Expression of circular and linearized bacterial chloramphenicol acetyltransferase genes with or without viral promoters after injection into fertilized eggs, unfertilized eggs and oocytes ofXenopus laevis.

Abstract

Circular and linearized plasmids containing bacterial chloramphenicol acetyltransferase (CAT) genes connected or not connected to viral promoters were injected into fertilized eggs, unfertilized eggs and oocyte nuclei ofXenopus laevis, and CAT enzyme expression was studied under different conditions. Circular DNAs injected into fertilized eggs did not change their molecular form greatly, but CAT enzyme activity was expressed by the blastula or gastrula stage depending on the strength of the enhancer contained within the promoter. Linearized plasmid DNAs injected into fertilized eggs were concatemerized and replicated extensively by the blastula stage, and were expressed also actively irrespective of whether DNAs contained the promoter or not. The CAT enzyme activity was roughly comparable to the level of CAT mRNA in injected embryos. Similar results were obtained for both circular and linearized DNAs in unfertilized eggs, although the level of CAT enzyme expressed here was quite low. In oocyte nuclei, by contrast, only circular DNAs were expressed, and the expression was independent of whether or not the DNAs contained the promoter. The large concatemers formed in embryos comigrated with host DNA, but the majority of them disappeared later, at the tadpole stage, suggesting the extrachromosomal nature of these DNAs. The pronounced decrease in the amount of comigrating DNAs was accompanied by the disappearance of both CAT mRNA and enzyme activity. Therefore, we conclude that active CAT enzyme expression induced by injection of linearized DNAs in embryos and early stage larvae is due mainly to transcription from the transiently existing extra-chromosomal concatermers rather than from long-lasting, probably genome-integrated DNAs.