Methods for studying the nuclei and chromosomes of dinoflagellates.

Dinoflagellates are unicellular eukaryotic organisms whose nuclear structure, chromosome architecture, chromatin organization, DNA composition, and mitosis show original features. It has been necessary to adapt techniques and to create innovative methods for growing cells, isolating nuclei, and studies of their chromosomes by transmission electron microscope (TEM). Among these are innovative squash and whole-mount preparations for light and TEM observations of chromosome architecture and the spatial organization of nucleofilaments. Particular attention was given to adapt high-pressure freezing (fast-freeze fixation) techniques for the best preservation of delicate antigenic sites, and good immunodetection. The study of DNA replication with or without incorporation of bromodeoxyuridine (BrdU) was also refined to use confocal laser scanning microscopy. In this chapter, we describe methods that we have invented and/or improved from existing techniques in order to better understand this fragile chromosome architecture and the mechanisms intervening during mitosis and the cell cycle. These methods allowed us to detect specific DNA-binding proteins and the distribution of B-and Z-DNA in chromosomes during the cell cycle and mitosis, and to focus on the indissoluble link between chromosome structure and function.