In vitro insertions and deletions in the G segment of phage Mu DNA do not abolish the inversion process

Abstract

The hybrid plasmid pKN56 which contains the right-end PstI·B fragment of phage Mu DNA with its invertible G segment has been used to test whether the physical integrity of the G segment is required for the inversion process. Insertion of a 4.1-Md fragment encoding resistance to kanamycin into the KpnI site located within the G segment did not abolish the invertibility of the G segment in the resulting plasmids (pKN72, pKN73). Insertion of additional non-Mu DNA up to a total molecular weight of about 10 × 10 6 also did not impair G inversion. In vitro shortening of the G segment to about half its size by removal of the internal HpaI fragment also failed to alter the inversion process. The inversion also occurs with normal frequency in E. coli mutants hip, himA, and himB, which affect integration of phage λ and Mu development. By measuring the curing efficiency of the enlarged and shortened plasmids in a polAts strain grown at 43°, it could be shown that no transposition of the G segment occurs at levels above 10 −5 for pKN72 and pKN73, and above 10 −7 for pKN119. However, transposition of Tn601 inserted within the G segment could not be detected. The KpnI and HpaI restriction sites within the G segment have been partially mapped by marker rescue experiments with amber mutants in Mu genes S and U. From the complementation results it can be concluded that genes S and U are expressed from different promoters.