Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.
Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).
Marini
N J
NJ
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Benbow
R M
RM
D000818:Animals; D002451:Cell Compartmentation; D002843:Chromatin / Q000648:ultrastructure; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000494:pharmacology; D008845:Microinjections; D010957:Plasmids; D014982:Xenopus laevis / Q000378:metabolism*
D002843:Chromatin; D004270:DNA, Circular; D008836:Micrococcal Nuclease