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The EMBO Journal Nov 2022Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The...
Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.
Topics: Paramecium; DNA Transposable Elements; Chromatin Assembly and Disassembly; Nucleosomes; DNA, Protozoan
PubMed: 36221862
DOI: 10.15252/embj.2022111839 -
Zoological Research Sep 2022During faunal studies of psammophilic ciliates along the coast of Qingdao, China, several marine karyorelictean species were isolated. Among them, four species within...
During faunal studies of psammophilic ciliates along the coast of Qingdao, China, several marine karyorelictean species were isolated. Among them, four species within the genus were investigated, including two species new to science: i.e., , , , and has been reported several times, but this study is the first to provide detailed morphological characters and phylogenetics. can be distinguished from its congeners by the presence of complex cortical granules, fewer macronuclei, and longer body size. differs from its congeners by having 14-17 right lateral ciliary rows and 24-37 dikinetids of intrabuccal kinety. Poorly known var. (Kahl, 1933) Foissner, 1996 should be elevated from subspecies to species level, (Foissner, 1996) , based on detailed redescriptions with statistical data, living morphology, infraciliature, and species definitions. Small subunit (SSU) rDNA was sequenced for the four species, and phylogenetic analysis revealed that all known taxa in formed the outline branch to the genus with moderate to high bootstrap support among lineages.
Topics: Animals; China; Ciliophora; DNA, Ribosomal; Phylogeny
PubMed: 35993134
DOI: 10.24272/j.issn.2095-8137.2022.014 -
Veterinary Clinical Pathology Dec 2022Epithelial cells show varying degrees of cytologic atypia in dogs with nonmalignant lesions (NML) and carcinomas (ubC) of the bladder, making histopathologic examination...
BACKGROUND
Epithelial cells show varying degrees of cytologic atypia in dogs with nonmalignant lesions (NML) and carcinomas (ubC) of the bladder, making histopathologic examination necessary for a definitive diagnosis.
OBJECTIVES
This study aimed to investigate the diagnostic performance of squash preparation cytology and identify several cytomorphologic features of ubC to assist in diagnoses.
METHODS
Squash preparations were made and reviewed in dogs that underwent transurethral cystoscopy. The results were compared with histopathologic diagnoses. Two cytopathologists performed blinded assessments using a scoring system established for 11 cytologic features, including the presence of macronuclei, abnormal nucleoli, atypical mitoses, signet ring cells, multinucleated cells, nuclear molding, anisokaryosis, cytoplasmatic microvacuolization, cell arrangements, and neutrophil and lymphocyte infiltrations. Based on cytologic and histopathologic diagnoses, dogs were divided into ubC and NML groups. Associations between cytologic and histopathologic diagnoses were investigated, and agreement between the cytopathologists was calculated. Cytologic features were analyzed with multivariate logistic regression models. The performance of predictors in the final model was evaluated in terms of Sensitivity (Se), Specificity (Sp), accuracy, positive predictive value (PPV), negative predictive value (NPV), likelihood ratio positive (LR+), and negative (LR-) values, and the diagnostic odds ratio (DOR).
RESULTS
Forty-four dogs diagnosed with ubC, and 17 with NML were included in the study. Cytologic and histopathologic diagnoses were significantly associated with each cytopathologist. There was an almost perfect agreement between cytopathologists (κ = 0.88). The absence of neutrophilic infiltration, the presence of multinucleated cells, and nuclear molding were associated with ubC; using a combination of these features in parallel testing resulted in Se = 0.98, Sp = 0.65, accuracy = 0.89, PPV = 0.88, NPV = 0.92, LR + =2.77, LR- = 0.04, and DOR = 7.7.
CONCLUSIONS
Squash preparation cytology could be a reliable technique to diagnose ubC in dogs. The best diagnostic combination was the absence of neutrophilic infiltration, multinucleated cells, and nuclear molding.
Topics: Dogs; Animals; Urinary Bladder; Urinary Bladder Neoplasms; Cytodiagnosis; Cytological Techniques; Carcinoma; Sensitivity and Specificity; Dog Diseases
PubMed: 35854403
DOI: 10.1111/vcp.13151 -
Methods in Molecular Biology (Clifton,... 2022Tetrahymena is a fascinating organism for studying the nuclear pore complex because it has two structurally and functionally distinct nuclei (macronucleus and...
Tetrahymena is a fascinating organism for studying the nuclear pore complex because it has two structurally and functionally distinct nuclei (macronucleus and micronucleus) within a cell, and there are two compositionally distinct nuclear pore complexes (NPCs) with different functions in each nucleus. Therefore, it is possible to link the function of a specific constituent protein with the nuclear function of the macronucleus and micronucleus. Additionally, these NPCs undergo dynamic changes in their structures and compositions during nuclear differentiation. Live CLEM imaging, a method of correlative light and electron microscopy (CLEM) combined with live cell imaging, is a powerful tool for visualizing these dynamic changes of specific molecules/structures of interest at high resolution. Here, we describe Live CLEM that can be applied to the study of the dynamic behavior of NPCs in Tetrahymena cells undergoing nuclear differentiation.
Topics: Electrons; Macronucleus; Microscopy, Electron; Nuclear Pore; Tetrahymena
PubMed: 35412257
DOI: 10.1007/978-1-0716-2337-4_30 -
The Journal of Eukaryotic Microbiology Sep 2022The term epigenetics is used for any layer of genetic information aside from the DNA base-sequence information. Mammalian epigenetic research increased our understanding... (Review)
Review
The term epigenetics is used for any layer of genetic information aside from the DNA base-sequence information. Mammalian epigenetic research increased our understanding of chromatin dynamics in terms of cytosine methylation and histone modification during differentiation, aging, and disease. Instead, ciliate epigenetics focused more on small RNA-mediated effects. On the one hand, these do concern the transport of RNA from parental to daughter nuclei, representing a regulated transfer of epigenetic information across generations. On the other hand, studies of Paramecium, Tetrahymena, Oxytricha, and Stylonychia revealed an almost unique function of transgenerational RNA. Rather than solely controlling chromatin dynamics, they control sexual progeny's DNA content quantitatively and qualitatively. Thus epigenetics seems to control genetics, at least genetics of the vegetative macronucleus. This combination offers ciliates, in particular, an epigenetically controlled genetic variability. This review summarizes the epigenetic mechanisms that contribute to macronuclear heterogeneity and relates these to nuclear dimorphism. This system's adaptive and evolutionary possibilities raise the critical question of whether such a system is limited to unicellular organisms or binuclear cells. We discuss here the relevance of ciliate genetics and epigenetics to multicellular organisms.
Topics: Animals; Cell Proliferation; Chromatin; Ciliophora; DNA; Epigenesis, Genetic; Genome, Protozoan; Mammals; Paramecium; RNA
PubMed: 35363910
DOI: 10.1111/jeu.12914 -
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 -
Molecular Biology and Evolution Apr 2022Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there...
Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there are two distinct nuclei in ciliates with separate germline and somatic cell functions. Here, we assembled a near-complete macronuclear genome of Fabrea salina, which belongs to one of the oldest clades of ciliates. Its extremely minimized genome (18.35 Mb) is the smallest among all free-living heterotrophic eukaryotes and exhibits typical streamlined genomic features, including high gene density, tiny introns, and shrinkage of gene paralogs. Gene families involved in hypersaline stress resistance, DNA replication proteins, and mitochondrial biogenesis are expanded, and the accumulation of phosphatidic acid may play an important role in resistance to high osmotic pressure. We further investigated the morphological and transcriptomic changes in the macronucleus during sexual reproduction and highlighted the potential contribution of macronuclear residuals to this process. We believe that the minimized genome generated in this study provides novel insights into the genome streamlining theory and will be an ideal model to study the evolution of eukaryotic heterotrophs.
Topics: Ciliophora; DNA, Protozoan; Genome, Protozoan; Introns; Macronucleus; Sequence Analysis, DNA
PubMed: 35325184
DOI: 10.1093/molbev/msac062 -
The Journal of Eukaryotic Microbiology Sep 2022This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of... (Review)
Review
This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of Paramecium caudatum is a programmed process conducted by a specific gene and its product protein. In Paramecium tetraurelia, autogamy was revealed to depend on the number of DNA syntheses rather than the number of cell divisions in clonal aging. The cytoplasmic complementarity test established that microinjection of wild-type cytoplasm can correct genetic defects of mutants. The concept of complementarity together with protein chemistry revealed compounds that control membrane excitability. In non-Mendelian inheritance, noncoding small RNAs made from the parental micronucleus regulate the rearrangement of the progeny's macronuclear DNA. The macronucleus has the potential to be used as a factory for genetic engineering. The development and differentiation of progeny's nuclei in mating pairs are controlled by the parental macronucleus. The chemical reaction processes associated with exocytosis have been revealed by microinjection of various enzymes and antibodies. Using the fusion gene of histone H2B and yellow-fluorescence protein, it was revealed that the fusion gene-mRNA is transferred between cells during mating. Experiments with endosymbiotic bacteria and the host shed light on the conditions needed to establish sustainable symbiotic relationships.
Topics: Cytoplasm; Macronucleus; Micromanipulation; Paramecium; Paramecium tetraurelia
PubMed: 35318763
DOI: 10.1111/jeu.12909 -
The Journal of Eukaryotic Microbiology Jul 2022Amitosis is widespread among eukaryotes, but the underlying mechanisms are poorly understood. The polyploid macronucleus (MAC) of unicellular ciliates divides by...
Amitosis is widespread among eukaryotes, but the underlying mechanisms are poorly understood. The polyploid macronucleus (MAC) of unicellular ciliates divides by amitosis, making ciliates a potentially valuable model system to study this process. However, a method to accurately quantify the copy number of MAC chromosomes has not yet been established. Here, we used droplet digital PCR (ddPCR) to quantify the absolute copy number of the MAC chromosomes in Tetrahymena thermophila. We first confirmed that ddPCR is a sensitive and reproducible method to determine accurate chromosome copy numbers at the single-cell level. We then used ddPCR to determine the copy number of different MAC chromosomes by analyzing individual T. thermophila cells in the G1 and the amitotic (AM) phases. The average copy number of MAC chromosomes was 90.9 at G1 phase, approximately half the number at AM phase (189.8). The copy number of each MAC chromosome varied among individual cells in G1 phase and correlated with cell size, suggesting that amitosis accompanied by unequal cytokinesis causes copy number variability. Furthermore, the fact that MAC chromosome copy number is less variable among AM-phase cells suggests that the copy number is standardized by regulating DNA replication. We also demonstrated that copy numbers differ among different MAC chromosomes and that interchromosomal variations in copy number are consistent across individual cells. Our findings demonstrate that ddPCR can be used to model amitosis in T. thermophila and possibly in other ciliates.
Topics: Chromosomes; Ciliophora; DNA Copy Number Variations; Humans; Macronucleus; Polyploidy; Tetrahymena; Tetrahymena thermophila
PubMed: 35313044
DOI: 10.1111/jeu.12907 -
Genome Research Apr 2022The unicellular ciliate contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy....
The unicellular ciliate contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy. As macronculear chromatin is devoid of heterochromatin, our study characterizes the functional epigenomic organization necessary for gene regulation and proper Pol II activity. Histone marks (H3K4me3, H3K9ac, H3K27me3) reveal no narrow peaks but broad domains along gene bodies, whereas intergenic regions are devoid of nucleosomes. Our data implicate H3K4me3 levels inside ORFs to be the main factor associated with gene expression, and H3K27me3 appears in association with H3K4me3 in plastic genes. Silent and lowly expressed genes show low nucleosome occupancy, suggesting that gene inactivation does not involve increased nucleosome occupancy and chromatin condensation. Because of a high occupancy of Pol II along highly expressed ORFs, transcriptional elongation appears to be quite different from that of other species. This is supported by missing heptameric repeats in the C-terminal domain of Pol II and a divergent elongation system. Our data imply that unoccupied DNA is the default state, whereas gene activation requires nucleosome recruitment together with broad domains of H3K4me3. In summary, gene activation and silencing in run counter to the current understanding of chromatin biology.
Topics: Chromatin; Histone Code; Histones; Nucleosomes; Paramecium; RNA Polymerase II
PubMed: 35264449
DOI: 10.1101/gr.276126.121