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A fundamental difference between somatic nuclei (macronuclei) of ciliates and cell nuclei of higher eukaryotes is that the macronuclear genome is a huge number (up to tens or hundreds of thousands) of gene-sized (0.5-25 kb) or subchromosomal (up to 2000 kb) minichromosomes. Electron microscopy shows that macronuclear chromatin usually looks like chromatin bodies or fibrils 200-300 nm thick in the interphase. However, the question of how many DNA molecules are contained in an individual chromatin body remains open. The organization of chromatin in macronuclei was studied in the ciliates Didinium nasutum and three Paramecium sp, which differ in pulsed-field gel electrophoresis (PFGE) karyotype, and compared with the model of topologically associated domains (TADs) of higher eukaryotic nuclei. PFGE showed that the sizes of macronuclear DNAs ranged from 50 to 1700 kb, while the majority of the molecules were less than 500 kb in length. A comparative quantitative analysis of the PFGE and electron microscopic data showed that each chromatin body contained one minichromosome in P. multimicronucleatum in the logarithmic growth phase, while bodies in the D. nasutum macronucleus contained two or more DNA molecules each. Chromatin bodies aggregated during starvation, when activity of the macronuclei decreased, leading to an increase of chromatin body size or the formation of 200- to 300-nm fibrils of several chromatin bodies. A model was proposed to explain the formation of such structures. In terms of topological characteristics, macronuclear chromatin bodies with subchromosomal DNA molecules were found to correspond to higher eukaryotic TADs.

Pulsed-field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short "gene-sized" DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80-265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.

The nucleolar organization in ciliate Didinium nasutum somatic interphase nuclei was studied using serial ultrathin sections and compared for various physiological states of the cell, namely, fed ciliates, starved ciliates, and dormant cysts. It has been shown that the interphase nucleoli are large structures with a complex architecture: the fibrillar component forms an intricate network in the macronucleus space, while the granular component is located inside this network. The structures looking as individual nucleoli in single sections are actually parts of branched nucleolar networks. The intricate nucleolar networks do not disintegrate after a 30-h starvation; however, the granular component becomes denser and develops numerous cavities filled with fine fibrils of a nonribonucleoprotein nature. In fed D. nasutum, the fibrillar structures on the periphery of nucleoli contain numerous pores (virtually absent in starved cell nucleoli), which can potentially serve for transporting newly synthesized rRNP. Branched nucleolar networks are undetectable in cysts. Their nucleoli are individual structures consisting mainly of the fibrogranular component.

The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.