Abstract
Dinoflagellates possess some of the largest known genomes. However, the study of their chromosomes is complicated by their similar size and their inability to be distinguished by traditional banding techniques. Dinoflagellate chromosomes lack nucleosomes and are present in a liquid crystalline state. In addition, approaches such as fluorescent in situ hybridization (FISH) are problematic because chromosomes are difficult to isolate from the nuclear membrane, which in dinoflagellates remains intact, also during mitosis. Here we describe a novel, reliable and effective technique to study dinoflagellate chromosomes by physical mapping of repetitive DNA sequences in chromosomes in suspension (FISH-IS), rather than on a microscope slide. A suspension of non-fixed chromosomes was achieved by lysing the cells and destabilizing the nuclear envelope. This treatment resulted in the release of the permanently condensed chromosomes in a high-quality chromosomal suspension. Nevertheless, slide preparations of the chromosomes were not suitable for conventional FISH because the nuclear integrity and chromosomal morphology was destroyed. Our newly developed, simple and efficient FISH-IS technique employs fluorescently labeled, synthetic short sequence repeats that are hybridized with suspended, acetic-acid-pretreated chromosomes for 1 h at room temperature. The method can be successfully used to discriminate single chromosomes or specific chromosomal regions, depending on the specificity of the repeat sequences used as probes. The combination of FISH-IS and flow sorting will improve genomic studies of dinoflagellates, overcoming the difficulties posed by their huge genomes, including long stretches of non-coding sequences in multiple copies and the presence of high-copy-number tandem gene arrays.
Highlights
Dinoflagellates are a large and diverse group of protists that encompasses extremely diverse organisms with photosynthetic, symbiotic, mixotrophic or parasitic life styles [1]
In the first series of experiments, the conditions for the preparation of high-quality suspensions of dinoflagellate chromosomes to be used in the fluorescent in situ hybridization (FISH) experiments were defined (Fig 1)
We evaluated different methods to achieve the release of the chromosomes from the synchronized dinoflagellate cells, choosing a 15-min incubation in hypotonic solution at room temperature (RT) to swell the cells, followed by centrifugation for 5 min and resuspension of the pellet in lysis buffer for 15 min on ice to release the chromosomes
Summary
Dinoflagellates are a large and diverse group of protists that encompasses extremely diverse organisms with photosynthetic, symbiotic, mixotrophic or parasitic life styles [1]. Divalent cations and HCc proteins, a family of DNAbinding proteins with homologies to bacterial and eukaryotic histone H1, are involved in chromatin compaction [6] Another unique feature is that dinoflagellate chromosomes remain condensed throughout the cell cycle and are permanently visible but without Q, G or C banding. As it does not include either the breakdown of the nuclear envelope (endomitosis) [7] or a typical metaphase plate, such that chromosomal alignment is absent Based on these features dinoflagellates constitute a unique biological model for the study of genome organization and expression [8] and an extremely interesting model for evolutionary comparisons in the rising field of the 4D nucleome research [9]. Additional challenges to studies of the organization, function and evolution of dinoflagellate chromosomes relate to the difficulty in culturing dinoflagellates in vitro
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