Gene rearrangements leading to the expression of an insertion-inactivated tetracycline resistance gene in pBR322.

Cloning into the HindIII site of plasmid pBR322 inactivates the tetR promoter and usually prevents the expression of the tetR gene. The corresponding clones revert to tetracycline resistance at a low frequency. Such reversion is caused by gene rearrangement within the plasmids. DNA sequence analysis reveals three classes of revertants. The first class contains plasmids with partial duplications, which result in the fusion of the promoter of the RNA I species to the tetR gene. The event itself destroys the region encoding the RNA primer for replication and thus the plasmids would be replication defective if the duplication did not also include this region of the molecule. The plasmids from the second class are simple deletions which again fuse the tetR region to the RNA I promoter. In one case, the junction takes place at the end of the RNA I transcript, leaving RNA I and the RNA primer virtually intact. However, it removes the promoter of the RNA primer, the latter now being read from the cloned material. The second member of this class has fused the tetR gene well upstream of the RNA I region so that the RNA primer is still read from its own promoter. The low-level tetracycline resistance is probably due to partial read-through of the RNA I terminator. The third class of revertants differs from the previous two by the acquisition of foreign DNA in the form of an IS2-type insertion element which is known to promote transcription.