Amplification of an Integration Plasmid Introduced into the tmr A Amplification Unit of A Bacillus subtitis Tunicamycin-resistant Mutant

Abstract

An integration plasmid for Bacillus subtilis, pTUE128 (8.2kb) which contained the amyE'-'bla fused gene, the cat gene, and the DNA replication origin of the E. coli plasmid μBR322, was constructed and was inserted into the amyE region of the chromosome of a tunicamycin-resistant B. subtilis mutant (tmr A7 mutation). After cultivation of the transformants with 100μg/ml of chloramphenicol and/or 10μg/ml of tunicamycin, amplification of pTUE128 was analyzed by the increase of β-lactamase activity, the gene for which was included in pTUE128 and of α-amylase activity from the chromosomal gene, and by the method of DNA-DNA hybridization. This was done using the 24.5 kb amplification unit of tmr A7 either by the addition of the high concentration of chloramphenicol or by the addition of tunicamycin.