Ultrastructural analysis of nucleolar transcription in cells microinjectedwith 5-bromo-UTP

In situ sites of nucleolar transcription in cells microinjected with 5-brom o-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or process ing factors. This high resolution approach allowed us to identify significa nt signal as early as after 4-min incubation periods following BrUTP microi njection. It revealed the dense fibrillar component (DFC) as being the firs t nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreo ver, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. F rom 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreov er, it demonstrates that BrUTP microinjection followed by postembedding det ection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.