Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus.
A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization. We cloned the nuc1+ gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I. We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (approximately 190 kD and 2 x 10(4) copies/cell) in the S. pombe nuclear fraction. By immunofluorescence microscopy, anti-nuc1+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus. The nucleolar immunofluorescence by anti-nuc1+ was faded in nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II. Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.
Hirano
T
T
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Konoha
G
G
Toda
T
T
Yanagida
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002466:Cell Nucleolus / Q000648:ultrastructure*; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D012318:RNA Polymerase I / Q000235:genetics / Q000502:physiology*; D004718:Saccharomycetales / Q000648:ultrastructure*; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure*
D012318:RNA Polymerase I; D004264:DNA Topoisomerases, Type I