Random sequencing of Paramecium somatic DNA

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.     

5-Azacytidine enhances transient expression of a transfected gene in cultured mammalian cells

The level of transient expression of the Escherichia coli β-galactosidase (β-Gal) gene transfected into three mammalian cell lines was enhanced by culturing the cells with 1–5 μM 5-azacytidine (5-azaC) after the transfection. This enhancement was similarly observed in four different transfection procedures tested. The enhancement of β-Gal expression was primarily due to the increase in the level of its mRNA. Since 5-azaC did not exhibit any obvious effect on the levels of endogenous gene products examined, it is suggested that 5-azaC may preferentially affect the transient expression of an exogenous gene.     

Centrin deficiency in Paramecium affects the geometry of basal-body duplication

BACKGROUND: Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated centrin function in Paramecium, whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication. RESULTS: We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b, orthologs of HsCEN2 and HsCEN3, respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies. CONCLUSIONS: Our observations show that centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.     

High coding density on the largest Paramecium tetraurelia somatic chromosome

Paramecium, like other ciliates, remodels its entire germline genome at each sexual generation to produce a somatic genome stripped of transposons and other multicopy elements. The germline chromosomes are fragmented by a DNA elimination process that targets heterochromatin to give a reproducible set of some 200 linear molecules 50 kb to 1 Mb in size. These chromosomes are maintained at a ploidy of 800n in the somatic macronucleus and assure all gene expression. We isolated and sequenced the largest megabase somatic chromosome in order to explore its organization and gene content. The AT-rich (72%) chromosome is compact, with very small introns (average size 25 nt), short intergenic regions (median size 202 nt), and a coding density of at least 74%, higher than that reported for budding yeast (70%) or any other free-living eukaryote. Similarity to known proteins could be detected for 57% of the 460 potential protein coding genes. Thirty-two of the proteins are shared with vertebrates but absent from yeast, consistent with the morphogenetic complexity of Paramecium, a long-standing model for differentiated functions shared with metazoans but often absent from simpler eukaryotes. Extrapolation to the whole genome suggests that Paramecium has at least 30,000 genes.     

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin- section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.     

RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia

The germ line genome of ciliates is extensively rearranged during development of the somatic macronucleus. Numerous sequences are eliminated, while others are amplified to a high ploidy level. In the Paramecium aurelia group of species, transformation of the maternal macronucleus with transgenes at high copy numbers can induce the deletion of homologous genes in sexual progeny, when a new macronucleus develops from the wild-type germ line. We show that this trans-nuclear effect correlates with homology-dependent silencing of maternal genes before autogamy and with the accumulation of approximately 22- to 23-nucleotide (nt) RNA molecules. The same effects are induced by feeding cells before meiosis with bacteria containing double-stranded RNA, suggesting that small interfering RNA-like molecules can target deletions. Furthermore, experimentally induced macronuclear deletions are spontaneously reproduced in subsequent sexual generations, and reintroduction of the missing gene into the variant macronucleus restores developmental amplification in sexual progeny. We discuss the possible roles of the approximately 22- to 23-nt RNAs in the targeting of deletions and the implications for the RNA-mediated genome-scanning process that is thought to determine developmentally regulated rearrangements in ciliates.     

Changes in micro RNA expression in a wild tuber-bearing Solanum species induced by 5-Azacytidine treatment

Phenotypic plasticity is often postulated as a principal characteristic of tuber-bearing wild Solanum species. The hypotheses to explore this observation have been developed based on the presence of genetic variation. In this context, evolutionary changes and adaptation are impossible without genetic variation. However, epigenetic effects, which include DNA methylation and microRNAs expression control, could be another source of phenotypic variation in ecologically relevant traits. To achieve a detailed mechanistic understanding of these processes, it is necessary to separate epigenetic from DNA sequence-based effects and to evaluate their relative importance on phenotypic variability. We explored the potential relevance of epigenetic effects in individuals with the same genotype. For this purpose, a clone of the wild potato Solanum ruiz-lealii, a non-model species in which natural methylation variability has been demonstrated, was selected and its DNA methylation was manipulated applying 5-Azacytidine (AzaC), a demethylating agent. The AzaC treatment induced early flowering and changes in leaf morphology. Using quantitative real-time PCR, we identified four miRNAs up-regulated in the AzaC-treated plants. One of them, miRNA172, could play a role on the early flowering phenotype. In this work, we showed that the treatment with AzaC could provide meaningful results allowing to study both the phenotypic plasticity in tuber-bearing Solanum species and the inter-relation between DNA methylation and miRNA accumulations in a wide range of species.     

5-Azacytidine facilitates osteogenic gene expression and differentiation of mesenchymal stem cells by alteration in DNA methylation

Mesenchymal stem cells (MSCs) are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages. The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage. In this study, we investigated the effect of 5-azacytidine, a DNA demethylating agent, on osteogenic differentiation of MSCs. The cells were exposed to 5-azacytidine in culture medium for 24 h prior to osteogenic induction. Osteogenic differentiation was determined by several the appearance of a number of osteogenesis characteristics, including gene expression, ALP activity, and calcium mineralization. Pretreatment of MSCs with 5-azacytidine significantly facilitated osteogenic differentiation and was accompanied by hypomethylation of genomic DNA and increased osteogenic gene expression. Taking dlx5 as a representative, methylation alterations of the “CpG island shore” in the promoter caused by 5-azacytidine appeared to contribute to osteogenic differentiation.     

Non-Mendelian inheritance induced by gene amplification in the germ nucleus of Paramecium tetraurelia

A genetic investigation of strain d4-95, which carries a recessive mutant allele (pwB(95)) of pawn-B, one of the controlling elements of voltage-dependent calcium channels in Paramecium tetraurelia, revealed a non-Mendelian feature. Progeny of the cross between d4-95 and wild type often expressed a clonally stable mutant phenotype, even when they had a wild-type gene. The mutant phenotype was also expressed after self-fertilization of theoretical wild-type homozygotes recovered from the cross. Our molecular analysis demonstrated that the copy number of the mutant pwB gene in the micro- and macronucleus of d4-95 was much greater than that of the wild type. Most of the amplified, extra pwB gene copies in d4-95 were heritable independently from the original pwB locus. Repeated backcrossing of d4-95 with the wild type to dilute extra pwB genes in the strain produced segregants with a completely normal Mendelian trait in testcrosses. These results strongly suggest that a non-Mendelian inheritance of d4-95 was induced by gene amplification in the micronucleus.     

Synthesis and Biological Study of the Cyclopentenyl Carbocyclic Nucleoside Analogue of 5-Azacytidine

Abstract

Cyclopentenyl cytosine (CPE-C, 3) possesses excellent antitumor and antiviral activity. The synthesis of the analogous cyclopentenyl triazine nucleoside, 5-aza-CPE-C (4), was accomplished by a novel approach that utilized a key 1-cyclopentenyl-4-methylisobiuret intermediate (7) produced from the corresponding cyclopentenylamine 5. 5-Aza-CPE-C was more than six-hundred times less potent than CPE-C both in its capacity to reduce CTP levels as well as in its antitumor and antiviral activity.     

The somatic function of the micronucleus in asexual reproduction of Paramecium: thermosensitivity of amicronucleates

It has been established that following removal of the micronucleus in Paramecium tetraurelia, the amicronucleate cell line enters a depression period, characterized by slow growth rate and oral abnormalities, at normal growth temperature (27°C). Such cell lines gradually recover in growth rate and stomatogenesis. In the present study, 4 recovered amicronucleate cell lines were challenged with high temperatures (35°C, 36°C, and 36.5°C). They exhibited growth rate reduction and abortive cytokinesis at 35°C and 36°C, and died at 36.5°C. In addition, they demonstrated oral defects similar to those observed in the depression period: disruption of the regular oral membranellar pattern, reduction in the length of the oral apparatus, and impaired phagocytosis (food vacuole formation). These high temperature-induced abnormalities were largely restricted to amicronucleates, and were rare or seen to a much lesser extent in sister micronucleate cell lines. This study demonstrates the participation of the micronucleus in conferring thermotolerance on the cells. It is hypothesized that the micronucleus specifies heat-shock proteins to maintain the integrity of oral and somatic cytoskeletal elements at high temperature.     

Stimulation of Plastidogenesis Induced by 5-Azacytidine in Euglena gracilis Klebs

When etiolated Euglena gracilis was treated with 10 mM 5-azacytidine (5-azaC), an inhibitor of DNA methylation, stimulation of plastidogenesis in both dark and light conditions was observed. The phenomenon occurred in 10–15% of the cells possibly due to the asynchronicity of the cultures. The main features of this sub-population, as evaluated by electron and fluorescence microscopy, were the following: 1. the presence in darkness of differentiating proplastids that were red fluorescent under UV, positive to TCNBT cytochemical reaction (specific for PSII) and negative to DAB (specific for PSI); 2. the acceleration of proplastid differentiation during the first 20–30 h of illumination; 3. the occurrence in both culture conditions of concentric lamellar bodies (LB_S). These structures were considered to be proplastids blocked in the first step of evolution, since they emitted a red fluorescence, were contained within compartments limited by a triple-layered envelope, were reactive to TCNBT in darkness and to both TCNBT and DAB in light conditions. Even if the action mechanism of 5-azaC on plastidogenesis in Euglena remains to be defined, the induced stimulatory effect on plastid differentiation pointed to a relationship between DNA methylation and plastid development. Furthermore, the presence of LB_S opens the possibility of studying early aspects of plastid development in Euglena.     

Disturbance of the determination of germinal and somatic nuclei by heat shock in Paramecium caudatum

During conjugation of Paramecium caudatum, nuclear determination occurs soon after the third postzygotic division: one of the four anterior nuclei becomes the micronucleus and the remaining three degenerate, while four posterior nuclei differentiate into macronuclear anlagen. Macronuclear differentiation is supposed to be dependent on a cytoplasmic differentiation factor. In this study, postzygotic cells were subjected to heat shock for 30 min and nuclear changes were observed by staining with carbol fuchsin solution. When heat shock was initiated during the period from metaphase to telophase of the third postzygotic division, cells showed an excess of macronuclear anlagen and were typically amicronucleate. Abnormal nuclear localization around the end of the third (last) postzygotic division may explain the origin of these kinds of cells. A similar phenomenon appeared after treatment with actinomycin D or emetine. Since heat shock did not inhibit macronuclear differentiation but destroyed the formation of micronuclei, some factor(s) probably plays an essential role in nuclear determination, especially in the protection of the micronuclei.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.