Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA
AUTOR(ES)
Lin, Yun-Long
RESUMO
Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55°C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Kmr) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Kmr transformants.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=241604Documentos Relacionados
- Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication.
- Site-specific in vitro binding of plasmid pUB110 to Bacillus subtilis membrane fraction.
- Functional analysis of the leading strand replication origin of plasmid pUB110 in Bacillus subtilis.
- Replication origins of single-stranded-DNA plasmid pUB110.
- A DNA sequence outside the pUB110 minimal replicon is required for normal replication in Bacillus subtilis.