Two transgenic clones X3 and X15 of Nicotiana tabacum var. Xanthi, heterozygous in two genes (a1 and a2) for chloroplast differentiation and transformed with the E. coli DNA repair gene ada cloned downstream from the 1' direction of the dual mas promoter, differed in the expression of the ada gene, in the number of copies of integrated T-DNA and in the response to the mutagenic action of alkylating and non-alkylating agents. The X3 genome contained four copies and the X15 genome one copy of T-DNA, nevertheless the expression of the ada gene, measured by the activity of O6-alkylguanine DNA alkyltransferase (ATase), was about six times higher in X15 than in X3. ATase activity in both clones was highest in extracts from callus whereas very low (X15) or no (X3) activity was detected in leaf extracts. This may explain the lack of difference between X15 and non-transformed tobacco (NTX) in the frequency of N-methyl-N-nitrosourea (MNU)-induced somatic mutations in leaves. In contrast, the frequency of somatic mutations in X3 was about 2-5 times higher than in NTX and X15 after the same doses of MNU, methyl methanesulfonate, maleic hydrazide and gamma-rays. Alteration of plant gene(s) essential in mutation pathway(s) by insertion of T-DNA or by somaclonal variation may explain the higher sensitivity of the X3 clone.

The protein coding region of theE. coli DNA repair geneada combined with the CaMV 35S promoter has been transferred to tobacco by means ofAgrobacterium tumefaciens Ti plasmid. In transgenic plants having theada gene in a sense orientation, detectable amounts of O6-alkylguanine-DNA-alkyltransferase has been found whereas in non-transformed plants this activity is absent. Cell suspension cultures derived from the former plants showed lower sensitivity to the toxic (growth inhibiting) effects of the bifunctional alkylating agent 1-(2-chloroethyl)-1-nitroso-3-(aminomethyl-1,3-diazinylo)-methylurea compared with cell cultures derived from a control non-transformed plant or from transgenic plants harbouring theada gene in an opposite, non-sense orientation.

The protein coding region of the E. coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid. Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity. The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants. This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.

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.

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.

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.

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

Lysates of barley chloroplasts release more radioactivity into acid soluble form from UV-irradiated and alkylated-depurinated E. coli [3H] DNA than from intact DNA. By means of affinity chromatography on depurinated DNA-cellulose and/or UV irradiated DNA-cellulose and by electrophoresis in polyacrylamide gels, four activities on depurinated DNA were separated. One of these contained activity against heavily UV-irradiated /270 J.m-2/ native DNA. In addition, two other nucleases specific towards UV-DNA were separated. One of them was active on native and heat denatured DNA irradiated with 10 J . m-2 UV, whereas the other was predominantly active on native UV-irradiated DNA.

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.

In sterile cultures of free barley embryos, N-methyl-N-nitrosourea (MNU) caused a decrease in the size of both template [14C]-labeled DNA and of daughter [3H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [3H]thymidine incorporation. In addition, duplexes containing [3H]-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 [3H]-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 [3H]-labeled strands. If [3H]-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.

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.

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.

[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.

Diphenylamine-formic acid hydrolyzate of Bacillus subtilis DNA (a mixture of pyrimidine oligodeoxyribonucleotides) was chromatographed using an ionic strength gradient on seven ion exchangers: five DEAE-Spherons 300 of various capacities, two types of BD-Spheron 300 and DEAE-Sephadex A-25. The suitability of ion-exchange derivatives of Spheron for the chromatography of oligonucleotides is discussed. The separation of mono- to undeca-deoxyribonucleotides was achieved.