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Microorganisms Jan 2023The Gram-negative bacterium is a macronucleus-specific symbiont of the ciliate . It is known that an infection of this bacterium induces high level expressions of the...
A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host .
The Gram-negative bacterium is a macronucleus-specific symbiont of the ciliate . It is known that an infection of this bacterium induces high level expressions of the host and genes, and the host cell acquires both heat-shock and high salt resistances. In addition, an infectious form of -specific 63-kDa periplasmic protein with a DNA-binding domain in its amino acid sequence is secreted into the host macronucleus after invasion into the macronucleus and remain within the nucleus. These facts suggest that binding of the 63-kDa protein to the host macronuclear DNA causes changes in the host gene expressions and enhances an environmental adaptability of the host cells. This 63-kDa protein was renamed as periplasmic region protein 1 (PRP1) to distinguish it from other proteins with similar molecular weights. To confirm whether PRP1 indeed binds to the host DNA, SDS-DNA PAGE and DNA affinity chromatography with calf thymus DNA and DNA were conducted and confirmed that PRP1 binds weakly to the DNA with a monoclonal antibody raised for the 63-kDa protein.
PubMed: 36677447
DOI: 10.3390/microorganisms11010155 -
Theoretical Population Biology Jun 2022Cell division is a necessity of life which can be either mitotic or amitotic. While both are fundamental, amitosis is sometimes considered a relic of little importance...
Cell division is a necessity of life which can be either mitotic or amitotic. While both are fundamental, amitosis is sometimes considered a relic of little importance in biology. Nevertheless, eukaryotes often have polyploid cells, including cancer cells, which may divide amitotically. To understand how amitosis ensures the completion of cell division, we turn to the macronuclei of ciliates. The grand scheme governing the proliferation of the macronuclei of ciliate cells, which involves chromosomal replication and amitosis, is currently unknown, which is crucial for developing population genetics model of ciliate populations. Using a novel model that encompasses a wide range of mechanisms together with experimental data of the composition of mating types at different stages derived from a single karyonide of Tetrahymena thermophila, we show that the chromosomal replication of the macronucleus has a strong head-start effect, with only about five copies of chromosomes replicated at a time and persistent reuse of the chromosomes involved in the early replication. Furthermore the fission of a fully grown macronucleus is non-random with regard to chromosome composition, with a strong tendency to push chromosomes and their replications to the same daughter cell.
Topics: Cell Division; Chromosomes; Ciliophora; Macronucleus; Tetrahymena thermophila
PubMed: 35331774
DOI: 10.1016/j.tpb.2022.03.004 -
Genome Research 2022With its nuclear dualism, the ciliate constitutes a unique model to study how host genomes cope with transposable elements (TEs). harbors two germline micronuclei...
With its nuclear dualism, the ciliate constitutes a unique model to study how host genomes cope with transposable elements (TEs). harbors two germline micronuclei (MICs) and a polyploid somatic macronucleus (MAC) that develops from one MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of approximately 45,000 TE-derived internal eliminated sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by noncoding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with high-throughput sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), whereas TEs are eliminated at a later stage. We show that DNA elimination proceeds independently of replication. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends, and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision by acquiring stronger sequence determinants and escaping epigenetic control.
Topics: Paramecium tetraurelia; DNA, Protozoan; RNA, Untranslated; DNA Transposable Elements; Germ Cells
PubMed: 36418061
DOI: 10.1101/gr.277027.122 -
Genome Biology and Evolution Jan 2021Polyploidy can provide adaptive advantages and drive evolution. Amitotic division of the polyploid macronucleus (MAC) in ciliates acts as a nonsexual genetic mechanism...
Polyploidy can provide adaptive advantages and drive evolution. Amitotic division of the polyploid macronucleus (MAC) in ciliates acts as a nonsexual genetic mechanism to enhance adaptation to stress conditions and thus provides a unique model to investigate the evolutionary role of polyploidy. Mutation is the primary source of the variation responsible for evolution and adaptation; however, to date, de novo mutations that occur in ciliate MAC genomes during these processes have not been characterized and their biological impacts are undefined. Here, we carried out long-term evolution experiments to directly explore de novo MAC mutations and their molecular features in the model ciliate, Tetrahymena thermophila. A simple but effective method was established to detect base-substitution mutations in evolving populations whereas filtering out most of the false positive base-substitutions caused by repetitive sequences and the programmed genome rearrangements. The detected mutations were rigorously validated using the MassARRAY system. Validated mutations showed a strong G/C→A/T bias, consistent with observations in other species. Moreover, a progressive increase in growth rate of the evolving populations suggested that some of these mutations might be responsible for cell fitness. The established mutation identification and validation methods will be an invaluable resource to make ciliates an important model system to study the role of polyploidy in evolution.
Topics: Ciliophora; Evolution, Molecular; Genome, Protozoan; Macronucleus; Mutation; Polyploidy; Tetrahymena thermophila
PubMed: 33146387
DOI: 10.1093/gbe/evaa232 -
PLoS Genetics Jan 2015The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm,...
The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development.
Topics: Cell Cycle; Cell Division; Cell Nucleus; Chromosomes; DNA Replication; DNA, Ribosomal; Gene Expression Regulation, Developmental; Genomic Instability; Origin Recognition Complex; S Phase; Tetrahymena thermophila
PubMed: 25569357
DOI: 10.1371/journal.pgen.1004875 -
International Journal of Molecular... Jun 2023Mismatch repair (MMR) is a conserved mechanism that is primarily responsible for the repair of DNA mismatches during DNA replication. Msh2 forms MutS heterodimer...
Mismatch repair (MMR) is a conserved mechanism that is primarily responsible for the repair of DNA mismatches during DNA replication. Msh2 forms MutS heterodimer complexes that initiate the MMR in eukaryotes. The function of Msh2 is less clear under different chromatin structures. contains a transcriptionally active macronucleus (MAC) and a transcriptionally silent micronucleus (MIC) in the same cytoplasm. Msh2 is localized in the MAC and MIC during vegetative growth. Msh2 is localized in the perinuclear region around the MIC and forms a spindle-like structure as the MIC divides. During the early conjugation stage, Msh2 is localized in the MIC and disappears from the parental MAC. Msh2 is localized in the new MAC and new MIC during the late conjugation stage. Msh2 also forms a spindle-like structure with a meiotic MIC and mitotic gametic nucleus. knockdown inhibits the division of MAC and MIC during vegetative growth and affects cellular proliferation. knockdown mutants are sensitive to cisplatin treatment. knockdown also affects micronuclear meiosis and gametogenesis during sexual development. Furthermore, Msh2 interacts with MMR-dependent and MMR-independent factors. Therefore, Msh2 is necessary for macronuclear stability, as well as micronuclear mitosis and meiosis in .
Topics: Tetrahymena thermophila; MutS Homolog 2 Protein; DNA Mismatch Repair; Cell Nucleus; Macronucleus
PubMed: 37445734
DOI: 10.3390/ijms241310559 -
Biochimica Et Biophysica Acta.... Jun 2022Developmental DNA elimination in Paramecium tetraurelia occurs through a trans-nuclear comparison of the genomes of two distinct types of nuclei: the germline...
Developmental DNA elimination in Paramecium tetraurelia occurs through a trans-nuclear comparison of the genomes of two distinct types of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC). During sexual reproduction, which starts with meiosis of the germline nuclei, MIC-limited sequences including Internal Eliminated Sequences (IESs) and transposons are eliminated from the developing MAC in a process guided by noncoding RNAs (scnRNAs and iesRNAs). However, our current understanding of this mechanism is still very limited. Therefore, studying both genetic and epigenetic aspects of these processes is a crucial step to understand this phenomenon in more detail. Here, we describe the involvement of homologs of classical meiotic proteins, Spo11, Msh4-1, and Msh5 in this phenomenon. Based on our analyses, we propose that proper functioning of Spo11, Msh4-1, and Msh5 during Paramecium sexual reproduction are necessary for genome reorganization and viable progeny. Also, we show that double-strand breaks (DSBs) in DNA induced during meiosis by Spo11 are crucial for proper IESs excision. In summary, our investigations show that early sexual reproduction processes may significantly influence later somatic genome integrity.
Topics: Germ Cells; Macronucleus; Meiosis; Paramecium tetraurelia; RNA, Untranslated
PubMed: 35181406
DOI: 10.1016/j.bbamcr.2022.119239 -
Molecular Biology of the Cell Jul 2021Endogenous RNA interference (RNAi) pathways regulate a wide range of cellular processes in diverse eukaryotes, yet in the ciliated eukaryote, , the cellular purpose of...
Endogenous RNA interference (RNAi) pathways regulate a wide range of cellular processes in diverse eukaryotes, yet in the ciliated eukaryote, , the cellular purpose of RNAi pathways that generate ∼23-24 nucleotide (nt) small (s)RNAs has remained unknown. Here, we investigated the phenotypic and gene expression impacts on vegetatively growing cells when genes involved in ∼23-24 nt sRNA biogenesis are disrupted. We observed slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants Δ, Δ, and Δ. In addition, Δ and Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in Δ and Δ strains, with Rad51 and double-stranded DNA break marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. Together, our findings suggest that an evolutionarily conserved role for RNAi pathways in maintaining genome integrity may be extended even to the early branching eukaryotic lineage that gave rise to .
Topics: DNA; DNA Breaks, Double-Stranded; DNA Repair; Evolution, Molecular; Gene Expression Profiling; Gene Expression Regulation; Protozoan Proteins; RNA, Small Interfering; Rad51 Recombinase; Recombinational DNA Repair; Sequence Analysis, RNA; Tetrahymena thermophila
PubMed: 34010017
DOI: 10.1091/mbc.E20-10-0631 -
European Journal of Protistology Aug 2023Two already known representatives of Holospora-like bacteria, "Candidatus Gortzia yakutica" from Paramecium putrinum and Preeria caryophila, originally retrieved from...
Two already known representatives of Holospora-like bacteria, "Candidatus Gortzia yakutica" from Paramecium putrinum and Preeria caryophila, originally retrieved from the Paramecium aurelia complex, were found in new hosts: Paramecium nephridiatum and Paramecium polycaryum, respectively. In the present study, these bacteria were investigated using morphological and molecular methods. For "Ca. G. yakutica", the first details of the electron microscopic structure in the main and new hosts were provided. Regarding Pr. caryophila, the ultrastructural description of this species was implemented by several features previously unknown, such as the so called "membrane cluster" dividing periplasm from cytoplasm and fine composition of infectious forms before and during its releasing from the infected macronucleus. The new combinations of these Holospora-like bacteria with ciliate hosts were discussed from biogeographical and ecological points of view. Host specificity of symbionts as a general paradigm was critically reviewed as well.
Topics: Holosporaceae; Symbiosis; Bacteria; Macronucleus; Paramecium; Phylogeny
PubMed: 37356197
DOI: 10.1016/j.ejop.2023.125998 -
The Journal of Eukaryotic Microbiology Jan 2018Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. Blepharisma...
Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. Blepharisma americanum is distinguishable by its pink coloration derived from the autofluorescent pigment blepharismin and tends to have a single somatic macronucleus with 3-6 nodes and multiple germline micronuclei. We used fluorescence confocal microscopy to explore the DNA content and amplification between the somatic and germline nuclei of B. americanum through its life cycle. We estimate that the DNA content of the macronucleus and micronucleus are 43 ± 8 Gbp and 83 ± 16 Mbp respectively. This correlates with an approximate DNA content difference of 500-fold from micronucleus to macronucleus and a macronuclear ploidy of ~1,100 N as compared to the presumably diploid micronucleus. We also investigate a previously reported macronuclear inclusion, which is present sporadically across all life cycle stages; this inclusion looks as if it contains blepharismin based on its fluorescent properties, but its function remains unknown. We also provide additional detail to our understanding of life cycles changes in B. americanum by analyses of fluorescent images. Overall, the data analyzed here contribute to our understanding of the diversity of nuclear architecture in ciliates by providing details on the highly polyploid somatic macronucleus of B. americanum.
Topics: Ciliophora; DNA, Protozoan; Fluorescent Dyes; Gene Amplification; Genome, Protozoan; Indoles; Life Cycle Stages; Macronucleus; Microscopy, Confocal; Staining and Labeling
PubMed: 28460157
DOI: 10.1111/jeu.12422