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Mutational Specificity of Photosynthetic Organism, Cyanobacterium Synechococcus at Molecular Level

光合生物、蓝藻聚球藻在分子水平上的突变特异性

基本信息

批准号：
09680525
负责人：
TAKIMOTO Koichi
金额：
$ 2.18万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680525/
关键词：
cyanobacteria mutation oxidative damage UV senstivity DNA damage base change reactive oxygen species photosensitization 酸化的傷害らん藻 Synechococcus pUC303 活性酵素遺伝子 DNAの傷害光合成

项目摘要

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 … More 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

由于蓝藻等原始原核生物在25亿年前获得了产生氧气的光合作用，地球就变成了氧化状态。虽然氧气是生物的先决条件，但另一方面，在呼吸、光合作用和代谢过程中产生的活性氧会攻击DNA、蛋白质等大分子。脂质和糖。由活性氧引起的DNA氧化损伤引起的突变可能导致肿瘤发生和衰老。蓝藻在光合作用过程中产生大量活性氧，可能是研究氧毒性的模式生物。蓝藻聚球菌培养、转化、DNA提取、抗生素敏感性、突变检测等技术开发。为了获得活性氧突变的基本信息，我们研究了8-oxoCr的致突变活性，8-oxoCr是一种典型的氧损伤碱基，已知是由核黄素光敏产生的，并使用E.coli.1测定了突变。通风对于蓝藻的生长是必要的，除了光和手术胶带是有利的胶带琼脂板。2. 早期对数培养有利于质粒转化。3. 聚珠球菌对抗生素、链霉素和氯霉素非常敏感。转化细胞存活和抑制许多未转化细胞的临界浓度为0.5-0.7 IOTA.tg/m1.4。将crp基因插入质粒pUC3O3中检测大肠杆菌coii突变，该基因在大肠杆菌coii和聚血球菌中均能复制。将重组质粒pUCRPk6导入聚球菌中进行复制后，在cip阴性的大肠杆菌中检测其crp基因的突变。突变克隆的分离需要进一步提高突变效率。5. 虽然聚球菌比大肠杆菌对紫外线有超抗性，但其切除修复能力不如大肠杆菌。光活化可能是一种主要的修复系统。6.8-oxoG与腺嘌呤错配导致G-C向TA翻转。在野生型和mutM突变体中也观察到GC到CG的转换，但在mutY中没有。在mutY突变体中，延迟转染光敏DNA可以使GC增加到CG横向。在有氧条件下，致病性病变可能增加，并且可能是MutY蛋白的底物。少