The Response Of Saccharomyces Cerevisiae Oxidative Damage Repair System To Environmental Genotoxic Factors And Construction Of Temperature-sensitive Mutator Saccharomyces Cerevisiae

Heavy metal ions and UV light radiation in the enviroment have carcinogenicity because they could cause gene toxicity to the cells.Relevant studies showed that the gene toxicity is related with the increase of reactive oxygen species(ROS).ROS indicates the active oxygen free radicals and other oxygen-containing substances with the response characteristics of oxygen free radicals,which can lead to oxidative damage of DNA.Saccharomyces cerevisiae has three pathways to respond the DNA damage caused by ROS,including anti-oxidation pathway,DNA damage checkpoint pathway and DNA damage repair pathway.These pathways form the oxidative damage repair system of Saccharomyces cerevisiae.The anti-oxidation pathway could remove ROS and maintain balance of redox.The DNA damage checkpoint pathway can detect DNA damage and abnormal DNA structure,so the cells could have enough time to repair DNA damage by delay cell cycles or induce apoptosis when unable to repair DNA damage.And the DNA damage repair pathway can repair DNA damage in the cells.In this thesis,the essential relation between environmental genotoxic factors and yeast oxidative damage repair system was studied by measuring the mutation rate of oxidative damage repair system gene deletion strains.Furthermore, temperature-sensitive mutator strains were also constructed for correcting the shortcoming of mutator strains.The major results of the thesis are as follows:1.Study of essential relation between environmental genotoxic factors and yeast oxidative damage repair system by measuring the mutation rate of oxidative damage repair system gene deletion strains.The response of yeast oxidative damage repair system to environmental genotoxic factors was studied by measuring the mutation rates of can1 gene in oxidative damage repair system gene deletion strains under the stress of heavy metal and under the stress of UV light.The results showed that the increased extent between mutation rates of msh2/rad51 gene deletion strains and wild type strain raised much more higher in the stress of heavy metal ions than in the stress of UV light.It indicated that DNA mismatch mutation and DNA strand breaks was more serious in the DNA damage caused by heavy metal ions than which caused by UV light.The mutation rate of tsa1 gene deletion strain also raised much more higher than that of wild type strain in the stress of heavy metal ions,however,the increased extent in the stress of UV was not as high as that of heavy metal ions,which illuminated that the increase of ROS played an important role in the gene toxicity of heavy metal ions.2.The construction of Saccharomyces cerevisiae temperature-sensitive mutator strains.By using the long flanking homology PCR,temperature-sensitive homologous recombination fragment which homologous areas were the promoter and ORF sequence of target gene was cloned,and the homologous recombination fragment of msh2 gene was obtained.Then the Saccharomyces cerevisiae JD54 strain was transformed by this homologous recombination fragment,and the correct transformants were coated on the 5-FOA plate for screening strains without the URA3 marker.Saccharomyces cerevisiae temperature-sensitive mutator strain was constructed successfully,however,when the mutagenic effects were analysesd,the result showed that the mutation rate of mutator strain was not increased at the high temperature as supposed.The reason we speculated was that Msh2 protein was not enough degraded in the high temperature.