The requirements for a high level of transposition of bacteriophage Mu

To analyse the functional and structural requirements of Mu transposition a special mini-Mu transposon was constructed. This mini-Mu, situated on a derivative of pBR322, has an easily selectable marker gene conferring chloramphenicol resistance (Cam) cloned between the ends of the mini-Mu. The genes required for transposition are cloned outside the ends and are under control of the PL promoter of bacteriophage lambda. To obtain a high frequency of transposition the A and B products are required. Under B- conditions both cointegrate formation and simple insertions are strongly reduced. The role of B in the transposition process is unknown and is discussed. Deletion analysis of the ends of the mini-Mu has revealed the existence of multiple sites required for transposition. The same sites were found to be strong A-binding sites. In addition two weak A-binding sites were found. Point mutations introduced into these last sites show that they are at least as important for transposition as the strong binding sites. The organization of the binding sites is asymmetric in Mu. At the left end two binding sites are situated more than 100 bp away from the end. These affect specifically the frequency of transposition. The distance between these two sites and the site close to the left end is extremely critical. At the right end also three A-binding sites are found, but only two seem to be important for transposition. Transposition with low frequency can be obtained with a mini-Mu with only one end. Secondary att sites are selected which all show some homology with an A-binding site. An absolute requirement for a 5'T at the artificial end is observed. Based on these results a simple scheme for the evolution of a transposon is discussed.