Replication of plasmid DNA in fertilized Xenopus eggs is sensitive to both the topology and size of the injected template.
The behavior of various plasmid templates was examined following their microinjection into fertilized eggs of the frog Xenopus laevis using an assay that permits the examination of both replicated and unreplicated plasmids in single eggs. Our results show that both the size and the topology of the template drastically affect the fate of the injected plasmid. Only a small proportion of injected monomeric supercoiled plasmids underwent replication during 6 h of incubation, although not all injected cells supported replication. Nicked circles were less stable than supercoiled molecules, and we could not detect their replication. Linear monomeric molecules polymerized into large, randomly oriented multimers which were extensively, but not entirely, replicated. Similar results were obtained when linear templates were ligated into polymeric forms in vitro prior to injection. Thus large molecules or molecules which, due to their topology, could be converted into high molecular weight forms following injection were preferred templates for replication. On rare occasions tandemly repeated, high molecular weight DNA was generated following the injection of supercoiled plasmid monomers. This large DNA was shown to be almost entirely replicated.
Endean
D J
DJ
Department of Genetics, University of Wisconsin, Madison 53706.
Smithies
O
O
D000818:Animals; D004247:DNA / Q000302:isolation & purification; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D005306:Fertilization; D008845:Microinjections; D010957:Plasmids; D011108:Polymers; D013698:Templates, Genetic; D014982:Xenopus laevis; D015053:Zygote
D011108:Polymers; D004247:DNA; D004262:DNA Restriction Enzymes