Summary: DNA from ATCC 12475 was partially digested with 3A and fragments were ligated into /II-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4·45 and 11·55 kbp respectively, each retransformed to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of chromosomal DNA in Southern blot experiments. deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1·95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of Streptomycin phosphotransferase activity appeared in extracts of , TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1·5 rather than 2 d, and reached a level over twice that of the original strain.


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