Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP

Abstract 

In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4–90 min at 37°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 processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 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. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br- labeled RNA is a useful technique for high resolution studies of structure–function associations in the nucleolus.