The size of DNA molecules and chromatin organization in the macronucleus of the ciliate Didinium nasutum (Ciliophora).

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.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora).

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.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora)

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.

Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens.

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.

Nucleolar apparatus in the macronucleus of Didinium nasutum (Ciliata): EM and 3D reconstruction.

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.