Molecular dissection of a specific nuclear domain: the chromatin region of the ribosomal gene cluster in Xenopus laevis.

ABSTRACT

Molecular dissection of the nuclear domain corresponding to the ribosomal chromatin cluster was investigated. The experimental scheme was based on the ability of restriction enzymes to digest the whole genome without affecting this region (several megabases in length). Such a strategy involved the judicious choice of restriction enzymes, which is possible in Xenopus laevis, where the rDNA sequence is known and the repeated units are organized into one unique cluster. SalI, XhoI, and EcoRV digestion produced frequent cutting of the genome leaving the ribosomal cluster intact. Isolation of the rDNA cluster was confirmed by separation of the digested DNA by pulsed-field electrophoresis. When applied to purified nuclei, this approach allowed the isolation of the ribosomal chromatin cluster under very mild conditions no cleavages (either enzymatic or mechanical) were detectable. Since the purification scheme depends only on the DNA sequence outside of the rDNA cluster, it permits the obtention of this domain in different functional states. Electron microscopic analysis demonstrated that the domain organization is substantially preserved and maintains its looped organization (the size and the full number of loops were preserved). This purification scheme provides a powerful tool for studying the structure-function relationships within the ribosomal nuclear domain.