Drug-induced dispersal of transcribed rRNA genes and transcriptional products: immunolocalization and silver staining of different nucleolar components in rat cells treated with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole.

Abstract

Upon incubation of cultured rat cells with the adenosine analog 5,6-dichloro-1-.beta.-D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar necklaces. This experimental system, which does not inhibit transcription of the rRNA genes, used immunocytochemistry to study the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosome protein (S1) of the small ribosomal subunit permit localization of nucleolar preribosome particles. Under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase I and argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. Although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. Each RNA polymerase-positive granular unit represents a single transcription unit and each continuous array of granules (string of nucleolar beads) reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules, a minimum of 60 rRNA genes are estimated to be active during interphase of DRB-treated rat cells.