Amplification of bacteriophage Mu DNA by rolling circle DNA replication in vitro.
When mini-Mu DNA was allowed to transpose and replicate in vitro over a prolonged period, the products consisted not only of simple inserts and cointegrates but also high molecular weight DNA many times the unit length of mini-Mu. This high molecular weight product contained predominantly full-length mini-Mu DNA and relatively little non-Mu DNA (the vector harboring the mini-Mu element and the target for transposition in the reaction system). It arose from rolling circle DNA replication of templates created by intramolecular strand transfer, which is catalyzed by Mu transposition proteins. A donor substrate, which is a supercoiled plasmid bearing a mini-Mu element, gave rise to large amounts of the high molecular weight product provided that the vector segment outside the mini-Mu element was 2 kilobase pairs or more. When a donor substrate had a vector segment of only 600 base pairs, the mini-Mu element first had to transpose to a larger circular target before giving rise to the high molecular weight product. These results suggest a mechanism by which Mu DNA can be amplified for lytic development without transposing multiple times. By establishing a circular template, multiple copies of Mu can be processively generated from a single initiation event.
Nakai
H
H
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, District of Columbia 20007.
D010583:Bacteriophage mu / Q000235:genetics*; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D008970:Molecular Weight; D013698:Templates, Genetic; D014764:Viral Proteins / Q000502:physiology
D004270:DNA, Circular; D004279:DNA, Viral; D014764:Viral Proteins