In vitro transposition of bacteriophage Mu: A biochemical approach to a novel replication reaction

Abstract

The transposition-replication reaction of phage Mu has been reproduced in a cell-free reaction system. Two assay methods were used for the detection of transposition products. The first method uses λ DNA as the target of transposition and a plasmid containing the ends of Mu DNA and an ampicillin-resistance gene as the donor; after the reaction, in vitro X packaging allows the scoring of amp′ transducing phages generated by transposition. In the second method, the products made in the presence of a radioactive precursor for DNA synthesis are directly analyzed by gel electrophoresis and unique product species are identified. The reaction requires a donor DNA carrying the two Mu ends in their proper relative orientation, extracts containing the A and B gene products of Mu, and host factor(s). RNA synthesis by E. coli RNA polymerase is not required for the reaction. The products include both cointegrates and simple inserts. Both types of products show incorporation of radioactive DNA precursors; however, simple inserts do not seem to undergo a full round of DNA replication.