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PubMed1995

The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae.

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.

Animals↗
PubMed1992

Induction of specific macronuclear developmental mutations by microinjection of a cloned telomeric gene in Paramecium primaurelia.

In Paramecium, the differentiation of a highly polyploid macronucleus from a diploid nucleus is accompanied by an extensive reorganization of the genome, involving reduction in chromosome size and formation of new telomeres at heterogeneous, but reproducible, positions. The results presented here, as well as work by others, indicate that telomere addition regions are not strictly determined by the micronuclear sequence, but are at least partially controlled by the old macronucleus. It is shown that microinjecting a high copy number of a plasmid containing the G surface antigen gene into the macronucleus of wild-type cells specifically modifies the processing of the G gene-bearing micronuclear chromosome at the following autogamy. Telomeric repeats are added upstream of the gene, rather than at their wild-type position 5 kb downstream of its 3' end, resulting in the deletion of the gene from the new macronucleus. This macronuclear mutation is unstable at the following autogamy, giving rise to many different telomere addition regions in different postautogamous clones. However, after several successive autogamies, cell lines can be obtained in which the telomeres reproducibly form in the same region. In crosses with wild-type cells, these macronuclear mutations show cytoplasmic inheritance; the micronuclei of the mutants are shown to be fully functional. The implications for the mechanism of choice of telomere addition sites are discussed.

Animals↗
PubMed1994

A weak germ-line excision mutation blocks developmentally controlled amplification of the rDNA minichromosome of Tetrahymena thermophila.

During development of the somatic macronucleus of Tetrahymena thermophila, the rDNA is excised from its germ-line chromosome, rearranged into a palindrome, and amplified to 10(4) copies. We have identified a cis-acting germ-line mutation, rmm11/6, that prevents amplification of the rDNA in all but approximately 1 in 10(5) cells when it is the only rDNA allele in the developing macronucleus. The rmm11/6 mutation resides in a conserved element required for excision, the chromosome breakage sequence (Cbs) flanking the 3' end of the rDNA. Surprisingly, the rmm11/6 mutation only weakly affects excision of the rDNA from its germ-line location; at least 25% of cells heterozygous for this mutation correctly excise the affected rDNA allele. In heterozygotes, when this rDNA allele is excised, it is also poorly amplified. The rDNA amplification defect caused by this mutation is not overcome by delaying amplification with the DNA synthesis inhibitor aphidicolin, indicating that rDNA excision and amplification are not experimentally separable. Our experiments provide the first evidence that the capacity to amplify the rDNA is restricted in the developing macronucleus. We propose that the rmm11/6 mutation delays excision of the rDNA and that the developmental progression of the macronucleus past a restricted window for amplification is responsible for the severe amplification defect caused by this weak rDNA excision mutation.

Animals↗
PubMed1991

Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

Animals↗
PubMed1997

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences.

Animals↗
PubMed2004

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.

Animals↗
PubMed1986

Induced change in a non-mendelian determinant by transplantation of macronucleoplasm in Paramecium tetraurelia.

Mutant strain d48 of Paramecium tetraurelia lacks the gene for antigen A in the macronucleus, whereas this gene is present in the micronucleus. Transfer of macronucleoplasm from the wild type to strain d48 caused d48 to revert to the wild type after autogamy. Transfer of cytoplasm was not as effective as transfer of macronucleoplasm. It was also found that the micronucleus of d48 developed normally when it was transplanted to wild-type cells, whereas the micronucleus of the wild type formed a macronucleus that lacked the antigen A gene when this micronucleus was transplanted into d48. It was concluded that the micronucleus of d48 has a normal antigen A gene and that the hereditary determinants responsible for the d48 trait are located in the macronucleus. Molecular analysis of d48 clones that had been induced to revert to the wild type revealed that they possessed the antigen A gene in the macronucleus.

Animals↗
PubMed1999

Localization of microtubules during macronuclear division in Tetrahymena and possible involvement of macronuclear microtubules in 'amitotic' chromatin distribution.

The ciliated protozoa Tetrahymena contains two nuclei, a micronucleus and a macronucleus. In the vegetatively growing cell, the macronucleus divides amitotic while the micronucleus divides by mitosis. It has been indicated that microtubules are involved in macronuclear division and microtubules are observed to exist in the dividing macronucleus. To clarify the localization and the organization of microtubules in the amitotic dividing macronuclei, we used immunofluorescent staining technique. The microtubules were observed in the cytoplasm and macronucleus. The microtubules were organized and dynamically changed their distribution throughout the macronuclear division. We suggest a possibility that these microtubules are involved in 'amitotic' distribution of chromatin throughout the macronuclear division.

Animals↗
PubMed2005

A possible role of mitochondria in the apoptotic-like programmed nuclear death of Tetrahymena thermophila.

The ciliated protozoan Tetrahymena has a unique apoptosis-like process, which is called programmed nuclear death (PND). During conjugation, the new germinal micro- and somatic macro-nuclei differentiate from a zygotic fertilized nucleus, whereas the old parental macronucleus degenerates, ensuring that only the new macronucleus is responsible for expression of the progeny genotype. As is the case with apoptosis, this process encompasses chromatin cleavage into high-molecular mass DNA, oligonucleosomal DNA laddering, and complete degradation of the nuclear DNA, with the ultimate outcome of nuclear resorption. Caspase-8- and caspase-9-like activities are involved in the final resorption process of PND. In this report, we show evidence for mitochondrial association with PND. Mitochondria and the degenerating macronucleus were colocalized in autophagosome using two dyes for the detection of mitochondria. In addition, an endonuclease with similarities to mammalian endonuclease G was detected in the isolated mitochondria. When the macronuclei were incubated with isolated mitochondria in a cell-free system, DNA fragments of 150-400 bp were generated, but no DNA ladder appeared. Taking account of the present observations and the timing of autophagosome formation, we conclude that mitochondria might be involved in Tetrahymena PND, probably with the process of oligonucleosomal laddering.

Acid Phosphatase↗
PubMed2005

Class I histone deacetylase Thd1p affects nuclear integrity in Tetrahymena thermophila.

Class I histone deacetylases (HDACs) participate in the regulation of DNA-templated processes such as transcription and replication. Members of this class can act locally at specific sites, or they can act more globally, contributing to a baseline acetylation state, both of which actions may be important for genome maintenance and organization. We previously identified a macronuclear-specific class I HDAC in Tetrahymena thermophila called Thd1p, which is expressed early in the development of the macronucleus when it initially becomes transcriptionally active. To test the idea that Thd1p is important for global chromatin integrity in an active macronucleus, Tetrahymena cells reduced in expression of Thd1p were generated. We observed phenotypes that indicated loss of chromatin integrity in the mutant cells, including DNA fragmentation and extrusion of chromatin from the macronucleus, variable macronuclear size and shape, enlarged nucleoli, and reduced phosphorylation of histone H1 from bulk chromatin. Macronuclei in mutant cells also contained more DNA. This observation suggests a role for Thd1p in the control of nuclear DNA content, a previously undescribed role for class I HDACs. Together, these phenotypes implicate Thd1p in the maintenance of macronuclear integrity in multiple ways, probably through site-specific changes in histone acetylation since no change in the acetylation levels of bulk histones was detected in mutant cells.

Acetylation↗
PubMed1992

Developmental expression of macronuclear specific antigen in Paramecium caudatum.

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudatum and P. dubosqui. Immunoblotting showed that the antigen was a polypeptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macronuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus.

Animals↗
PubMed1992

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: early expression of the zygotic genotype.

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1-2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10 mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macronuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time.

Animals↗
PubMed1995

Differential amplification of pheromone genes of the ciliate Euplotes raikovi.

In hypotrich ciliates, the entire silent chromosomal genome of the germinal nucleus (micronucleus) undergoes extensive DNA rearrangements that, during the development of the somatic nucleus (macronucleus) at the beginning a new cell life cycle, eventually result in the production of linear DNA molecules. These molecules represent functional genes, each one consisting of a central coding region flanked by two shorter regions, which apparently lack canonical elements for regulation of replication and transcription. These are amplified to thousands of copies in the "adult" macronucleus of the vegetative cell. We defined the extent of this amplification for allelic codominant genes which, in the macronucleus of Euplotes raikovi, encode polypeptide cell recognition factors (pheromones). This amplification was shown to be allele-specific. The copy numbers of genes coding for pheromones Er-1, Er-2, and Er-10 were determined to be 2.5-2.9 x 10(4), 0.9-1.2 x 10(4), 1.6-1.85 x 10(4) respectively, and these numbers did not appreciably vary during the vegetative cell proliferation. This differential amplification of pheromone genes was (i) independent of whether two genes coexisted in the same heterozygous cell or were separated in the corresponding homozygotes, and (ii) directly correlated with quantitative variations in mRNA synthesis and pheromone secretion. On the basis of these results, it is suggested that a mechanism of gene-specific amplification may be used by hypotrich ciliates to modulate gene expression.

Animals↗
PubMed1976

Extra cytoproct mutant in Paramecium tetraurelia: morphogenetical analysis of proters and opisthes.

To account for the differences between proters and opisthes with regard to extra cytoproct morphogenesis in Paramecium tetraurelia, two hypotheses have been proposed and tested. (i) The differences may be a result of different actions of proter-macronucleus and opisthe-macronucleus. This hypothesis has been tested by cytoplasmically connecting the proter with the opisthe in the form of chains, some of which have only one macronucleus per chain. However, the connected proters and opisthes remain different in extra cytoproct morphogenesis, thus arguing against the hypothesis. (ii) The differences may be a result of differences between the proter and the opisthe with regard to the development of their posterior-ventral cortex: proters have a newly-developed posterior-ventral cortex whereas opisthes receive the posterior-ventral cortex from the pre-fission mother animal. This hypothesis has been tested by surgically producing an opisthe with a newly-regenerated posterior cortex. Such opisthes, however, remain different from proters in extra cytoproct morphogenesis. Thus no direct support for the second hypothesis is obtained. Also, proter-opisthe difference in morphogenesis may be understood in terms of Wolpert's positional information hypothesis, by assuming that the anterior and posterior ends of a dividing animal serve as reference points for establishing a gradient and that positional information before separation of the two daughter animals leads to differences in extra cytoproct morphogenesis between them after separation.

Animals↗
PubMed1996

DNA digestion and chromatin condensation during nuclear death in Tetrahymena.

DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organism Tetrahymena. We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agreement with a previous study, indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn2+ and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion in Tetrahymena, suggesting a requirement here for an endonuclease which is Ca2+-independent and Zn2+-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration.

Animals↗
PubMed1977

A single integrated gene for ribosomal RNA in a eucaryote, Tetrahymena pyriformis.

The macronucleus of the protozoan, Tetrahymena, is known to contain multiple rRNA genes which are not linked to the chromosomes. Here we present evidence that the germinal micronucleus of this organism contains a single gene for rRNA integrated into the chromosomal DNA. Unlike the extrachromosomal copies of the macronucleus, which are composed of a pair of reversely repeated sequences (a palindrome), the integrated copy of rDNA is nonrepetitive or half the size of the extrachromosomal rDNA. Furthermore, we have failed to detect such an integrated copy of rDNA in the macronucleus. The implications of these observations for the amplification and evolution of rDNA are discussed.

Animals↗
PubMed2005

Translocation of an 89-kDa periplasmic protein is associated with Holospora infection.

The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.

Amino Acid Sequence↗
PubMed2004

Small RNAs in genome rearrangement in Tetrahymena.

Small RNAs produced by an RNAi-related mechanism are involved in DNA elimination during development of the somatic macronucleus from the germline micronucleus in Tetrahymena. The properties of these small RNAs can explain how the primary sequence of the parental macronucleus epigenetically controls genome rearrangement in the new macronucleus and provide the first demonstration of an RNAi-mediated process that directly alters DNA sequence organization. Methylation of histone H3 on lysine 9 and accumulation of chromodomain proteins, hallmarks of heterochromatin, also occur specifically on sequences undergoing elimination and are dependent on the small RNAs. These findings contribute to a new paradigm of chromatin biology: regulation of heterochromatin formation by RNAi-related mechanisms in eukaryotes.

Animals↗