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Cell Reports Apr 2024In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA...
In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.
Topics: Paramecium; DNA Cleavage; DNA Breaks, Double-Stranded; Genome, Protozoan; Ku Autoantigen; DNA Repair; Protozoan Proteins; DNA End-Joining Repair
PubMed: 38547127
DOI: 10.1016/j.celrep.2024.114001 -
International Journal of Molecular... Dec 2023DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in or its eukaryotic homolog, the MutSα (Msh2-Msh6)...
DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6 localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6 only localized in MICs and newly developing MACs. knockout led to aberrant nuclear division during vegetative growth. The mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6 interacted with Msh2 and MMR-independent factors. Downregulation of expression affected the stability of Msh6. In addition, knockout led to the upregulated expression of several homologs at different developmental stages. Msh6 is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in .
Topics: DNA Mismatch Repair; Tetrahymena thermophila; MutS Homolog 2 Protein; Escherichia coli; DNA-Binding Proteins; Meiosis; Gametogenesis
PubMed: 38139447
DOI: 10.3390/ijms242417619 -
Cells Dec 2023Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex...
Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex mediate replication-couple (RC) and replication-independent (RI) nucleosome assembly, respectively. CHAF1B and HIRA share a similar domain but play different roles in nucleosome assembly by binding to the different interactors. At present, there is limited understanding for the similarities and differences in their respective functions. contains transcriptionally active polyploid macronuclei (MAC) and transcriptionally silent diploid micronuclei (MIC). Here, the distribution patterns of Caf1b and Hir1 exhibited both similarities and distinctions. Both proteins localized to the MAC and MIC during growth, and to the MIC during conjugation. However, Hir1 exhibited additional signaling on parental MAC and new MAC during sexual reproduction and displayed a punctate signal on developing anlagen. Caf1b and Hir1 only co-localized in the MIC with Pcna1 during conjugation. Knockdown of impeded cellular growth and arrested sexual reproductive development. Loss of led to MIC chromosome defects and aborted sexual development. Co-interference of and led to a more severe phenotype. Moreover, knockdown led to the up-regulation of expression, while knockdown of also led to an increase in expression. Furthermore, Caf1b and Hir1 interacted with different interactors. These results showed that CAF-1 and Hir1 have independent and complementary functions for chromatin assembly in .
Topics: Nucleosomes; Tetrahymena thermophila; Chromatin Assembly and Disassembly; Chromatin; Histones
PubMed: 38132148
DOI: 10.3390/cells12242828 -
Frontiers in Microbiology 2023Ciliates serve as excellent indicators for water quality monitoring. However, their utilization is hindered by various taxonomic confusions. The ciliate genus Bory de...
Ciliates serve as excellent indicators for water quality monitoring. However, their utilization is hindered by various taxonomic confusions. The ciliate genus Bory de Saint-Vincent, 1824 is commonly found in different aquatic habitats, but its taxonomy has been sparsely investigated using state-of-the-art methods. This study investigated two new species from Nanhui Wetland, Shanghai, China, using living observation, protargol staining, and molecular phylogeny methods. sp. nov. is 180-340 × 20-25 μm in size and possesses 12-16 somatic kineties, 1 terminal contractile vacuole, 2 macronuclear nodules, and 2 types of rod-shaped extrusomes. sp. nov. is distinguished from its congeners by its cell size of 210-400 × 25-35 μm, 14-17 somatic kineties, 1 terminal contractile vacuole, 1 macronucleus, and 2 types of rod-shaped extrusomes. Phylogenetic analyses based on SSU rRNA gene sequences indicate that Lacrymariidae is monophyletic but is not. Additionally, a brief review of the genus is provided in this study. We suggest that Alekperov, 1984, Kahl, 1930, and Burkovsky, 1970 be removed from the genus and propose nov. comb. and nov. comb. for the latter two. ZooBank registration: Present work: urn:lsid:zoobank.org:pub:CDFB1EBD-80BD-4533-B391-CEE89F62EDC4 sp. nov.: urn:lsid:zoobank.org:act:417E7C2D-DAEC-4711-90BB-64AB3CD2F7D5 sp. nov.: urn:lsid:zoobank.org:act:8778D6B0-1F2E-473C-BE19-3F685391A40D.
PubMed: 38029185
DOI: 10.3389/fmicb.2023.1259653 -
Nucleic Acids Research Dec 2023Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliate...
Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliate Paramecium tetraurelia, two micronuclei (MIC) serving sexual reproduction coexist with a somatic macronucleus (MAC) dedicated to gene expression. During sexual processes, the MAC is progressively destroyed while still ensuring transcription, and new MACs develop from copies of the zygotic MIC. Several gene clusters are successively induced and switched off before vegetative growth resumes. Concomitantly, programmed genome rearrangement (PGR) removes transposons and their relics from the new MACs. Development of the new MACs is controlled by the old MAC, since the latter expresses genes involved in PGR, including the PGM gene encoding the essential PiggyMac endonuclease that cleaves the ends of eliminated sequences. Using RNA deep sequencing and transcriptome analysis, we show that impairing PGR upregulates key known PGR genes, together with ∼600 other genes possibly also involved in PGR. Among these genes, 42% are no longer induced when no new MACs are formed, including 180 genes that are co-expressed with PGM under all tested conditions. We propose that bi-directional crosstalk between the two coexisting generations of MACs links gene expression to the progression of MAC development.
Topics: Gene Expression; Gene Rearrangement; Genome; Paramecium tetraurelia; Macronucleus
PubMed: 37953377
DOI: 10.1093/nar/gkad1006 -
Marine Life Science & Technology Aug 2023During a study on the diversity of ciliated protists in Lake Weishan Wetland, the largest wetland in northern China, four epibiotic sessilid peritrichs were isolated...
On four epibiotic peritrichous ciliates (Protozoa, Ciliophora) found in Lake Weishan Wetland: morphological and molecular data support the establishment of a new genus, gen. nov., and two new species.
During a study on the diversity of ciliated protists in Lake Weishan Wetland, the largest wetland in northern China, four epibiotic sessilid peritrichs were isolated from aquatic host animals. Two of them, i.e., Kellicott, 1885 and Fauré-Fremiet, 1943, were known species whereas the other two, i.e., gen. nov., sp. nov. and sp. nov., are new to science. The new genus gen. nov. is characterized by its branched non-contractile stalk, everted peristomial lip, obconical macronucleus and transverse silverlines. Two species are assigned to the new genus, namely sp. nov. and (Fauré-Fremiet, 1905) comb. nov. Morphologically, sp. nov. is recognized by its goblet-shaped zooids, single-layered peristomial lip, dichotomously branched stalk, and infundibular polykinety 3 (P3) containing three equal-length rows. sp. nov. is characterized by its slender zooid, curved macronucleus, and three equal-length rows in infundibular P3. Improved diagnoses and redescriptions of and are provided including, for the first time, data on the ciliature of . Phylogenetic analyses based on SSU rDNA, ITS1-5.8S rDNA -ITS2, and LSU rDNA sequence data strongly support the assertion that the family Epistylididae comprises morphospecies with different evolutionary lineages and indicate that gen. nov. may represent a new taxon at family level.
PubMed: 37637257
DOI: 10.1007/s42995-023-00184-z -
Journal of Indian Society of... 2023Periodontitis characterized by mild symptoms in the early stages, which makes diagnostics problematic. The gingival epithelium can be used for micronucleus assay since...
CONTEXT
Periodontitis characterized by mild symptoms in the early stages, which makes diagnostics problematic. The gingival epithelium can be used for micronucleus assay since gums are the area affected by the disease.
AIMS
The aim of the study was to study the frequency of occurrence and the range of nuclear anomalies in gingival epithelium of healthy people and people with periodontitis.
SETTINGS AND DESIGN
Scrapings of the gingival epithelium were made next to the central incisors (1.1) and molar teeth (1.7) in control and experimental groups (ten healthy males 35-50 years old and 10 males with periodontitis).
MATERIALS AND METHODS
The preparations were stained by Romanowsky-Giemsa. The frequency of nuclear aberrations (‰), the accumulation index, and the repair index were determined.
STATISTICAL ANALYSIS USED
The differences in the medians of nuclear aberrations were determined using Wilcoxon and the Van-der-Waerden tests. The pathology proportions were compared using the -test. To determine the predictors of periodontitis, receiver operator characteristic analysis was used. For multiple comparisons, the Bonferroni correction was used.
RESULTS
In the experimental group, the range of nuclear aberrations was wider, the ratio of karyolysis in the unaffected area was higher, than that in control; perinuclear vacuoles were fewer and macronuclei were more in the affected area. The frequency of cells with micronuclei over 1.33‰ in the affected area is the periodontitis marker.
CONCLUSIONS
Gingival epithelium can be used in micronucleus assay. Micronucleus test revealed a wider range of nuclear aberrations in the cells of the gingival epithelium and a higher frequency of occurrence of micronuclei in patients with periodontal disease compared to healthy subjects. Therefore, cytological signs of the inflammation appear earlier than the clinical ones and are verified more clearly. The markers of apoptosis and destruction of nuclei, and low repair index indicate normal elimination of damaged cells. An increased accumulation index in people with periodontitis may indicate the risk of malignant tumors.
PubMed: 37593557
DOI: 10.4103/jisp.jisp_18_22 -
IScience Aug 2023provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape...
provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism. When the nuclear transport factor, CSE1, is knocked down by RNAi, the shape and volume changes of the macronucleus are attenuated, and nuclear morphology is altered. CSE1 protein undergoes a dynamic relocalization correlated with nuclear shape changes, being mainly cytoplasmic prior to nuclear coalescence, and accumulating inside the macronucleus during coalescence. At the end of regeneration, CSE1 protein levels are reduced as the macronucleus returns to its pre-coalescence volume. We propose a model in which nuclear transport via CSE1 is required to increase the volume of the macronucleus, thereby decreasing the surface-to-volume ratio and driving coalescence of the nodes into a single mass.
PubMed: 37520736
DOI: 10.1016/j.isci.2023.107318 -
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 -
Microorganisms May 2023The genus Dujardin, 1841, with nearly 40 morphospecies, is one of the largest genera in the well-known subclass Scuticociliatia. In the present study, two species were...
Taxonomic and Phylogenetic Studies of Two Brackish Species (Protista, Ciliophora, Scuticociliatia) from Subtropical Coastal Waters of China, with Report of a New Species.
The genus Dujardin, 1841, with nearly 40 morphospecies, is one of the largest genera in the well-known subclass Scuticociliatia. In the present study, two species were collected from subtropical coastal waters of the East China Sea. The morphology and molecular phylogeny were investigated using modern standard methods. n. sp. is mainly characterized by an elliptical body in outline with the right ventrolateral side straight, 16-22 somatic kineties, 3-5 preoral kineties, and the posterior end of the membranelle 2a hook-like. An improved diagnosis of Pan et al., 2015 was provided: body size in vivo usually 90-135 × 45-85 μm, right ventrolateral side convex, 36-51 somatic kineties, 1-5 preoral kineties, one to three spherical macronuclei, membranelle 2a arranged in a zig-zag pattern in middle portion, posterior region hook-like, both membranelle 1 and membranelle 3 composed of three rows of basal bodies. The small subunit ribosomal DNA (SSU rDNA) of two species is sequenced, and their molecular phylogeny is analyzed. The new species n. sp. clusters with KF840519, JX310015, KF206430, and cf. KF848875, basically in accord with the morphological characteristics.
PubMed: 37374924
DOI: 10.3390/microorganisms11061422