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New mutation detection system of repackaged λ gt11 DNA containing LacZ gene

New mutation detection system of repackaged λ gt11 DNA containing LacZ gene
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摘要 Objective: To establish a reformative detection system which has sound ability of providing information 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: G and A: T→T: A transversion. Conclusion: Mutation detection system of λgt11 DNA containing LacZ gene is proven better than that of λDNA without LacZ gene. The combination of survival, mutant frequency and sequence spectrum can not only increase the sensitivity and specificity of the new method, but also provide a better understanding of the molecular mechanism of mutation for ultimate extrapolation to risk assessment. 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.
出处 《Journal of Medical Colleges of PLA(China)》 CAS 2002年第3期162-166,共5页 中国人民解放军军医大学学报(英文版)
基金 National Science Foundation of China (NS-FC:No: 3880680 No: 39670643)
关键词 MUTATION λgt11 DNA LacZ gene 9-aminoacridine 1-ethyl-1-nitrosourea 再包装λgt11DNA LacZ基因 新突变检测系统 报告基因
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