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PubMed1998

Homology-dependent maternal inhibition of developmental excision of internal eliminated sequences in Paramecium tetraurelia.

Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5'-TA-3' direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.

Animals↗
PubMed2000

A novel chromodomain protein, pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila.

Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

Amino Acid Sequence↗
PubMed1984

Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia.

A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.

Animals↗
PubMed2005

Exploiting nuclear duality of ciliates to analyse topological requirements for DNA replication and transcription.

Spatial and temporal replication patterns are used to describe higher-order chromatin organisation from nuclei of early metazoan to mammalian cells. Here we demonstrate evolutionary conserved similarities and differences in replication patterns of micronuclei and macronuclei in the spirotrichous ciliate Stylonychia lemnae. Since this organism possesses two kinds of morphologically and functionally different nuclei in one cell, it provides an excellent model system to analyse topological requirements for DNA replication and transcription. Replication in the heterochromatic micronucleus occurs in foci-like structures showing spatial and temporal patterns similar to nuclei of higher eukaryotes, demonstrating that these patterns are inherent features of nuclear architecture. The 'nanochromosomes' of the macronucleus are replicated in the propagating replication band. We show that it consists of hundreds of replication foci. Post-replicative macronuclear chromatin remains organised in foci. These foci are not randomly distributed throughout the macronucleus, indicating a higher-order organisation of macronuclear chromatin above the level of 'nanochromosomes'. Both telomerase and proliferating cell nuclear antigen (PCNA) occur as foci-like structures in the rear zone of the replication band, suggesting that a wave of chromatin modification driven by a short or continuous exogenous signal permits the assembly of replication factories at predicted sites. We further show that transcription occurs at discrete sites colocalised with putative nucleoli and dispersed chromatin. Common principles of functional nuclear architecture were conserved during eukaryotic evolution. Moreover nuclear duality inherent to ciliates with their germline micronucleus and their somatic macronucleus may eventually provide further insight into epigenetic regulation of transcription, replication and nuclear differentiation.

Animals↗
PubMed1996

Involvement of active cellular mechanisms on the disorganization of oral apparatus in amicronucleate cells in Tetrahymena thermophila.

Ciliated protozoa, a group of unicellular eukaryotes, have two kinds of nuclei, a macronucleus (somatic nucleus) and a micronucleus (germinal nucleus) in a single cell. We previously reported that amicronucleate cells of Tetrahymena thermophila induced by nocodazole gradually lost their oral apparatus (OA) and ciliary rows but that amacronucleate cells did not. Since the macronucleus is responsible for the gene expression in the vegetative phase, the effects of actinomycin D and cycloheximide on the disorganization of the OA in amicronucleate cells induced by nocodazole were investigated. These inhibitors prevented the disorganization of the OA in amicronucleate cells. Amicronucleate cells did not grow even in the medium supplemented with high concentration of Fe, Cu and folinic acid which allow cells to grow without formation of food vacuoles. The results suggest that the macronucleus in the amicronucleate cells plays an active role in the induction of disorganization of the OA and malfunctions of nutrient uptake from the cell surface and/or in the fundamental cell division mechanisms, resulting in the death of amicronucleate cells.

Animals↗
PubMed2006

Death harmony played by nucleus and mitochondria: nuclear apoptosis during conjugation of tetrahymena.

Tetrahymena programmed nuclear death or nuclear apoptosis is a unique process during conjugation in which only the parental macronucleus is eliminated from the progeny cytoplasm, and other nuclei such as new micro- and macronuclei are unaffected. The nuclear death process consists of three successive steps: chromatin cleavage into high-molecular mass DNA, oligonucleosomal laddering concomitant with nuclear condensation, and complete degradation of the nuclear DNA. Following the first step of the death process, the parental macronucleus is engulfed by a large autophagosome in which many mitochondria are incorporated. Those sequestered mitochondria simply break down and release endonuclease similar to mammalian endonuclease G that is responsible for the generation of the DNA ladder, leading to the conclusion that mitochondria play a crucial role in the execution of the death program. Thus, the parental macronucleus is subject to final death by autophagy in collaboration with caspase-like enzymes, resulting in the ultimate outcome of nuclear resorption.

Animals↗
PubMed1999

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.

Animals↗
PubMed2005

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum.

Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.

Actin Cytoskeleton↗
PubMed1959

Macronuclear duplication in the ciliated protozoan Euplotes.

The ribbon-like macronucleus of Euplotes eurystomus pinches in half amitotically at each cell division. Several hours before the actual division two lightly staining duplication bands (reorganization bands) appear at the ends of the nucleus and approach each other slowly, finally meeting near the middle. Distal to the bands, that is, in regions through which the bands have already passed, the concentration of DNA (Feulgen) and "histone" (alkaline fast green) is greater than in the central zone. These facts suggest the hypothesis that DNA-histone synthesis takes place in a sequential fashion starting at the tips of the nucleus and proceeding to the middle. That this hypothesis is correct is shown by autoradiographic and photometric observations. Tritium-labelled thymidine is incorporated only in a limited region immediately distal to the bands. The average amount of Feulgen dye bound by the nucleus rises as the duplication bands approach each other, and is double the presynthesis value by the time the bands meet. A similar rise in the alkaline fast green dye is seen in duplicating nuclei, although no completely post-synthesis values were obtained in this study. The quantitative data are consistent with the assumption that the macronucleus contains a number of DNA-histone "units," presumably chromosomes, each of which duplicates once and only once.

Animals↗
PubMed2006

The germ line limited M element of Tetrahymena is targeted for elimination from the somatic genome by a homology-dependent mechanism.

A RNA interference (RNAi) like mechanism is involved in elimination of thousands of DNA segments from the developing somatic macronucleus of Tetrahymena, yet how specific internal eliminated sequences (IESs) are recognized remains to be fully elucidated. To define requirements for DNA rearrangement, we performed mutagenesis of the M element, a well-studied IES. While sequences within the macronucleus-retained DNA are known to determine the excision boundaries, we show that sequences internal to these boundaries are required to promote this IES's rearrangement. However, this element does not contain any specific sequence required in cis as removal of its entire left or right side was insufficient to abolish all rearrangement. Instead, rearrangement efficiency correlated with the overall size of the M element sequence within a given construct, with a lower limit of nearly 300 bp. Also, the observed minimal region necessary to epigenetically block excision supports this size limit. Truncated M element constructs that exhibited impaired rearrangement still showed full transcriptional activity, which suggests that their defect was due to inefficient recognition. This study indicates that IESs are targeted for elimination upon their recognition by homologous small RNAs and further supports the idea that DNA elimination is a RNAi-related mechanism involved in genome surveillance.

Animals↗
PubMedDate not supplied

Isolation of a Tetrahymena thermophila strain which induced metaphase I meiotic arrest: new pathway of abortive conjugation.

A hypodiploid strain of Tetrahymena thermophila has been obtained that shows arrest at the stage of condensed nuclei, corresponding to metaphase I of normal conjugants and induced arrest at meiotic metaphase I (i.e. at the stage of condensed, bivalent chromosomes) in its wt partner mate. The metaphase I arrested conjugants retained their old macronuclei and most of them underwent cell fusion, instead of separation of exconjugants. The doublets were viable and cortically integrated. When the arrest inducing strain was crossed to the haploid tester strain, the haploid micronuclei were arrested in the meiotic metaphase I as the diploid ones had been; the monovalent, chromosomes were condensed, the arms of sister chromatids were not separated, and they were not segregated. Separation of the arms of sister chromatids and disjunction of bivalent chromosomes were not prerequisite for the formation of microtubular spindles in those cells that were arrested in meiotic metaphase I. After re-feeding, the doublet cells resumed cell divisions, segregating two macronuclei and micronuclei at random. One macronucleus was derived from the arrest inducing strain and the other from the tester strain. Heterokaryon strains with macronuclei derived from the parental arrest inducing strain and with the micronucleus derived from the parental wt tester strain were obtained. Surprisingly, these heterokaryons did not induce meiotic arrest. Thus, the arrest in the melotic metaphase I was induced by the micronucleus and not by the macronucleus of the arrest inducing strain.

Animals↗
PubMed2005

snRNA and heterochromatin formation are involved in DNA excision during macronuclear development in stichotrichous ciliates.

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant "X" is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

Animals↗
PubMed1992

Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated, heterozygous macronuclei of Tetrahymena thermophila.

Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4-8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1992

Rate of phenotypic assortment in Tetrahymena thermophila.

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf, the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with approximately 45 assorting units, as has been asserted.

Animals↗
PubMed1992

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement.

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed.

Alleles↗
PubMed1999

Macronuclear division and cytokinesis in Tetrahymena.

Tetrahymena contains a micronucleus and a macronucleus. The micronucleus divides with typical mitosis, while the macronucleus divides amitotically. Although the mechanism responsible for macronuclear division was previously unknown, we clarified the organization of microtubules during macronuclear division. The macronuclear microtubules dynamically changed their distribution in an organized way throughout the macronuclear division. The macronuclear microtubules and the cytoplasmic microtubules cooperatively carried out the macronuclear division. When the micronuclear division was finished, p85 appeared at the presumptive division plane prior to the cytokinesis. The p85 directly interacted with calmodulin in a Ca(2+)-dependent manner, and p85 and CaM colocalized to the division furrow during cytokinesis. Moreover, the Ca(2+)/CaM inhibitor, W7, inhibited the direct interaction between p85 and CaM, the localization of both proteins to the division plane, and the formation of the division furrow. Thus, Ca(2+)/CaM and p85 have important roles in initiation and progression of cytokinesis in Tetrahymena.

Amino Acid Sequence↗
PubMed1996

Effects of the transcription inhibitor actinomycin D on postzygotic development of Tetrahymena thermophila conjugants.

During Tetrahymena thermophila conjugation, new somatic macronuclei develop from a common zygotic nucleus derived from meiotic products of the germline, and the old parental somatic nucleus is destroyed. The transcription inhibitor actinomycin D disrupts many events of postzygotic conjugation (cycloheximide causes indistinguishable effects). Early treatment causes a block of all postzygotic development, suggesting a transcription requirement for conjugants to pass a checkpoint, allowing entry into postzygotic development. Thereafter, pair separation, resorption of the old macronucleus, and elimination of one of the new micronuclei are blocked if actinomycin D is added at least 1.5 hr before each of these events normally occurs. Treatment just before DNA rearrangements in the developing macronuclei (anlagen) causes aberrant anlage DNA loss, suggesting that this DNA loss may be caused by inhibition of gene expression involved in genome rearrangements. DNA loss, and correlated lethality, appear to require previous gene expression, since actinomycin D added earlier causes cells to arrest in development without anlage DNA loss, and these conjugants can (at some frequency) complete conjugation and make viable progeny once actinomycin D is removed. The old macronucleus already had been inactivated before most actinomycin D treatments were initiated, indicating that the various induced defects we observed are the result of inhibition of postzygotic gene expression, presumably in anlagen. The defects induced by actinomycin D are similar to defects previously observed in conjugants harboring nullisomic germline deficiencies but proficient old macronuclei.

Animals↗