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Cloning of Pseudomonas plasmid pMG7 and its restriction-modification system in Escherichia coli
Abstract:
Plasmid pMG7 of Pseudomonas aeruginosa codes for a type II DNA restriction-modification (r-m) system, PaeR7. This plasmid has not been observed to transfer to Escherichia coli either by conjugation or by transformation. We have cloned BglII linears (42 kb) and the BamHI large fragment (37 kb) of pMG7 into cosmid pHC79 (6.4 kb) and introduced the recombinant molecules into E. coli by in vitro packaging. Several clones were obtained which demonstrated in vivo restriction of phage phi 80. One of these clones, GT138, was further tested and showed in vivo modification of phi 80. Extracts from two clones, GT138 and GT125, yielded a restriction endonuclease activity which produced fragments of phi 80 DNA identical to those produced by PaeR7. Cosmid cloning should be useful for obtaining substantial yields of large fragments of plasmids that are difficult to purify in their native strains.
Insights
Researchers cloned the PaeR7 restriction-modification system from Pseudomonas aeruginosa into E. coli using cosmid cloning. This method successfully enabled the study of this difficult-to-transfer plasmid
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Pseudomonas aeruginosa plasmid pMG7 encodes the PaeR7 type II restriction-modification system.
- Direct transfer of pMG7 into Escherichia coli is challenging via conjugation or transformation.
Purpose of the Study:
- To enable the functional analysis of the PaeR7 restriction-modification system by cloning it into E. coli.
- To evaluate the utility of cosmid cloning for studying large, uncooperative plasmids.
Main Methods:
- Cloning of large fragments (37-42 kb) of pMG7 into the cosmid vector pHC79.
- Introduction of recombinant cosmids into E. coli via in vitro packaging.
- Assessment of restriction and modification activity against phage phi 80 in E. coli clones.
Main Results:
- Several E. coli clones exhibiting in vivo restriction of phage phi 80 were obtained.
- Clone GT138 demonstrated both in vivo restriction and modification of phi 80.
- Enzyme extracts from clones GT138 and GT125 produced PaeR7-identical phi 80 DNA cleavage patterns.
Conclusions:
- Cosmid cloning provides a viable strategy for expressing and analyzing large plasmid-borne DNA restriction-modification systems in heterologous hosts.
- This approach facilitates the study of genes from plasmids that are difficult to isolate or transfer using traditional methods.