Abstract
During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell’s two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 μm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.
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Acknowledgments
We thank Jennifer Grünert (LMU, Munich, Germany), Zuzana Zubáčová, Miroslav Hyliš, Ondřej Šebesta (Charles University in Prague, Czech Republic), and Veronika Marešová (Masaryk University in Brno, Czech Republic) for technical assistance; Alexander Schleiffer (CSF Vienna Biocenter, Austria) for bioinformatics assistance; and Elizabeth Schroeder-Reiter (LMU, Munich, Germany) and Jiří Král (Charles University in Prague, Czech Republic) for comments on the manuscript. This study was funded by Grant No. P305/12/1248 and partly funded by student Grant No. SVV-260 026 (MU), which were both from the Czech Science Foundation (GAČR).
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Supplemental Fig. 1
Scanning electron micrograph of a Giardia intestinalis trophozoite cell fixed with methanol/acetic acid fixative (3:1) following “drop/cryo“ preparation and glutaraldehyde fixation. Two interphase nuclei remined attached to flagellar axonemes. (GIF 209 kb)
Supplemental Fig. 2
Controls for discriminating chromosomes/chromatin from contamination on chromosome preparations. (a) SEM micrograph of DNA–richand DNA–free material following PtBlue staining (a, left = SE image; a, right = BSE image). Arrows indicate a DNA-free object (contamination) that is not visible following PtBlue staining. The other object with a faint PtBlue staining in a BSE image contains DNA and represents a loosened interphase nucleus. (b) Correlative light- and scanning electron microscopy: SEM micrograph of a chromosome complement (b, left) and fluorescence LM micrograph of the same chromosome complement with DAPI staining (b, right). Circles indicate a non-chromosomal structure (contamination) present but not visible under DAPI staining. (GIF 348 kb)
Supplemental Fig. 3
The alignment of Giardia intestinalis histone H4 and a human H4 indicating the conserved position of H4 lysine 16 (arrow). (PDF 201 kb)
Supplemental Fig. 4
Putative condensin subunits (kleisins) found in Giardia intestinalis (GL 50803_15239, GL 50803_102874). (A) Sequential steps followed to search for Giardia kleisins with the final conserved-domain identification of retrieved hits. A putative function of the found hypothetical proteins is proposed. (B) Alignments of putative Giardia kleisins and of other eukaryotic kleisins that were selected according to information provided by Schleiffer et al. (2003). Giardia hypothetical proteins (GL 50803_15239, GL 50803_102874) were aligned with β- and γ-kleisins. The conserved parts of N- and C-termini of kleisin protein families are indicated in boxes and represent their binding domains to “head“ domains of SMC proteins (Fennell-Fezzie et al. 2005). In the alignment, the kleisin family of other eukaryotic kleisins is indicated by a capital letter added to the sequence name (B or G for β- or γ-kleisins, respectively). (PDF 430 kb)
Supplemental Fig. 5
Putative condensin non-SMC subunits (HEAT proteins) found in Giardia intestinalis. The table displays the sequential search for Giardia HEAT proteins with the conserved domain identification of retrieved hits. A resulting putative function of the found hypothetical proteins is proposed. (PDF 418 kb)
Supplemental Fig. 6
Stereomicrographs of Giardia metaphase chromosomes allowing visualization of chromosomes in three dimensions (a, b). Seven chromosomes out of the nine of Giardia strain HP-1 nucleus are imaged. The asterix indicates a contaminating non-chromosomal material, which was DAPI-negative in light microscope, as indicated by correlative light- and electron microscopy (a). The dotted line indicates a slight groove between two sister chromatids (b). (GIF 201 kb)
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Tůmová, P., Uzlíková, M., Wanner, G. et al. Structural organization of very small chromosomes: study on a single-celled evolutionary distant eukaryote Giardia intestinalis . Chromosoma 124, 81–94 (2015). https://doi.org/10.1007/s00412-014-0486-5
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DOI: https://doi.org/10.1007/s00412-014-0486-5