Abstract
The chloramphenicol-resistance (CPr) plasmid pTZ12 (2.55kb) in Bacillus subtilis was genetically analyzed in detail, and the CPr determinant and the functional unit of replication were mapped. The plasmids pTZ12 and pBR322 were digested with suitable restriction endonucleases and Hgated with T4 ligase. The ligated DNAs were introduced into E. coli by transformation and CP-resistant transformants were selected. In conclusion, the CPr determinant was mapped between a TaqI site and a BclI site (about 900 base pairs) on pTZ12. A set of pTZ12-pBR322 recombinant plasmids isolated from E. coli was introduced into B. subtilis by transformation to test for ability to replicate in B. subtilis. From the results, the region of the functional unit of pTZ12 replication was mapped. It was also proved that the gene product of this CPr determinant was chloramphenicol acetyltransferase (CAT) and the native CAT in the cells carrying pTZ12 was a dimeric protein with two identical subunits having a molecular weight of approximately 24, 000 (24 K).
