Plasmid cloning vectors that can be nicked at a unique site.
We describe ColEl-type plasmids, with relaxed DNA replication, based on pMB9, and carrying the CmR determinant of R1, in addition to the TcR determinant of pMB9. One of the plasmids, pPH207, has unique sites for EcoRI, HindIII, BamI, SalI and HpaI. Insertion of foreign DNA into all but the last of these inactivates either the CmR or the TcR determinant. The original CmR TcR plasmid (pCM2) contains a copy of IS1 which produces deletions to left and to right. Most of these inactivate either the CmR or the TcR determinant. An internal 280 bp deletion of IS1 DNA in pPH207 greatly reduces the frequency at which deletions are observed. The main feature of these plasmids is a site that is cleaved by some preparations of EcoRI in only one strand of the DNA duplex (the EcoRIn site). This site facilitates strand separation of sequences inserted at the HindIII, BamI and SalI sites of the TcR gene, and also of any inserted at the true EcoRI site by a method that destroys that site. Since the orientation of the EcoRIn site is known, the orientation of sequences inserted at the neighbouring sites can be easily determined. Plasmid pPH207 is not mobilised by a Hfr, but its mobilisation is promoted by ColEl. It is therefore Mob- bom+. Experiments with minicells show that it directs the copious synthesis of chloramphenicol transacetylase.
D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D010641:Phenotype; D010957:Plasmids; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004264:DNA Topoisomerases, Type I