Inactivation of the replication genes of a pSC101 derivative by IS1-mediated integration of the plasmid pSM1.

Abstract

The plasmid pSM1 carrying a single copy of IS1 has been shown to integrate into various sites on another plasmid pHS1, a temperature sensitive replication mutant of the tetracycline resistance plasmid pSC101. The resulting cointegrates (named pMZ plasmids) contain two IS1 sequences in a direct orientation, each at a junction of the integration. This paper describes a detailed analysis of such cointegrates and shows that the integration of pSM1 occurs frequently at a region of pHS1, resulting in inactivation of the pHS1 replication system. To do this, we attempted to isolate pHS1 containing IS1 (namely pHS1::IS1) from eight independently isolated cointegrates using the restriction endonuclease PstI, which cleaves at a single site within IS1 and no site within pHS1. After ligation of the PstI digests of a pMZ plasmid at DNA concentrations favorable for recircularization of each fragment in the digests and subsequent transformation of the ligated DNA, tetracycline resistant transformants were selected at 30°. Two cointegrates (pMZ3 and 7) did indeed give rise to the pHS1::IS1 plasmids which showed temperature sensitive DNA replication like pHS1, while the other six cointegrates (pMZ1, 2, 4, 5, 8 and 9) did not. This suggests that the cointegrates pMZ3 and 7 contain a functional pHS1 replication system, while the others contain an inactive system. The inactivation as a result of integration of pSM1 at its IS1 into sites within pHS1 is like translocation of the IS1 sequence itself which has been shown to inactivate various bacterial genes.We also describe the nucleotide sequence in a pHS1:: IS1 (named pMZ71), generated from pMZ7, of the junction region between the pHS1 and IS1 sequences as well as the entire IS1 sequence. PMZ71 would be a useful plasmid with which to study the IS1 sequence genetically and biochemically.