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Microorganisms Feb 2023The conjusome plays an important role in the conjugation events that occur in . The conjusome appears in the anterior of conjugant pairs during the early stages of new...
The conjusome plays an important role in the conjugation events that occur in . The conjusome appears in the anterior of conjugant pairs during the early stages of new macronuclei (anlagen) development. It lacks a membrane, and is composed of a network of fibrous, electron dense material, containing background cytoplasm and ribosomes. Several proteins localize to this organelle, including Pdd1p, a chromodomain protein that participates in the formation of chromatin-containing structures in developing macronuclear anlagen, and is associated with the elimination of specific germ-line sequences from developing macronuclei. Conjugants lacking the allele in the parental macronucleus do not show Pdd1p antibody staining in conjusomes. Investigations were performed using mutant cell lines, uniparental cytogamy and drug treatment, and show that the conjusome appears to be dependent on parental macronuclei condensation, and is a transitory organelle that traffics nuclear determinants from the parental macronucleus to the developing anlagen. These data, taken together with Pdd1p knockout experiments, suggest the conjusome is involved in the epigenetic phenomena that occur during conjugation and sexual reorganization. This is likely a conserved organelle. Conjusome-like structures were also observed in another Ciliate, . In general, conjusomes have features that resemble germ line P-granules.
PubMed: 36838383
DOI: 10.3390/microorganisms11020418 -
Microorganisms Jan 2023Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The...
Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The transcriptionally active somatic genome, contained within the physically larger macronucleus, is both structurally and functionally different from the silent germline genome housed in the smaller micronucleus. This difference in genome architecture is particularly exaggerated in , in which the somatic genome comprises tens of thousands of gene-sized nanochromosomes maintained at a high and variable ploidy, while the germline has a diploid set of megabase-scale chromosomes. To examine the compositional differences between the nuclear structures housing the genomes, we performed a proteomic survey of both types of nuclei and of macronuclear histones using quantitative mass spectrometry. We note distinct differences between the somatic and germline nuclei, with many functional proteins being highly enriched in one of the two nuclei. To validate our conclusions and the efficacy of nuclear separation, we used protein localization through a combination of transformations and immunofluorescence. We also note that the macronuclear histones strikingly display only activating marks, consistent with the conclusion that the macronucleus is the hub of transcription. These observations suggest that the compartmentalization of different genome features into separate structures has been accompanied by a similar specialization of nuclear components that maintain and facilitate the functions of the genomes specific to each nucleus.
PubMed: 36838311
DOI: 10.3390/microorganisms11020343 -
European Journal of Protistology Apr 2023Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates...
Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.
Topics: Genomics; Germ Cells; Gene Rearrangement; Macronucleus; Micronucleus, Germline; Ciliophora
PubMed: 36822126
DOI: 10.1016/j.ejop.2023.125969 -
European Journal of Protistology Apr 2023Although the river nerite Theodoxus fluviatilis (Gastropoda: Neritimorpha) has an exceptionally broad geographical and ecological distribution, it carries only four...
Re-discovery and novel contributions to morphology and multigene phylogeny of Protospirella mazurica (Raabe, 1968) Aescht, 2001 (Ciliophora: Pleuronematida), an obligate symbiont of the river nerite Theodoxus fluviatilis Linnaeus, 1758 (Mollusca: Gastropoda).
Although the river nerite Theodoxus fluviatilis (Gastropoda: Neritimorpha) has an exceptionally broad geographical and ecological distribution, it carries only four ciliate species: Hypocomella quatuor, Protospirella mazurica, Scyphidia sp., and Trichodina baltica. After more than a half-century gap, we re-discovered P. mazurica in a Danubian population of T. fluviatilis (haplotypes F31 and F34) and characterized it using an integrative morpho-molecular approach. Protospirella mazurica is distinguished by (i) a small, elongate-ellipsoidal to ovoidal body, (ii) a broadly ellipsoidal macronucleus accompanied by a single globular micronucleus, (iii) a subterminal contractile vacuole, (iv) about 24 somatic kineties, (v) thigmotactic ciliature composed of about 10 kineties shortened posteriorly to form a parenthetical system, and (vi) a long inverted J-shaped paroral membrane associated with three unequally long membranelles. According to the present phylogenetic analyses of two mitochondrial and three nuclear markers, P. mazurica robustly clusters within the order Pleuronematida (Oligohymenophorea: Scuticociliatia) along with other symbiotic members of the families Hemispeiridae and Thigmophryidae as well as free-living representatives of the paraphyletic family Cyclidiidae. In light of the present phylogenetic analyses, we consider the family Ancistridae to be a junior synonym of the family Hemispeiridae, which collates 14 genera in our classification framework.
Topics: Humans; Animals; Phylogeny; Gastropoda; Rivers; Oligohymenophorea; Ciliophora; Mollusca; China
PubMed: 36805973
DOI: 10.1016/j.ejop.2023.125956 -
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 -
Microorganisms Dec 2022Fertilization-initiated development and adult-onset aging are standard features in the life history of eukaryotes. In , the number of cell divisions after the birth of a...
Fertilization-initiated development and adult-onset aging are standard features in the life history of eukaryotes. In , the number of cell divisions after the birth of a new generation is an essential parameter of sexual phase transition and aging. However, the gene driving this process and its evolutionary origin have not yet been elucidated. Here we report several critical outcomes obtained by molecular genetics, immunofluorescence microscopy, transformation by microinjection, and enzymological analysis. The cloned immaturin gene induces sexual rejuvenation in both mature and senescent cells by microinjection. The immaturin gene originated from proteobacteria's glutathione-S-transferase (GST) gene. However, immaturin has been shown to lose GST activity and instead acquire nuclease activity. In vitro substrates for immaturin-nuclease are single- and double-stranded DNA, linear and circular DNA, and single-stranded viral genome RNA such as coronavirus. Anti-immaturin antibodies have shown that the subcellular localizations of immaturin are the macronucleus, cytoplasm, cell surface area, and cilia. The phase transition of sexuality is related to a decrease in the intracellular abundance of immaturin. We propose that sexual maturation and rejuvenation is a process programmed by the immaturin gene, and the sexual function of each age is defined by both the abundance and the intracellular localization mode of the immaturin-nuclease.
PubMed: 36677375
DOI: 10.3390/microorganisms11010082 -
Proceedings of the National Academy of... Jan 2023During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the...
During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of , a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.
Topics: Humans; Ciliophora; DNA Transposable Elements; DNA, Protozoan; Gene Editing; Germ Cells; Transposases
PubMed: 36669106
DOI: 10.1073/pnas.2213985120 -
Journal of Fish Diseases Mar 2023Cryptocaryon irritans causes one of the most serious diseases in various wild and cultured marine fish, leading to mass mortality and economic loss. In this study,...
Cryptocaryon irritans causes one of the most serious diseases in various wild and cultured marine fish, leading to mass mortality and economic loss. In this study, hydroxyl radical (•OH) solution produced by strong ionization discharge combined with water jet cavitation effect was injected into orange-spotted grouper (Epinephelus coioides) aquaculture tanks for C. irritans control. The results showed that all C. irritans theronts were inactivated by •OH solution at concentrations of 0.5 mg/L within 2 min. •OH could induce alteration of shape, the absence of motility and macronucleus dispersion in theronts. A possible explanation was that the macronucleus of C. irritans might be damaged by •OH; as a result, its metabolism and life activities were disturbed. The •OH treatment increased the survival rate of E. coioides challenged with C. irritans from 64.7 ± 8.0% (mean ± SD) to 100% and reduced their infection intensity significantly. Stress response biomarkers such as malonaldehyde, glutathione, glutathione peroxidase, superoxide dismutase (SOD) and catalase levels in the gills of E. coioides at different time points were analysed. The SOD activity in the •OH group first decreased and then recovered to the initial level at the end of the experiment. The other stress response biomarkers had no significant difference from that in the uninfected control group after •OH treatment. Additionally, the gill of E. coioides in the •OH group exhibited slight and reversible transformation compared with the uninfected control group. Compared with •OH treatment, chlorine dioxide and formalin treatment reduced the survival rate, induced oxidative damage and changed the histological gill structure in E. coioides. In conclusion, •OH could be applied effectively to control C. irritans infection without affecting the normal physiological condition of E. coioides.
Topics: Animals; Bass; Ciliophora; Ciliophora Infections; Fish Diseases; Hymenostomatida; Superoxide Dismutase; Fish Proteins
PubMed: 36519440
DOI: 10.1111/jfd.13736 -
Molecular Biology of the Cell Feb 2023harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the "cell cycles" of the diploid micronucleus and polyploid...
harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the "cell cycles" of the diploid micronucleus and polyploid macronucleus are offset. Micronuclear S phase initiates just before cytokinesis and is completed in daughter cells before onset of macronuclear DNA replication. Mitotic micronuclear division occurs mid-cell cycle, while macronuclear amitosis is coupled to cell division. Here we report the first RNA-seq cell cycle analysis of a binucleated ciliated protozoan. RNA was isolated across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the 26,000+ predicted genes were shown to be cell cycle regulated. Proteins present in both nuclei exhibit a single mRNA peak that always precedes their macronuclear function. Nucleus-limited genes, including nucleoporins and importins, are expressed before their respective nucleus-specific role. Cyclin D and A/B gene family members exhibit different expression patterns that suggest nucleus-restricted roles. Periodically expressed genes cluster into seven cyclic patterns. Four clusters have known PANTHER gene ontology terms associated with G1/S and G2/M phase. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication, and cell division.
Topics: Tetrahymena thermophila; Cell Nucleus; Cell Cycle; Mitosis; Ciliophora; Gene Expression Profiling; Tetrahymena
PubMed: 36475712
DOI: 10.1091/mbc.E22-08-0326 -
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