH 7 and in basic solutions.3)The effectiveness (in relation to M1 seedling height reduction) of the tested nitrosoamides with a methyl group (MNG and MNH) is higher than of the nitrosoamides with an ethyl group (ENG and ENH).4)50% decomposition of aqueous solutions (at 25° C) of MNH and ENH takes about 60 hours, for MNG 120 hours and for ENG 420 hours.5)Aeration during treatment or a period in the cold (5° C) after treatment improved the germination of MNG treated seeds. Sucrose, KNO3 and thiourea had no effect.AbstractByla testována aktivita 1-methyl-1-nitrosomočoviny (MNH), 1-ethyl-1-nitrosomočovin (ENH), 1-methyl-3-nitro-1-nitrosoguanidinu (MNG) a 1-etyl-3-nitro-1-nitrosoguanidinu (ENG) po působení na semena ječmene aArabidopsis. Aktivita těchto nitrosoamidč byla vyjadřována klíčivostí a redukcí výšky semenáčků ječmene, respektive redukeí délky kořínkůArabidopsis.1.Po působení obou nítrosomočovin (MNH a ENH) zůstala klíčivost ječmene na úrovni kontroly i u koncentrací, které způsobily maximální redukci výšky semenáčků. Po působení obou nitrosoguanidinů (MNG a ENG) se snižovala klíčivost ječmene paralelně se snižováním výšky semenácků. Podobné vztahy mezi kl íčivostí a redukcí růstu kořinku Arabidopsis nebyly zjištěny. Tyto rozdíly mezi chovánim M1 generace u ječmene a Arabidopsis po působení nitrosoguanidinů mohou být příčinou nemutagenního působení MNG a ENG u ječmene.2.Efektivita (effectiveness) zkoušených nitrosoamidů ve vztahu redukci výşky semenácků u jeěmene byla většý u sloučenin s methylskupinou než s ethylskupinou.3.Všechny čtyři zkoušené nitrosoamidy byly aktivní v roztocich pH od 3 do 6 a téměř zcela neúcinné v neutrálním a alkalickém pH.4.5% rozklad sledovaných nitrosloučenin při 25° proběhne u MNH a ENH za 60 hodin, u MNG za 120 hod. a u ENG za 420 hod.5.Provzdušuování roztoku MNG během působení nebo několikadenní perioda chladu (5 °C) po pùsobení této látky zlepšila klícivost semen ječmene. Bez vlivu na ni zůstalo působení sacharosy, KNO3 a thiomočoviny.AbstractИспытываласъ активность 1-метил-1-нитрозомочевины (MNH), 1-этил-1-нитрозо-мочевины (ENH), 1-метил-3-нитро-1-нитрозогуанидина (MNG) и 1-этил-3-нитро-1-нитрозогуанидина (ENG) после действия на семена ячмена и Arabidopsis. Активность нитрозамидов выражалась всхожестью и по снижению роста проростков ячменя, у Arabidopsis укорочением корней.1.После действия обеих нитрозомочевин (MNH, ENH) всхожесть ячменя осталась на уровне контроля даже у концентраций, наиболее понижающих рост проростков. После воздействия обоих нитрозогуанидинов (MNG, ENG) всхожесть ячменя снижалась параллельно со снижением роста проростков. Сходные отношения между всхожестью и снижением роста корней у Arabidopsis не были установлены. Эти различия в по-ведении M1 генерации у ячменя и Arabidopsis могут быть причиной немутагенного действия MNG и ENG у ячменя.2.Эффективность (effectiveness) испытанных нитрозоамидов по отношению к снижению роста проростков ячменя была более высокой у соединений с группой метиль-ной по сравнению с соединениями с этильной группой.3.Все четыре испытанные нитрозоамида были активными в растворах с pH от 3 до 6 и почти неактивны в щелочных растворах.4.50% разложение соединений происходит при 25 у MNH и ENH за 60 часов, у MNG за 120 часов и у ENG за 420 часов.5.Аэрация раствора MNG при применении или охлажнение (5° С) в течение нескольких дней после воздействия повысили всхожесть семян ячменя. Сахароза, KNO3 и тиомочевина не повлияли на всхожесть.

DNA repair synthesis stimulated by mutagenic N-methyl-N-nitrosourea in barley seeds and free embryos

Abstract Barley seeds and free embryos were treated with a non-lethal dose of N -methyl- N -nitrosourea (MNU) (6 mM, 5 h, 25°C), kept submersed in a solution of [ 3 H]-BUdR and bubbled with nitrogen for 2 days. In these conditions, repair of DNA single-strand breaks was observed only when the seeds were used, whereas in free embryos this repair was not detected. Both MNU-treated seeds and free embryos incorporated about 3 times more [ 3 H]BUdR into DNA of normal density (1.707 g/cm 3 ) than did the untreated samples. This was demonstrated by isopynic centrifugation in caesium chloride gradients. The radioactivity peak at higher density (1.715 g/cm 3 ) observed in experiments with seeds represented a foreign DNA coming from microbial contamination. In experiments with free embryos the radio-activity peak of higher density was absent. 5-Fluorodeoxyuridine (2 × 10 −5 M), present during the incubation of embryos, increased the incorporation of [ 3 H]BUdR into light DNA of both treated and untreated samples at the same rate. In similar experiments, about 3 times more radio-active [ 3 H]thymidine was incorporated into DNA of MNU-treated embryos than into untreated ones.

DNA synthesis during the early stage of germination in the barley embryo meristems and its inhibition by N-methyl-N-nitrosourea

Abstract3 peaks of DNA synthesis were observed in the barley embryo of seeds, germinating for 49 h in running tap water at 25°C. The first peak, found after 22h, was formed by S-cells in the roots and in the 1st leaf meristem. The second peak (after 34–37h) and third peak (after 46–49 h) represents the S-cells in the roots, apex and 1st, 2nd and 3rd leaf meristems. Application of N-methyl-N-nitrosourea for 3 h at the onset of germination inhibited the rate of DNA synthesis and postponed the peaks of DNA synthesis in individual meristems of the embryo.AbstractPři klíčení semen ječmene po dobu 49 h v tekoucí vodě teploty 25°C, byly zjištěny 3 vrcholy syntézy DNA v embryu. První vrchol, zjištěný po 22 h, tvořily S-buňky v kořínku a v prvním listovém meristému. Na druhém vrcholu, zjištěném po 34–37 h a na třetím vrcholu, zjištěném po 46–49 h, se podílely S-buňky v kořenovém a vrcholovém meristému a v prvním, druhém a třetím listovém meristému. Po 3 h působení N-methyl-N-nitrosomoěoviny na začátku klíčení, dochází kinhibici syntézy DNA a ke zpoždění v začátku této syntézy v jednotlivých meristémech embrya.

The effect of inhibitors on the mutagenic activity of N-methyl-N-nitrosourea and N-ethyl-N-Nitrosourea inArabidopsis thaliana

NaN3—a respiratory inhibitor and anaerobic conditions (nitrogen) decrease the mutagenic activity of N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea inArabidopsis thaliana. DNP-an uncoupling agent increases the effect of both nitrosoureas, but this may not be caused by the specific action of DNP.AbstractDýchací inhibitor NaN3 a anaerobní podmínky (dusík) snižují mutační aktivitu N-methyl-N-nitrosomočoviny a N-ethyl-N-nitrosomočoviny uArabidopsis thaliana. Inhibitor oxydační fosforylace DNP zvyšuje účinek obou nitrosomočovin, ale toto zvýšení nemusí být způsobeno specifickým účinkem DNP.AbstractИнгибитор дыхания NaN3 и анаэробные условия (азот) снижают мутагенную активность И-этил-И-нитрозомочевины и И-метил-И-нитрозомочевины yArabidopsis thaliana. ДНП повышает эффект обоих нитрозомочевин, однако, это не обусловлено специфическим влиянием ДНП.

Post-replication DNA repair in barley embryos treated with N-methyl-N-nitrosourea

Abstract In sterile cultures of free barley embryos, N -methyl- N -nitrosourea (MNU) caused a decrease in the size of both template [ 14 C]-labeled DNA and of daughter [ 3 H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [ 3 H]thymidine incorporation. In addition, duplexes containing [ 3 H]-daughter DNA analyzed in BND cellulose contained more single-stranded regions in MNU-treated embryos than in the corresponding control. Incubation of MNU-treated embryos in nutrient medium for up to 18 h after the [ 3 H]-labeling permitted the recovery of small-sized daughter DNA to full-sized strands and led to the enhancement of double-strandedness of DNA duplexes containing [ 3 H]-labeled strands. If [ 3 H]-labeling had been carried out 8–10 h after the MNU treatment, the size of daughter DNA, the proportion of double-strandedness and the rate of thymidine uptake into DNA partially increased in comparison with rates observed when labeling had been done just after or 3 h after the MNU treatment, but these variables did not reach the values of the corresponding controls.