Abstract Objective:To establish a reformative detection system which has sound ability of providing infor-mation on molecular mutagenesis spectrum and the specificity of detection system of repackaged λ phage.Methods...Abstract Objective:To establish a reformative detection system which has sound ability of providing infor-mation on molecular mutagenesis spectrum and the specificity of detection system of repackaged λ phage.Methods:LacZ gene,as mutational target gene and reporter gene, was applied into the detection system.The λ gt11 DNA treated with ENU(1-ethyl-1-nitrosourea)and 9-AA(9-aminoacridine)was repackaged in vitro.The packaged λ phage was then grown in E.coli Y1090 on a selective plate containing X-gel and IPTG.The survival and mutation frequencies were determined by counting the clear-plaque and blue-plaque,and the molecular mutation mechanism was studied by extracting and sequencing the LacZ gene of mutants.Results:The survival of repackaged λ phages treated with 9-AA and ENU apparently decreased in consistent dose-dependence.The mutation frequency of clear-plaque mutants showed a linear dose-related increase.The predominant mutations induced by 9-AA were ±1 frameshift mutation, and 9-AA induced -1 frameshift was much more effective than induced +1 frameshift.9-AA also induced substitutions with transversions more common.ENU -induced mutations were chiefly occurred at G:C sites .Substitutions induced by ENU were mainly G:C→A:T,G:C→C:Gand A:T→T:A transversion.Conclusion;Mutation detection sys-tem of λgt 11 DNA containing LacZ gene is proven better than that of λDNA without LacZ gene.The combi-nation of survival, mutant frequency and sequence spectrum can not only increase the sensitivity and specifici-ty of the new method, but also provide a better understanding of the molecular mechanism of mutation for ul-timate extrapolation to risk assessment.展开更多
基金National Science Foundation of China (NS-FC:No: 3880680 No: 39670643)
文摘Abstract Objective:To establish a reformative detection system which has sound ability of providing infor-mation on molecular mutagenesis spectrum and the specificity of detection system of repackaged λ phage.Methods:LacZ gene,as mutational target gene and reporter gene, was applied into the detection system.The λ gt11 DNA treated with ENU(1-ethyl-1-nitrosourea)and 9-AA(9-aminoacridine)was repackaged in vitro.The packaged λ phage was then grown in E.coli Y1090 on a selective plate containing X-gel and IPTG.The survival and mutation frequencies were determined by counting the clear-plaque and blue-plaque,and the molecular mutation mechanism was studied by extracting and sequencing the LacZ gene of mutants.Results:The survival of repackaged λ phages treated with 9-AA and ENU apparently decreased in consistent dose-dependence.The mutation frequency of clear-plaque mutants showed a linear dose-related increase.The predominant mutations induced by 9-AA were ±1 frameshift mutation, and 9-AA induced -1 frameshift was much more effective than induced +1 frameshift.9-AA also induced substitutions with transversions more common.ENU -induced mutations were chiefly occurred at G:C sites .Substitutions induced by ENU were mainly G:C→A:T,G:C→C:Gand A:T→T:A transversion.Conclusion;Mutation detection sys-tem of λgt 11 DNA containing LacZ gene is proven better than that of λDNA without LacZ gene.The combi-nation of survival, mutant frequency and sequence spectrum can not only increase the sensitivity and specifici-ty of the new method, but also provide a better understanding of the molecular mechanism of mutation for ul-timate extrapolation to risk assessment.
