Mutational Specificity of Photosynthetic Organism, Cyanobacterium Synechococcus at Molecular Level

由于原始的原核生物，如蓝藻，在25亿年前获得了光合作用，导致氧气的产生，地球变成了氧化环境。氧是生物体生存的必要条件，但另一方面，在呼吸、光合作用和新陈代谢过程中产生的反应性氧也会攻击DNA、蛋白质等大分子。脂质和糖。氧化性DNA损伤导致的活性氧突变可能导致肿瘤发生和衰老。蓝藻由于能产生大量的活性氧来维持光合作用，可能是研究氧毒害的模式生物。我们进行了蓝细菌聚球藻的培养、转化、DNA提取、抗生素敏感性和突变检测等技术开发。为了获得活性氧突变的基本信息，还对紫外敏感性和DNA修复能力进行了研究。 ...更多信息 种，我们研究了8-oxoCr的诱变活性，8-oxoCr是碱基的代表性氧损伤，已知其由核黄素光敏化产生，并使用大肠杆菌1测定突变。通风是必要的生长蓝藻除了光和外科磁带是有利的磁带琼脂板。2.对数生长期早期培养有利于质粒转化。3.聚球藻对抗生素、链霉素和氯霉素非常敏感。转化细胞存活和抑制许多非转化细胞的临界浓度为0.5-0.7。IOTA.tg/m1.4为了检测突变，将大肠杆菌crp基因插入到质粒pUC 30 3中，该质粒能够在大肠杆菌和聚球藻中复制。将重组质粒pUCRPk 6导入聚球藻中进行复制，提取质粒，在cip阴性大肠杆菌中进行crp基因突变检测。突变体克隆的分离需要进一步提高突变效率。5.虽然聚球藻比大肠杆菌对紫外线具有更高的抗性，但切除修复能力不如大肠杆菌有效。光复活可能是一个主要的修复系统。6.8-oxoG由于与腺嘌呤错配而引起G-C到TA的颠换。GC到CG的颠换也观察到野生型和mutM突变体，但不是在mutY。光敏DNA的延迟转染可使mutY突变体中的GC向CG转化。致病性病变可能在有氧条件下增加，并且可能是MutY蛋白的底物。少

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