Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.     

Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.     

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).     

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.     

冠突伪尾柱虫（Pseudourostyla cristata）实验再生研究

冠突伪尾柱虫(Pseudvurostyla cristata) 含约70枚大核。我们用显微手术横切G1期细胞,得前后两块相等断片;分别培养。60小时后,断片再生完成。在再生过程中,随细胞体积增大,大核数目也增加。大核的数目和细胞体积存在着一定的均衡关系。在细胞无性分裂过程中,许多大核改组后,融合成一个融合大核。这个融合大核具两个仔虫的大核数目和DNA量。我们用显微手术得到含融合大核的后断片。在后断片再生后恢复的虫体内,我们发现本应分配到两个仔虫中去的大核数目,被限制在一个虫体的大核数目上。这说明了细胞质可以影响和调节大核的数目。并还证明了这种虫体大核DNA量较正常虫的大核DNA量约多一倍。其中大部分虫体分裂时,大核不经改组就开始融合和分裂;从而使DNA量回复正常。同讨还发现小部分虫体通过排出大核多余核物质方式来调节大核DNA量。这些现象说明了细胞核质之间存在着一种调节相对平衡和相互协调的机制。     

Culture Age, Intracellular Ca2+ Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus

In Colpoda cucullus, intracellular Ca²⁺ mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca²⁺ concentration (F₃₄₀/F₃₈₀ ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca²⁺-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.     

DNA methylation in ciliates: implications in differentiation processes.

Much experimental evidence on the role of DNA methylation in gene expression has been reported. Here we review reports on DNA methylation in ciliated protozoa, emphasizing its implications in cell differentiation processes. Both types of methylated bases (adenine and cytosine) can be found in macronuclear DNA. The division cycle and conjugation have been studied with regard to adenine methylation, and several different functions have been assigned to the methylation changes detected in these processes. Cytosine methylation changes were analyzed during stomatogenesis of Paramecium and encystment of Colpoda inflata. A comparative analysis with other similar microbial eukaryotic differentiation processes is carried out.     

Chromatin elimination and the genetic organisation of the macronucleus in Tetrahymena thermophila

In exponentially growing Tretrahymena thermophila the DNA content of the following structures was determined by cytophotometry: macronuclei of sister cells immediately after division; micronuclei; extranuclear chromatin in dividing cells and postdividers. Further, the development of macro-nuclear DNA amount in successive cell generations was determined. It was found that chromatin elimination is a frequent process reducing DNA content by about 4% per fission. This chromatin disappears within 20 min after division. The quantity of DNA extruded is highly variable and is different from the micronuclear DNA amount or multiples of it. The frequency of generations with two replication rounds as well as those without replication is estimated to be in the range of 2% each. These findings together with the qualitative difference between micro- and macronuclear DNAs suggest that the macronucleus of Tetrahymena is not entirely composed of complete genomes and that parts of the genetic material must be treated specifically for different sequences either during extrusion or during replication.     

The Nuclear Apparatus of the Ditransversal Ciliate Homalozoon vermiculare (Ciliophora,Rhabdophora) during Interphase and Division. I. The Macronucleus1

ABSTRACT. The nuclear apparatus of Homalozoon vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. The number of macronuclear segments depends (i) on the number of divisions of individual segments during the interphase and (ii) on the number of segments that arise prior to cytokinesis from the (temporary) filiform macronucleus. Precytokinetic changes of the macronucleus involve the fusion of individual segments followed by contraction and subsequent elongation of the entire macronucleus. The chromatin bodies uncoil into fine fibrils during macronuclear contraction. At the time when the division furrow appears, the macronucleus starts to renodulate. The interphase segment contains a more or less reticulated chromatin body partly attached to the nuclear envelope and about 30 polymorphous nucleoli. The latter consist of the pars granulosa, the pars fibrosa, and an additional fibrillar component. The nucleoli undergo drastic changes prior to division and the granular component disappears completely during macronuclear condensation. On the average, the macronucleus contains a 3,400-fold amount of DNA compared with a haploid micronucleus, but the intraspecific differences in the DNA content of the entire macronucleus are extremely large. In contrast, DNA content and size of an individual segment of the macronucleus are precisely regulated during interphase.     

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.     

包囊游仆虫包囊形成和解脱过程中大、小核的研究

包囊游仆虫形成包囊时大核除经历形态大小的变化外,大核DNA含量也低于正常大核水平;细胞脱包囊前,在大核一端或两端发生染色质粗浓集,是大核DNA复制的结果;染色质粒在整个大核内浓集时,大核DNA含量已达到正常大核水平,此时DNA复制结束,细胞脱包囊。小核在游仆虫形成包囊和脱包囊过程中,其形态大小、DNA含量等无明显变化。     

DNA elimination and its relation to quantities in the macronucleus of Tetrahymena.

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies. Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T. pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminated. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Macronuclear DNA Content in Tetrahymena thermophila*†

Synopsis.
Unequal macronuclear division in Tetrahymena thermophila introduces variance into G1 macronuclei; unless eliminated such variance would result in continuous variation in DNA content. Analysis of G1 and G2 macronuclear variances reveals that the added variance is eliminated by action on the extremes of macronuclear DNA content. In this model (Model II), macronuclei with small amounts of DNA have an additional complete S phase, while those with large amounts of DNA skip S. From available data, chromatin extrusion is shown not to contribute significantly, if at all, to the elimination of variance. Computer simulations utilizing haploid subunits indicate that model II predictions apply reasonably well to experimental data in terms of coefficients of variation, mean DNA content, and frequency of additional and skipped S phases. The simulations reveal also that within certain constraints, particularly the thresholds for additional and skipped S phases, macronuclear assortment is unaffected by Model II regulation. The relationships between Model II and other aspects of the cell cycle are briefly discussed.     

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.     

DNA content contributes to nuclear size control in Xenopus laevis

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.     

DNA elimination and its relation to quantities in the macronucleus of Tetrahymena

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminate. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes. The result indicates that in a given cell line at least one of the DNA molecules becoms extinct after 60 generations which we expect would cause the cell's extinction and restrict a clone's life to 60 generations. As this does not happen in nature, there must be some control of the copy numbers preventing their extinction during vegetative multiplication. Whether elimination increases or decreases the imbalance of genes remains to be investigated. © 1992 Wiley-Liss, Inc.