Abstract
In contrast to the chromosomal genome organization common to most eukaryotes, DNA in the macronucleus of spirotrichous ciliates like Stylonychia lemnae is organized into small gene-sized nanochromosomes. We intended to elucidate whether a spatial organization of nucleoli similar to other eukaryotes can be found in absence of typical chromosomes. Whereas micronuclei of Stylonychia exhibit homogenously stained heterochromatin and possess no nucleoli, macronuclear chromatin is compartmentalized and contains numerous putative nucleoli. Since the identity of these spherical structures has never been unequivocally demonstrated to date, we applied immunofluorescence techniques together with confocal laser scanning microscopy to identify nucleolar bodies in the macronucleus of Stylonychia and to analyse their spatial organization. We found that multiple spherical bodies, which fulfil nucleolar function, occupy a peripheral localization in mature macronuclei. Using fibrillarin/Nop1p as a nucleolar marker, we monitored the assembly of such nucleolar bodies during macronuclear differentiation. 3D-FISH experiments revealed that rRNA genes are mostly concentrated adjacent to but not inside of fibrillarin/Nop1p-containing bodies. We further showed that transcription sites for rRNA synthesis but also for mRNA synthesis occur predominantly at surfaces of nucleolar bodies and chromatin-poor spaces bordering condensed chromatin. Our data suggest that transcription of rRNA genes in the macronucleus of Stylonychia does not rely on a classical nucleolus-type organization. We assume that vectorial synthesis and processing of rRNA and mRNA is directed to a functional interchromatin compartment.
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Notes
Supplementary data may be found at <http://dx.doi.org/10.1007/s10577-005-1033-2>.
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft and the National Science Foundation (grant no: EIA-0121422). A special acknowledgement is dedicated to Thomas Cremer, Ludwig Maximilians University Munich, Department Biology II, Anthropology and Human Genetics. If he had not generously provided his microscopy facility, this work would not have been possible.
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Supplementary Data 1
DO00001033_ESM.avi
A 3D reconstruction illustrates that delimitated chromatin spots occasionally observed in the interior of nucleolar bodies in single optical sections are built up by chromatin invaginations into such a body, showing that they in fact are localized beyond the interior of these spherical structures. The surface was rendered using To-Pro-3 counterstaining. AVI movie file (2.9 Mb).
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Postberg, J., Alexandrova, O. & Lipps, H.J. Synthesis of pre-rRNA and mRNA is directed to a chromatin-poor compartment in the macronucleus of the spirotrichous ciliate Stylonychia lemnae. Chromosome Res 14, 161–175 (2006). https://doi.org/10.1007/s10577-006-1033-x
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DOI: https://doi.org/10.1007/s10577-006-1033-x