J. Šatava

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.

Induction of proteinase K-sensitive sites and M. luteus endonuclease-sensitive sites in DNA of barley embryos by sodium azide

Abstract DNA damage induced in germinating barley embryos by mutagenic and sublethal doses (0.1–2 mM, 2 h) of sodium azide, applied at pH 3, was measured by alkaline elution. Isolated nuclei were lysed at a high pH with either 2% SDS or 2 M NaCl on polyvinyl chloride filters and digested with proteinase K or with Micrococcus luteus endonuclease prior to elution. The azide treatments resulted in a dose-dependent increase of proteinase K-sensitive sites and an appearance of Micrococcus luteus endonuclease-sensitive sites. These sites were detected as DNA single-strand breaks after digestion of the DNA with either one or both of the enzymes. The two types of lesion were additive and occurred in a ratio of about 1:1. The additive effect suggested independent origin for the two types of lesion. Breaks independent of proteinase K digestion appeared only when DNA was analysed 24 h after the action of azide. The nature and significance of these DNA lesions are discussed.

Isolation from barley embryo of endonuclease specific for apurinic sites in DNA

The endonuclease activity specific for apurinic sites in DNA was detected in barley embryos. The enzyme was partially purified. It reveals high activity on partially depurinated DNA but low or nil activity on intact and alkylated DNA. The method used for the detection of enzyme activity was based on the changes in the sedimentation velocity of substrate DNA in neutral sucrose gradients with 80 % formamide.

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.

NUCLEASES OF BARLEY CHLOROPLASTS ACTING ON DNA MODIFIED BY UV-LIGHT AND BY METHYL METHANESULPHONATE

ABSTRACT By means of affinity chromatography and gel electrophoresis four forms of nuclease acting specifically on depurinated DNA, one nuclease predominantly active on native and denatured UV irradiated DNA, one nuclease predominantly acting on native UV irradiated DNA were partially purified from barley chloroplasts In addition a possible glycosylase activity for alkylated DNA was also detected in the soluble chloroplast material.

Similar degree of methyl methanesulphonate-induced DNA damage in two clones ofTradescantia differing in mutagenic sensitivity to alkylating agents

In two clones ofTradescantia (4430 and 02) differing in the sensitivity to the mutagenic action of alkylating agents, equimolar doses of [14C] methyl methanesulphonate (MMS) elicited a similar degree of protein, RNA and DNA alkylation and a similar amount of DNA-7-methylguanine and DNA-3-methyladenine in cells of inflorescence. Moreover, in the same clones and tissues the same doses of nonlabelled MMS produced a similar amount of DNA single strand breaks and/or alkali labile sites as measured in alkaline sucrose gradients. None of the DNA lesions followed is therefore decisive for explanation of the different mutagenic sensitivity ofTradescantia clones.

Nucleases in chloroplast of barley.

Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions were as active to UV-irradiated (270 J m-2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high concentrations of EDTA, NaCl, Mn2+, Ca2+, Zn2+ ions and by N-ethylmaleimide. They do not require Mg2+ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide pH range (5.5–9), the optimum for DNase action in the presence of Mg2+ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely that DNase acts in the endonucleolytic way.

Utilization of the label from bacterial [3H] DNA by isolated barley roots and embryos under different conditions

Abstract[3H] DNA fromEscherichia coli and [3H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [3H] thymidine and [3H] DNA treatment which shows that the activity of [3H] DNA is utilized during the S phase. After application of [3H] thymidine, only cell nuclei in S phase were labelled. After the application of [3H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [3H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [3H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [3H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA.

Nicotiana tabacum DNA: Isolation from cell culture

The adaptation of Marmur’s method, suitable for DNA isolation from plant cell culture, is described.

HETEROGENEITY OF AP-ENDONUCLEASE IN BARLEY CELLS

ABSTRACT Five to six distinct forms of AP-endonuclease were found in barley cells by means of phosphocellulose chromatography. Some of their properties are described and their origin is discussed.