Gene-directed mutagenesis in bacteriophage T7 provided by polyalkylating RNAs complementary to selected DNA sites.

摘要

Bacteriophage T7 early transcripts were used as carriers of alkylating groups to affect complementary T7 DNA sites for inducing mutations in preselected genes. A heterofunctional polyalkylating agent N,N,N'-tri-(beta-chloroethyl)-N'-(p-formylphenyl)propylene diamine-1,3 was attached to 3-5% of the transcript nucleotides. The controlled alkylating groups carried on RNA were activated after RNA . DNA hybridization. The modified transcripts were shown to hybridize only with the complementary H strand and to form covalently bound R loops in the appropriate T7 sites. The T7 DNA molecules locally alkylated by the modified transcripts of gene 1.3 coding for T7 ligase were packaged into T7 proteins and used to infect Escherichia coli B. As judged by plating efficiency and the ligase-deficient E. coli BL2 strain, 4 of 140 plaques obtained after infection contained mutants defective in gene 1.3 in amounts of 0.7-1.5%. The T7 DNA locally alkylated by the modified transcripts of gene 0.3 and 1.1 were used for the transfection of E. coli C1757. Analysis of 24 plaques produced by transfection indicated that 3 contained mutants defective in gene 0.3 in amounts of 2-10%. The mutants had also a second unidentified mutation. Complementation analysis data suggest that the second mutation is due to a defect in gene 1.1. The results obtained demonstrate the efficiency of the approach developed for gene-directed mutagenesis.