Structure of the interphase chromatin in the ciliata Bursaria truncatella macronucleus. II. Loop organization of inactive chromatin clumps

Electron microscopic study of chromatin organization in isolated macronuclei of a ciliate Bursaria truncatella showed macronuclear chromatin to be organized in compact clumps 120--180 nm in diameter linked with each other by one or several chromatin fibres. Macronucleus being dispersed in a solution of low ionic strength, radial loops basically of nucleosomal structure start appearing around chromatin clumps. Long-time dispersing of macronuclear chromatin brings complete decompactization of chromatin clumps into a set of nucleosome fibres. The way the fibres of interphase chromatin are packed in a chromatin clump is discussed.     

Macronuclear chromatin structure dynamics in Colpoda inflata (Protista, Ciliophora) resting encystment.

The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal.     

The structure of inactive interphase macronuclear chromatin of the ciliate Bursaria truncatella. Radial loops in the structure of chromatin clumps

The organization of chromatin in macronuclei of Bursaria truncatella cells that completed their growth and differentiation was electron microscopically studied. The data obtained showed that (1) inactive macronuclear chromatin was organized in compact chromatin clumps 120 to 180 nm in diameter linked by one or several chromatin fibres, and (2) in low salt buffer the chromatin clumps gradually unraveled, radial loops of supranucleosomal or, more often, nucleosomal structure appearing around chromatin clumps. Upon prolonged incubation in low salt buffer chromatin clumps were completely transformed into nucleosomal fibres. The data obtained evidenced in favour of a loop-packed structure of chromatin clumps.     

Chromatin structure in somatic nucleus of ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis>

The structural organization of macronuclear chromatin of the ciliate Didinium nasutum was studied. The macronuclear genome of D. nasutum is represented by DNA molecules of subchromosomal size. At interphase, macronuclear chromatin is organized into chromatin of 100–200-nm clumps. Some of these clumps form short, thick fibers that consist of several chromatin clumps. Using the differential staining of nucleic acids on ultrathin sections, we revealed perichromatin fibers and granules on the surface of many chromatin clumps. A 3D model of the spatial distribution of chromatin clumps in the macronucleus was built based on serial ultrathin sections and peculiar features of chromatin spatial organization were studied.     

Resting cysts of Parentocirrus hortualis Voß, 1997 (Ciliophora,Hypotrichia), with preliminary notes on encystation and various types of excystation

Resting cysts of Parentocirrus hortualis were investigated, using live observation, SEM and TEM. Processes during encystation and excystation were observed in vivo under the light microscope. During encystation, the trophic body becomes globular, the ciliature is resorbed in an anterior direction, the macronuclear nodules fuse into an elongated mass, and finally a cyst wall develops. As typical for oxytrichids, the resting cysts of P. hortualis are of the kinetosome-resorbing type and their wall is made of four layers: ectocyst, mesocyst, endocyst, and metacyst. The beginning of excystation is indicated by the formation of an excystation vacuole that helps the regenerating specimen to break the cyst wall. The excysting specimen leaves the resting cyst in a thin membrane that is gradually resorbed in the outer environment. Also two other excystation modes were observed. During the rare mode, the excystation vacuole breaks the thin membrane instead of the cyst wall that ruptures under the pressure of the body of the regenerating specimen. During the reproduction mode, the regenerating specimen divides within the resting cyst, producing two to four tomites. This is the first report of division in resting cysts of oxytrichids, but reproduction in division cysts was already described in keronopsids.     

Cortical and Nuclear Events During Cell Division and Resting Cyst Formation in Colpoda inflata

Nuclear and cortical phenomena during dividing and resting cyst formation of Colpoda inflata are described. Cell division forms a cyst and produces two or four tomites. In each tomite, the right oral field results from the proliferation of the anterior extreme of a single kinety, and the left oral field results from the proliferation of four, five, or six somatic kineties. After macronuclear division, each macronuclear mass undergoes a chromatinic extrusion process. During resting cyst formation, the oral infraciliature of the vegetative cell is resorbed. The somatic kineties dispose in a radial way and some pairs of kinetosomes disappear. As in cell division, there is an extrusion process. From these results we conclude that the resting cysts of Colpoda inflata cannot be included in any group of the previous classifications for hypotrich resting cysts. Thus, we propose a new additional group to Walker and Maugel's classification called PKR (partial-kinetosome-resorbing) cysts.     

Isolation and characterization of a cDNA encoding a putative high mobility group (HMG) – box protein from stored mRNA in resting cysts of the ciliate Oxytricha (Sterkiella) nova – Ciliate macronuclear gene encoding a putative HMG-box protein

We have isolated an cDNA after applying a DDRT-PCR analysis on mRNA from mature resting cysts of the ciliate Oxytricha (Sterkiella) nova. From this cDNA fragment the complete macronuclear minichromosome was obtained by using the Mac-End-PCR method. After cloning and sequencing, this cDNA shown certain similarity to HMG-like proteins. The analysis of the inferred amino acid sequence shown that this putative HMG-like protein has one HMG-box interrupted by a intron. The analysis of others characteristics (including a 3D model) confirms that it is a HMGB family protein. It is the first time that a macronuclear gene encoding a putative HMG-box protein is isolated from resting cysts of a stichotrich ciliate. The possible implications of this stored mRNA in the ciliate cryptobiotic stage are discussed.     

Amitotic division of the macronucleus in Tetrahymena thermophila: DNA distribution by genomic unit

The macronucleus of the ciliate Tetrahymena cell contains euchromatin and numerous heterochromatins called chromatin bodies. During cell division, a chromatin aggregate larger than chromatin body appears in the macronucleus. We observed chromatin aggregates in the dividing macronucleus in a living T. thermophila cell, and found that these were globular in morphology and homogeneous in size. To observe globular chromatin clearly, optimal conditions for making it compact were studied. Addition of Mg ion, benomyl and oryzalin, microtubule inhibitors, to cell suspension was effective. Globular chromatin appeared when the micronuclear anaphase began at the cell cortex, and disappeared long after cell separation. Using living cells with a small macronucleus at early log phase, we counted the number of globular chromatin per nucleus and measured the DNA content of globular chromatin in the macronucleus which was stained with Hoechst 33342 by using ImageJ. The number of globular chromatin per nucleus was reduced by half after division, indicating the globular chromatin is a distribution unit of DNA. A globular chromatin contained similar DNA content as that of the macronuclear genome. We developed methods for inducing and isolating a cell with an extremely small macronucleus with a DNA amount of one globular chromatin. These cells grew, divided, and give clones, suggesting that the macronuclear genome is not dispersed within the macronucleus and the globular chromatin may be a macronuclear genome. We named this globular chromatin "macronuclear genome unit" (MGU).     

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata.

The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.     

Template activity of macronuclear and micronuclear chromatin of the ciliate Stylonychia mytilus.

Template activity of macronuclear and micronuclear chromatin of the ciliate Stylonychia mytilus was studied under in vitro conditions. Micronuclear chromatin has a template activity which is about 50% of that of the macronucleus. The relative number of initiation sites is much smaller in micronuclear chromatin.     

The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus

The formation of polytene chromosomes during macronuclear development of the ciliate Stylonychia mytilus was examined in spread electron microscopical preparations. The chromatin organization of early macronuclear anlagen closely resembles the organization of micronuclear chromatin. In the course of polytenization 300 A chromatin fibers become organized in loop-like structures laterally attached to a thinner axial fiber. It is suggested that this reorganization of chromatin during polytenization is a necessary event for the subsequent chromatin elimination.     

Fine structure of excystment of the quadriflagellate algaPolytomella agilis

Summary Populations of mature resting cysts of the algaPolytomella agilis were purified from asynchronously encysting cultures and incubated in fresh culture medium to promote excystment. Up to 90 percent of the cysts germinated, with approximately 50 percent excysting between 3 and 7 hours of incubation. Each germinating cyst releases a single, fully differentiated, swimming cell. The entire excystment process of individual cysts was followed by light microscopy to establish the time course of release and cells at comparable stages of excystment were examined by electron microscopy. During the first 3 hours of incubation the cysts increase in size, presumably due to uptake of water, and a polarity is established in the cytoplasm which makes it possible to identify the site of subsequent release. Release involves a selective degradation of a portion of the cyst wall at this site followed by a physical rupturing of the weakened area. Details of the structural alterations in the wall and cytoplasm are described. The cytoplasmic organelles observed to dedifferentiate during encystment (preceding paper) are completely redifferentiated during excystment. The emergent cell is flagellated and possesses the elongate form typical of the swimming cell.This work was supported by grant A6353 from the National Research Council of Canada to D. L.Brown and by the Inland Waters Directorate of Environment Canada.     

Chromatin structure in the macronucleus of the ciliate Stylonychia mytilus.

Evidence is presented that macronuclear chromatin in the hypotrichous ciliate Stylonychia mytilus occurs in discrete fragments, each representing at least single genes. The size of these fragments varies between 3 and more than 70 nucleosomes with an average length of about 18 nucleosomes. This observation is discussed with respect to macronuclear structure of hypotrichous ciliates.