Distinguishing the sites of pre-rRNA synthesis and accumulation in Ehrlich tumor cell nucleoli

Abstract

The precise location of transcribing rRNA genes within Ehrlich tumor cell nucleoli has been investigated using two approaches: high-resolution autoradiography of cells pulse-labelled with tritiated uridine, varying the exposure time, and in situ-in vitro transcription coupled with an immunogold labelling procedure. When autoradiographic preparations are exposed for a short time, silver grains are found associated almost exclusively with interphasic cell nucleoli. Labelling of extranucleolar areas requires longer exposure. Within the nucleolus, the first sites to be revealed are in the dense fibrillar component. Prolonging exposure increases labelling over the dense fibrillar component, with label becoming more and more apparent over the fibrillar centers. Under these conditions, however, labelling does not extend into the granular component, and no background is observed. Initiation of transcription on ultrathin cell sections occurs preferentially at the borders of condensed chromatin blocks and in their close vicinity. The condensed chromatin areas themselves remain unlabelled. Inside most nucleoli, gold-particle clusters are mainly detected in the fibrillar centers, especially at their periphery, whereas the dense fibrillar component and the granular component remain devoid of label. These results, together with previous observations made on the same cell type, clearly indicate that the fibrillar centers are the sites of rRNA gene transcription in Ehrlich tumor cell nucleoli, while the dense fibrillar component is the site of pre-rRNA accumulation.