| Unscheduled DNA synthesis induced by gamma-radiation
in radicle meristem cells of Triticum aestivum L. Takaji IKUSHIMA Research Reactor Institute, Kyoto University, Kumatori-cho, sennan-gun, osaka 590-04, Japan DNA damages by physical or chemical agents can be repaired through various processes such as photoreactivation, excision repair, post-replication repair and de novo synthesis,22,24) and these repair processes, unseparably connected with other fundamental metabolism of DNA, i.e. replication and recombination, are essential to living cells, and casually related to mutagenesis.1,8,21) Also, it has been well demonstrated that DNA synthesis can be induced in cells at the non-DNA replicative phase by ionizing or ultraviolet radiation, or chemical mutagens for unicellular organisms3,23,26,28) as well as for mammalian cultured cells,4,7,9,11,27) and that this unscheduled DNA synthesis is commonly associated with the performance of DNA repair replication in excision repair process, one of the most universal repair processes.3,5,6,27,29) The existence of such synthesis in plant cells, however, is problematical, and it has been suggested that green plant cells in general might lack excision repair process.25,30,32,33,35,36) To examine the universality of repair synthesis in plants, a series of experiments have been conducted 15) and the present report deals with unscheduled DNA synthesis in cells in situ in the radicle meristem of germinating seeds of hexaploid wheat after gamma-ray irradiation. Germinating seeds of Triticum aestivum L. variety "Chinese spring" (2n=42) were irradiated with a sublethal dose, 5.5 kR, of 60Co gamma-radiation at 55 kR/h at 25C.14) Seeds were previously surface-sterilised by submersion in 1% sodium hypochlorite for 10 min followed by rinsing thoroughly in distilled water and germinated on moist filter paper in petri dishes containing aqueous solution of 0.1 mg/ml chloramphenicol and 1 mg/ml penicillin in the dark for 3 days at 25C. Immediately after irradiation, the radicles were submerged in (methyl-3H) thymidine solution (3H-TdR, 45 Ci/mM, 10 microCi/ml) containing 10-2 M hydroxyurea, a DNA replication inhibitor,31) for one or two hours, and then rinsed free of the radio-isotope in cool water and in a solution of unlabelled thymidine. Parallel controls with non-irradiated radicles were also run. The radicle tips were then fixed in a solution of acetic-alcohol (3:1 v/v). The Feulgen-stained radicle tips were used for preparing autoradiographs by the dipping technique.16,18) The number of labelled nuclei were counted for each of three radicles per lot on the autoradiograms after exposure and development of the emulsion. The synthesis of DNA was also monitered by measuring the radioactivity of 3H-TdR incorporated in to TCA (trichloroacetic acid)-insoluble fractions. Fixed roots were homogenized with a glass homogenizer and the homogenates were suspended into 10% cold TCA solution in test tubes, which were kept in ice-bath for at least one hour. The precipitates were trapped onto glass fiber discs and washed twice with 5% cold TCA solution, once with cold 95% ethyl alcohol and acetone. After drying, tritium radioactivity was counted in Packard Tri-Carb liquid scintilation spectrometer. |
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