Replication of Ribosomal DNA in Xenopus laevis

Abstract

The localization of the replication origins of r[ribosomal]DNA in X. laevis was studied by 2 different methods. The DNA of X. laevis larvae was fractionated by CsCl gradient centrifugation in bulk and ribosomal DNA and examined by EM. In bulk DNA, clusters of microbubbles, which are related with the origins of replication, appear to be spaced along the DNA molecules at intervals comparable with the size of the average replicon of X. laevis. In rDNA, the distance between adjacent clusters is much shorter and corresponds to the size of the rDNA repeating unit. When rDNA was submitted to digestion with restriction enzymes (EcoRI or HindIII), the microbubbles are observed in the nontranscribed spacer-containing fragment. Cultured cells of X. laevis were synchronized by mitotic selection and incubated with 5-fluoro-2-deoxyuridine for a time longer than the G1 phase. This treatment synchronizes the replicons and allows them to start replicating very slowly. It was thus possible to obtain a preferential labeling of the regions containing the origins. The analysis by gel electrophoresis of the EcoRI-digested rDNA showed that the radioactivity was preferentially incorporated in the fragments which contain the non-transcribed spacer. The rRNA gene cluster apparently consists of multiple units of replication, possibly one per gene unit. The origins of replication are localized into the nontranscribed spacer.