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

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Abstract
Plasmid pSM1 carrying a single copy of insertion sequence IS1 integrates into various sites on another plasmid pHS1, a temperature sensitive replication mutant of the tetracycline resistance plasmid pSC101. The resulting cointegrates (pMz plasmids) contain 2 IS1 sequences in a direct orientation, each at a junction of the integration. A detailed analysis of such cointegrates is described and shows that the integration of pSM1 occurs frequently at a region of pHS1 resulting in inactivation of the pHS1 replication system. Attempts were made to isolate pHS1 containing IS1 (pHS1::IS1) from 8 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 [Escherichia coli] were selected at 30.degree. C. Two cointegrates (pMZ3 and 7) gave rise to the pHS1::IS1 plasmids which showed temperature sensitive DNA replication like pHS1; the other 6 cointegrates (pMZ1, 2, 4, 5, 8 and 9) did not. Thus, cointegrates pMZ3 and 7 evidently contain a functional pHS1 replication system, while the others contain an inactive system. 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. The nucleotide sequence in a pHS1::IS1 (pMZ71), generated from pMZ7, of the junction region between the pHS1 and IS1 sequences is described as is the entire IS1 sequence. pMZ71 would be useful to study the IS1 sequence genetically and biochemically.