Molecular architecture of the amplified nucleoli of Xenopus oocytes

An understanding of the functional organization of nucleoli, the sites of r ibosome biosynthesis, is limited by the present uncertainty about the topol ogical arrangement of the transcribing rRNA genes. Since studies with 'stan dard' nucleoli from somatic cells produced conflicting results, we have exa mined the amplified nucleoli of Xenopus oocytes. These nucleoli are unique in that they contain high copy numbers of rRNA genes, are not attached to c hromosomes, lack non-ribosomal DNA and can be examined in light microscopic spread preparations of nuclear contents. By immunostaining and confocal mi croscopy we show that in growing stage IV oocytes the sites of rDNA are sur rounded by the dense fibrillar component. The rDNA is actively transcribed as revealed by BrUTP injection into oocytes and localization of components of the nucleolar transcription machinery (RNA polymerase I and the transcri ption factor UBF). At the ultrastructural level, the rDNA sites correlate w ith the fibrillar centers of amplified nucleoli fixed in situ. The results provide clear evidence that the transcriptionally active rRNA genes are con fined to the fibrillar centers of the oocyte nucleoli and open the possibil ity to analyze the protein composition of almost native, transcriptionally highly active nucleolar chromatin by immunofluorescence microscopy.