Localization of 5 S genes and transcripts in Pisum sativum nuclei

We have investigated the position of the 5 S gene clusters in the nuclei of Pisum sativum root tip cells, and of their transcripts in the nucleolus, using in situ hybridization and confocal microscopy. Single-stranded RNA probes were produced by in vitro transcription, incorporating fluorescein or digoxygenin label. There are known to be 3 pairs of 5 S gene clusters in this species, which would be expected to give 6 sites of hybridization in G1 cells and 12 sites in G2 cells. In presumed G2 cells, many of the sites appeared as paired spots very close together, which we interpret as the sites on sister chromatids. A clear association of one or more clusters with the nucleolar periphery was observed, and it is possible that this has a functional significance. The transcript labelling within the nucleolus showed a reproducible and highly ordered pattern, consisting of cavities devoid of transcript labelling surrounded by sheets of bright labelling, making a characteristic and often very symmetrical cage-work structure. This labelling pattern may represent an underlying organization of transcript processing within the nucleolus, and is highly reminiscent of the distribution of a previously described nucleolar matrix protein.