ANALYSIS OF THE SITE FOR 2ND-STRAND INITIATION DURING REPLICATION OF THE STREPTOMYCES PLASMID PIJ101

The indigenous plasmid pIJ101 is the parent of many cloning vectors us ed in Streptomyces. One early pIJ101 derivative, pIJ702, has been part icularly widely used. PIJ702 lacks sti:cop/korB and accumulates single -stranded DNA (ssDNA). The 1-2 kb BclI-BclI sti: cop/korB and 0.7 kb S peI-BclI sti regions were isolated from pIJ101 and cloned into pIJ702 at the PstI site in both orientations. No ssDNA was detected in constr ucts containing sti present in its correct orientation with respect to the basic replicon, with or without cop/korB. Constructs which contai ned sti in the reverse orientation did accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural ori entation. Furthermore, sti inserted in either orientation into the str ucturally unstable pIJ702-pUC8 shuttle vectors prevented them from rea rranging in S. lividans. The sti function was defined to a 0.53 kb Spe I-SacII fragment and the probable site for second-strand initiation (s si) was identified.