| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Since primodial prokaryotic organisms such as cyanobacteria bad acquired photosynthesis resulting in the generation of oxygen 25 hundred million years ago, the earth changed to oxidive conditions. Although oxygen is prerequisite for bioorganisms, on the other hand reacitvc oxygen species produced duriing respiration, photosynthesis and metabolism attack macromolcules such as DNA, protein. lipids and sugars. Mutations induced by oxidative DNA damage cue to reactive oxygen species possibly cause oncogenesis and aging. Cyanobacteria is probably a model organism for investigation of oxygen toxicity because of the generation of much amount of reactive oxygen species curing photosynthesis. We have perfomed technical deveIopment of the cuIture of cyanobacteria Synechococcus, transformation, extraction of DNA, sensitivity to antibiotics and detection of mutation. UV sensitivity and DNA repair capacity were also investigated To obtain fundamentaI information about mutations by reactive oxygen s
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pecies, we investigated mutagenic activity of 8-oxoCr, a representative oxygen damage of bases, which has been known to be produced by riboflavin photosenstization and assayed mutation using E.coli.
1. Ventilation is necessary for growth of cyanobacteria in addition to light and surgical tapes are advantage for taping agar plates. 2. Early log phase culture is good to transform with plasmids. 3. Synechococcus is very sensitive to antibiotics, streptomycin and chloramphenicol. Critical concentration for the survival of transformed cells and the suppress of many non-transformed cells was 0.5-0.7 IOTA.tg/m1.4. To detect mutation E.coIi crp gene was inserted into plasmid pUC3O3, which is able to replicate in both E.coIi and Synechococcus. After the reconstructed plasmid pUCRPk6 was introduced into Synechococcus and subjected to replication, extracted plasmid was assayed in cip negative E.coIi to determine the mutation on crp gne. Further inc7ease in mutation efficiency is required for isolation of mutant clones. 5. Although Synechococcus is hyper-resistant to UV rather than E.coIi, excision repair capacity was less effective than E.coIi. Photoreactivation may be a primary repair system. 6.8-oxoG caused G-C to TA transversion as the result of mismatch with adenine. GC to CG transversions were also observed in wild type and mutM mutator mutant although not in mutY . Delayed transfection of photosensitized DNA allowed to iucrease GC to CG transversious in mutY mutant. Causative lesions may increase in an aerobic conditions and may a substrate of MutY protein. Less
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