Novel rearrangements of IS30 carrying plasmids leading to the reactivation of gene expression

Abstract

Unusual DNA rearrangements involving the prokaryote mobile genetic element IS30 have been identified. In order to study the potential mechanisms for the reactivation of a gene after IS element insertion, IS30 was introduced between the lacUV5 promoter and the galK gene of the multicopy plasmid pFR100. In this plasmid terminators of RNA transcription in the sequence of IS30 prevent expression of the galK gene from the lacUV5 promoter. A number of independently isolated mutant plasmids re-expressing the galK gene were studied and shown to be tandemly repeated dimers of the original plasmid. However, the two copies of IS30 were tandemly repeated at one of the original sites of insertion, while at the other site IS30 and three base pairs were missing. The repeated copies of IS30 were separated by two base pairs, the same as those originally flanking the elements. An apparently identical mechanism generated cointegrates between a derivative of plasmid pFD51 carrying IS30 upstream of a promoterless galK gene and a derivative of plasmid pACYC177 carrying IS30 inserted into the beta-lactamase gene. This arrangement brought the galK gene under the control of the beta-lactamase promoter of pACYC177. A mechanism involving aborted conservative transposition of IS30 is discussed as a possible route for the generation of these novel cointegrates. In a third experiment we isolated an insert of IS30 which was also two base pairs away from an already resident IS30 element. This insertion of IS30 created a strong promoter of RNA transcription, which has the potential to increase the expression of the transposase in the downstream copy of the element.