ABNORMAL INTEGRITY OF THE NUCLEOLUS ASSOCIATED WITH CELL-CYCLE ARRESTOWING TO THE TEMPERATURE-SENSITIVE UBIQUITIN-ACTIVATING ENZYME E1

A mouse cell mutant, ts85, containing the temperature-sensitive ubiqui tin-activating enzyme was arrested in Ga phase at the non-permissive t emperature. In the arrested cells, azure C, a nucleolus-specific stain , revealed a U-shaped or ring-shaped arrangement of nucleolar lobes wi th an unstained region in the center. Silver staining of the nucleolar organizer region (NOR) and fluorescence in situ hybridization (FISH) with rDNA both gave signals in azure C-positive regions. Electron micr oscopic examination revealed a cloud of unidentified electron-dense pa rticles (diameter approximately 70 nm) in the azure C-negative center space. When the arrested cells were released into M-phase, we observed the association of NOR-bearing chromosomes with a pulverization-like abnormality FISH with rDNA and NOR silver staining demonstrated that t he pulverization-like abnormality was restricted to NORs. The frequent occurrence of persistent nucleolar material in prophase and prometaph ase of the stressed cells after release indicated a delayed dissociati on of the nucleolus that brought about the abnormal chromosomes in M-p hase. ts85 cells transfected with the mouse E1 cDNA recovered growth a t the non-permissive temperature and no longer showed abnormal nucleol ar morphology. It seems that the ubiquitin system plays a role in the dissolution of the nucleolus, possibly involving the NOR-bearing chrom osomes.