Nucleolar localization of rRNA coding sequences in Prorocentrum micans Ehr. (dinomastigote, kingdom Protoctist) by in situ hybridization

Abstract

To define the molecular mechanisms of ribosome biogenesis and to find out in which nucleolar compartment transcription of rDNA occurs, we have performed in situ hybridization (ISH) of RNase-treated cryosections using biotinylated rRNA coding sequences as a probe and the eukaryotic dinoflagellate nucleolar system as a model. Recent data from ISH of eukaryotic ribosomal genes by electron microscopy (EM) has so far failed to establish a consensus which clearly defines the function of the three compartments of the nucleolus. Dinomastigote protoctists are the only known eukaryotes whose chromatin is totally devoid of nucleosomes. Their chromosomes remain permanently condensed during the entire cell cycle and active nucleoli arise from an unwound part of some of the otherwise compact chromosomes. In this work, DNA-DNA hybrids were detected either by fluorescent avidin or by indirect immunogold staining procedures in EM; this is the first use of cryosections to detect hybrids in EM not only in the nucleolus sensu lato but also in a dinomastigote cell. Coding sequences of ribosomal genes were detected both in the periphery of the nucleolar organizer region (NOR), which corresponds to the unwound part of the nucleolar chromosome, and in the proximal part of the fibrillo-granular (FG) region. These results suggest that the rRNA gene transcription predominantly occurs at the periphery of the NOR where the coding sequences are located. A predictive model summarizes and allows discussions and comparisons with other eukaryotes in which nucleolar mechanisms were previously studied. This leads to the conclusion that dinoflagellate cells constitute an excellent model for the study of the functional structure of the eukaryotic nucleolus. In addition, the high level of labeling obtained on cryosections suggests that this procedure should be preferred for future ISH work.