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.     

Low molecular weight DNA in the heteromeric macronuclei of two cyrtophorid ciliates

Summary— The size range of the native DNA molecules in the heteromeric macronuclei of two cyrtophorid ciliates (Trithigmostoma cucullulus, Chilodonella uncinata) was mainly investigated by using agarose gel electrophoresis. Numerous bands superimposed on a continuous spectrum of molecular sizes between about 0.35 kb and 30 kb were resolved by conventional electrophoresis. Species-specific banding patterns indicate a variation between species in the copy number of individual DNA fragments. A slight intra-specific variability of banding patterns can exist. Electrophoretic distributions for two strains of T cucullulus were indeed found to differ by at least one more intense band (‘overamplified’ sequences?). Fractionation by contour-clamped homogeneous electric field (CHEF) gel electrophoresis revealed that the size continum of macronuclear DNA molecules does not extend beyond 60–70 kb. The average size was estimated to be around 4 kb. Unresolved DNA fraction (> 1000 kb) accounted for less than 10% of the mass of cellular DNA entering CHEF gels. Macronuclear ribosomal DNA of each cyrtophorid species was identified by Southern hybridization with a Tetrahymena rDNA probe. The hybridization signal was observed on a single band of low molecular weight DNA. The corresponding size was close to 14.5 kb in Trithigmostoma and 15.5 kb in Chilodonella, which is about twice the size of monomeric rDNA in hypotrichous ciliates. We showed that S1 nuclease resistant duplexes wit half the length of the native rDNA can be formed by rapid renaturation of heat-denatured molecules and hybridized with native rDNA. This strongly suggests that the nucleotide sequence of this rDNA is a large palindrome. Unlike the hypotrichs, macronuclear rDNA in cyrtophorids should be organized into palindromic dimers as in Tetrahymena species.     

Three-dimensional structure of the ciliate Didinium nasutum nucleoli

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.     

Dramatic diversity of ciliate histone H4 genes revealed by comparisons of patterns of substitutions and paralog divergences among eukaryotes

The accumulation of divergent histone H4 amino acid sequences within and between ciliate lineages challenges traditional views of the evolution of this essential eukaryotic protein. We analyzed histone H4 sequences from 13 species of ciliates and compared these data with sequences from well-sampled eukaryotic clades. Ciliate histone H4s differ from one another at as many as 46% of their amino acids, in contrast with the highly conserved character of this protein in most other eukaryotes. Equally striking, we find paralogs of histone H4 within ciliate genomes that differ by up to 25% of their amino acids, whereas paralogs in other eukaryotes share identical or nearly identical amino acid sequences. Moreover, the most divergent H4 proteins within ciliates are found in the lineages with highly processed macronuclear genomes. Our analyses demonstrate that the dual nature of ciliate genomes-the presence of a "germline" micronucleus and a "somatic" macronucleus within each cell-allowed the dramatic variation in ciliate histone genes by altering functional constraints or enabling adaptive evolution of the histone H4 protein, or both.     

Fine structure and cytochemistry of the nuclei of the primitive ciliateTracheloraphis crassus (Karyorelictida)

Summary The nuclei ofTracheloraphis crassus were studied using light and electron microscopy combined with Bernhard's RNP staining and pronase digestion. The nuclear apparatus of this species consists of a longitudinal row of 11–43 macronuclei and 4–16 micronuclei. Like in all karyorelictids, the macronuclei are unable to divide and become segregated during cytokinesis; their number is supplemented in every cell cycle by differentiation of several new macronuclei from micronuclei.Each adult macronucleus contains a single compact endonuclear aggregate of several large chromocenters, readily destained with EDTA, and several RNP containing nucleoli. There is continuity between the material of the chromocenters and the decondensed DNP fibrils in the nuclear matrix. The nucleoli contain NORs in the form of fibrillar centers. The endonuclear aggregate includes also groups of RNP granules which are especially resistant to EDTA destaining. A microfibrillar sphere, usually localized at the periphery of the aggregate, contacts one or several nucleoli. The sphere is not bleached with EDTA, and only its periphery becomes digested with pronase. The macronuclear matrix consists of both protein fibrils and pronase-resistant fibrils, the latter being localized at the nuclear periphery.Developing macronuclear primordia contain loose strands of decondensed chromatin; only later they form chromocenters and nucleoli.The micronuclei reproduce by mitosis with typical chromosomes (2n=66). During interphase, they are filled with condensed chromatin which can be bleached with EDTA; they form no nucleoli. Ring-like lamellae, existing in the cavities of the chromatin mass, stain for RNA (after Bernhard) and are pronase-sensitive. These lamellae resemble the kinetochore material conserved during interphase in another karyorelictid ciliate,Trachelocerca geopetiti.     

Ultrastructure of the nuclear apparatus of the lower ciliateRemanella granulosa Kahl (Karyorelictida)

Summary The nuclear apparatus ofRemanella granulosa has been investigated using conventional TEM methods and Bernhard's technique of preferential RNP staining. This species has two (rarely three) macronuclei and a single micronucleus (rarely two micronuclei). The nuclei always form a single group.The macronuclei contain a fibro-granular matrix resistant to EDTA destaining, and several nucleoli and chromatin bodies. The chromatin bodies are readily bleached with EDTA and are often clustered, or even fused, forming chromocenters. The nuclei are of the compact concentric type. Some macronuclei contain nuclear bodies, as finely fibrous spheres or bundles of coarse fibers, or both. Neither type of nuclear body is destained with EDTA. The spheres are frequently associated with nucleoli. There is no evidence of any transition between the two types of nuclear bodies. The macronuclear envelope contains numerous pore complexes and is strengthened with an electron dense layer. The micronucleus is filled with spongy condensed chromatin and surrounded by an envelope with occasional pores. This nucleus lacks nucleoli and nuclear bodies.     

Formation of Chromoneme-like Fibrils Induced in Infusorian Somatic Nuclei by Magnesium Ions

A study was made of the effect of Mg²⁺ on higher-order chromatin structure in macronuclei (Ma) of infusoria Paramecium aurelia and Bursaria truncatella. In infusorian Ma, inactive chromatin is commonly packed in chromatin bodies sized 60–200 nm. When isolated chromatin or Ma were treated with Mg²⁺ (about 3 mM), chromatin bodies arranged into fibrils 100–300 nm in diameter, which resembled higher eukaryotic chromonemes. The dynamics of chromoneme-like fibril formation was described. The results testified to the similarity of chromatin bodies to chromomeres of higher eukaryotes. Structurally intact central chromomere cores proved to be essential for the formation of chromoneme-like fibrils. Chromatin organization in infusoria was shown to follow the discrete-level model (nucleosomes–nucleomeres–chromomeres–chromonemes) assumed for higher eukaryotes.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

Conjugation in Hyalophysa chattoni Bradbury (Apostomatida): An adaptation to a symbiotic life cycle

Hyalophysa chattoni, borne as an encysted phoront on a crustacean's exoskeleton, metamorphoses to the trophont during the host's premolt. After the molt within 15 min to 2 h conjugants with food vacuoles appear in the exuvium, swimming along with the trophonts. Starvation in other ciliates usually precedes conjugation, but food vacuoles in conjugants do not preclude starvation. Only after ingestion and dehydration of vacuoles ceases, does digestion of exuvial fluid begin. Conjugants resorb their feeding apparatus as they fuse. A single imperforate membrane from each partner forms the junction membrane. In a reproductive cyst conjugants divide synchronously, but now the junction membrane is interrupted by pores and channels. After the last division the daughters undergo meiosis – two meiotic divisions and one mitotic division yielding two prokarya as they simultaneously differentiate into tomites. After fertilization, pairs separate and the synkaryon divides once into a macronuclear anlage and a micronucleus. Exconjugants leave the cyst and seek a host. The parental macronucleus remains active until the phoront stage when the anlage develops. Owing to random association of micronuclei during meiosis, Hyalophysa's exconjugants are more genetically diverse than exconjugants from conventional patterns of conjugation.     

Extrusion of DNA from the Macronucleus of Tetrahymena pyriformis GL

SYNOPSIS Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.     

The Invasive Nature of an Infectious Bacterial Symbiont

ABSTRACT. Xenosomes are infectious bacterial symbionts that exist exclusively in the cytoplasm of the small philasterine marine ciliate Parauronema acutum. We have used this host-symbiont system as a model to study infection. In the past we postulated that infection took place by a process in which the symbionts escaped digestion and entered into the host's cytoplasm through the food vacuole during phagocytosis. This is clearly not the case. We now present evidence based on electron microscopic observations that the symbionts infect in a manner involving direct penetration of the protozoan's cell membranes. We have obtained additional data that suggest that, following entrance of the symbionts into the cytoplasm, only a single xenosome is required to establish an infection.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

Morphological and Molecular Characterization of a New Protist Family, Sandmanniellidae n. fam. (Ciliophora, Colpodea), with Description of Sandmanniella terricola n. g., n. sp. from the Chobe Floodplain in Botswana

ABSTRACT. Sandmanniella terricola n. g., n. sp. was discovered in soil from the Chobe floodplain, Botswana, southern Africa. Its morphology and 18S rDNA gene sequence were studied with standard methods. Sandmanniella terricola is very likely an adversity strategist because it reaches peak abundances 6–12 h after rewetting the soil and maintains trophic food vacuoles with undigested bacteria in the resting cyst, a highly specific feature suggested as an indicator for an adversity life strategy. Possibly, the energy of the stored food vacuoles is used for reproduction and support of the cyst wall. Morphologically, Sandmanniella terricola is inconspicuous, having a size of only 50 × 40 μm and a simple, ellipsoidal shape. The main characteristics of the genus are a colpodid silverline pattern; a perioral cilia condensation; a flat, dish-shaped oral cavity, in the centre of which originates a long, conical oral basket resembling that of certain nassulid ciliates; and a vertically oriented left oral polykinetid composed of brick-shaped adoral organelles. This unique mixture of features and the gene sequence trees, where Sandmanniella shows an isolated position, suggest establishing a new family, the Sandmanniellidae n. fam., possibly related to the families Colpodidae or Bryophryidae. The curious oral basket provides some support for the hypothesis of a common ancestor of colpodid and nassulid ciliates.