[Design of hybrid plasmids based on the actinomycete plasmid pSB24.1 and plasmid prBR325 and a study of their stability in the Streptomyces-E. coli system].

Abstract

Actinomycete plasmid pSB24.1 was cloned on vector of the E. coli pBR325 system. The following bireplicon plasmids were obtained: pSU501 and pSU502 (by XhoI site of pSB24.1 and SalGI site of pBR325), pSU503 (by Bg1II (c) site of pSB24.1 and BamHI site of pBR325) and pSU504 (by Bg1II(b) site of pSB24.1 and BamHI site of pBR325). In the cells of E. coli C600 plasmids pSU501-504 determined phenotype AprCmrTcs and were stable. In the cells of Str. lividans the initial structure pSU501 selected by Ltz+ phenotype was maintained at a frequency of 12.5 per cent. Analysis of the deletion variants of pSU501 isolated from Str. lividans showed that the deletions were induced by both the pBR325 region and the pSB24.1 DNA fragment near XhoI site. The region from SacII(a) site to Bg1II(b) site clockwise in the map of plasmid pSB24.1 was not significant for its replication and maintenance in Str. lividans. There were detected unique sites of pSB24.1 and its derivatives useful for cloning. Possible shortening of plasmid pSB24.1 by 567 kb (the length between the terminator of the open frame reading translation and XhoI site) was revealed.