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G3 (Bethesda, Md.) May 2018Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a...
Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a germline nucleus (micronucleus; abbr. MIC). Furthermore, undergoes extensive genome rearrangements during sexual conjugation and post-zygotic development of daughter cells. These rearrangements are necessary because the precursor MIC loci are often both fragmented and scrambled, with respect to the corresponding MAC loci. Such genome architectures are remarkably tolerant of encrypted MIC loci, because RNA-guided processes during MAC development reorganize the gene fragments in the correct order to resemble the parental MAC sequence. Here, we describe the germline organization of several nested and highly scrambled genes in These include cases with multiple layers of nesting, plus highly interleaved or tangled precursor loci that appear to deviate from previously described patterns. We present mathematical methods to measure the degree of nesting between precursor MIC loci, and revisit a method for a mathematical description of scrambling. After applying these methods to the chromosome rearrangement maps of we describe cases of nested arrangements with up to five layers of embedded genes, as well as the most scrambled loci in .
Topics: Chromosomes; DNA; Gene Rearrangement; Genetic Loci; Macronucleus; Micronucleus, Germline; Oxytricha; Recombination, Genetic
PubMed: 29545465
DOI: 10.1534/g3.118.200176 -
Epigenetics & Chromatin Jul 2021Histone chaperones facilitate DNA replication and repair by promoting chromatin assembly, disassembly and histone exchange. Following histones synthesis and nucleosome...
Histone chaperones facilitate DNA replication and repair by promoting chromatin assembly, disassembly and histone exchange. Following histones synthesis and nucleosome assembly, the histones undergo posttranslational modification by different enzymes and are deposited onto chromatins by various histone chaperones. In Tetrahymena thermophila, histones from macronucleus (MAC) and micronucleus (MIC) have been comprehensively investigated, but the function of histone chaperones remains unclear. Histone chaperone Nrp1 in Tetrahymena contains four conserved tetratricopepeptide repeat (TPR) domains and one C-terminal nuclear localization signal. TPR2 is typically interrupted by a large acidic motif. Immunofluorescence staining showed that Nrp1 is located in the MAC and MICs, but disappeared in the apoptotic parental MAC and the degraded MICs during the conjugation stage. Nrp1 was also colocalized with α-tubulin around the spindle structure. NRP1 knockdown inhibited cellular proliferation and led to the loss of chromosome, abnormal macronuclear amitosis, and disorganized micronuclear mitosis during the vegetative growth stage. During sexual developmental stage, the gametic nuclei failed to be selected and abnormally degraded in NRP1 knockdown mutants. Affinity purification combined with mass spectrometry analysis indicated that Nrp1 is co-purified with core histones, heat shock proteins, histone chaperones, and DNA damage repair proteins. The physical direct interaction of Nrp1 and Asf1 was also confirmed by pull-down analysis in vitro. The results show that histone chaperone Nrp1 is involved in micronuclear mitosis and macronuclear amitosis in the vegetative growth stage and maintains gametic nuclei formation during the sexual developmental stage. Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila.
Topics: Cell Nucleus Division; Chromatin; Chromosomes; Histone Chaperones; Tetrahymena thermophila
PubMed: 34301312
DOI: 10.1186/s13072-021-00409-4 -
G3 (Bethesda, Md.) Oct 2019, like other ciliates, has separate germline and somatic nuclei. The diploid germline genome in the micronucleus is composed of long conventional chromosomes. The...
, like other ciliates, has separate germline and somatic nuclei. The diploid germline genome in the micronucleus is composed of long conventional chromosomes. The macronucleus contains a somatic genome which is naturally fragmented into thousands of kilobase-sized chromosomes. Here, we develop a method to stably incorporate artificial chromosomes into the macronucleus. We report two cases of successful transformation and demonstrate the use of somatic transformation to investigate gene regulation and gene function in We show that the transformed artificial chromosomes are maintained through multiple asexual divisions. Furthermore, they support the transcriptional regulation of the native chromosome from which they were derived and are translated to produce functional proteins. To test if transformed chromosomes are amenable to practical applications, we generated a tagged version of a representative gene (AL1) and used it to co-precipitate associated proteins. This revealed an association with nucleic acid binding proteins, specifically RNA-binding proteins, and RNA immunoprecipitation of AL1 revealed its association with multiple RNAs. The use of artificial chromosomes in enables an array of genetic and molecular biological assays, as well as new avenues of inquiry into the epigenetic programming of macronuclear development and genome rearrangement.
Topics: Chromosomes, Artificial; Fluorescent Antibody Technique; Gene Expression Regulation; Gene Rearrangement; Genes, Protozoan; Genome, Protozoan; Oxytricha; Protozoan Proteins
PubMed: 31506318
DOI: 10.1534/g3.119.400298 -
The Journal of Eukaryotic Microbiology Jan 2018Proteins that possess a chromo domain are well-known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a...
Proteins that possess a chromo domain are well-known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy-terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi-directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates.
Topics: Amino Acid Sequence; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Heterochromatin; Multigene Family; Protein Domains; Protozoan Proteins; Sequence Alignment; Tetrahymena thermophila
PubMed: 28692189
DOI: 10.1111/jeu.12443 -
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 -
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 -
Frontiers in Microbiology 2021A new species of , isolated from the deep-sea Pacific Ocean (>3,000-m depth) in the vicinity of the Mariana Trench, is described based on morphological and molecular...
A new species of , isolated from the deep-sea Pacific Ocean (>3,000-m depth) in the vicinity of the Mariana Trench, is described based on morphological and molecular data. The systematic status of the ciliate genus has long been highly ambiguous, and species have been assigned to an independent class until recently. In the present study, we described sp. n. as a small (25-32 × 14-17 μm ) drop-shaped ciliate, with longitudinal furrows along the ciliary rows on the right side, six adoral membranelles, eight somatic kineties, and one macronucleus comprising 7-11 nuclear globules. Phylogenetic analyses inferred from small subunit rRNA gene sequences revealed that seven species in the phylogenetic tree formed a fully supported clade representing an independent class. sp. n. was established to be most closely related to , with a sequence similarity of 96.64%, and was found to be able to survive at both atmospheric pressure and hydrostatic pressure of 320 bar, thereby indicating effective barotolerance.
PubMed: 34745044
DOI: 10.3389/fmicb.2021.743920 -
European Journal of Protistology Feb 2019The morphology and infraciliature of two haptorian ciliates, Phialina clampi nov. spec. and P. caudata (Kahl, 1933) nov. comb. (original combination: Lacrymaria...
The morphology and infraciliature of two haptorian ciliates, Phialina clampi nov. spec. and P. caudata (Kahl, 1933) nov. comb. (original combination: Lacrymaria caudataKahl, 1933), isolated from sandy sediments of an estuary in Yantai, northern China, were investigated using live observations and protargol preparations. Phialina clampi nov. spec. is distinguished from its congeners by the following traits: extended cells about 80-300 × 18-50 μm in vivo; a single oval-shaped macronucleus; caudally located contractile vacuole; two types of extrusomes: type I about 20-35 × 1-4 μm in size, type II 1.5-3.0 × 0.7-1.0 μm in size and attached to somatic cortex; 27-31 somatic kineties. Phialina caudata is characterized as follows: extended cells about 80-170 × 20-45 μm in vivo; body radish-shaped, with a sharp posterior end; a single globular to oval macronucleus and micronucleus; subterminal contractile vacuole; 20-24 somatic kineties. Phylogenetic analyses based on SSU rRNA gene sequences indicate that P. caudata clusters with an unidentified Phialina before grouping with Phialina clampi, which form a basal clade of the family Lacrymariidae.
Topics: China; Ciliophora; Geologic Sediments; Phylogeny; RNA, Ribosomal; Species Specificity
PubMed: 30453232
DOI: 10.1016/j.ejop.2018.10.002 -
Parasites & Vectors Mar 2023Chilodonella uncinata is an aerobic ciliate capable of switching between being free-living and parasitic on fish fins and gills, causing tissue damage and host...
BACKGROUND
Chilodonella uncinata is an aerobic ciliate capable of switching between being free-living and parasitic on fish fins and gills, causing tissue damage and host mortality. It is widely used as a model organism for genetic studies, but its mitochondrial metabolism has never been studied. Therefore, we aimed to describe the morphological features and metabolic characteristics of its mitochondria.
METHODS
Fluorescence staining and transmission electron microscopy (TEM) were used to observe the morphology of mitochondria. Single-cell transcriptome data of C. uncinata were annotated by the Clusters of Orthologous Genes (COG) database. Meanwhile, the metabolic pathways were constructed based on the transcriptomes. The phylogenetic analysis was also made based on the sequenced cytochrome c oxidase subunit 1 (COX1) gene.
RESULTS
Mitochondria were stained red using Mito-tracker Red staining and were stained slightly blue by DAPI dye. The cristae and double membrane structures of the mitochondria were observed by TEM. Besides, many lipid droplets were evenly distributed around the macronucleus. A total of 2594 unigenes were assigned to 23 functional classifications of COG. Mitochondrial metabolic pathways were depicted. The mitochondria contained enzymes for the complete tricarboxylic acid (TCA) cycle, fatty acid metabolism, amino acid metabolism, and cytochrome-based electron transport chain (ETC), but only partial enzymes involved in the iron-sulfur clusters (ISCs).
CONCLUSIONS
Our results showed that C. uncinata possess typical mitochondria. Stored lipid droplets inside mitochondria may be the energy storage of C. uncinata that helps its transmission from a free-living to a parasitic lifestyle. These findings also have improved our knowledge of the mitochondrial metabolism of C. uncinata and increased the volume of molecular data for future studies of this facultative parasite.
Topics: Animals; Alveolata; Parasites; Phylogeny; Ciliophora; Mitochondria
PubMed: 36882771
DOI: 10.1186/s13071-023-05695-3 -
European Journal of Protistology Feb 2018We described the infraciliature of Opisthotrichum janus, Epidinium ecaudatum, and Ophryoscolex purkynjei, belonging to the family Ophryoscolecidae, from pyridinated...
We described the infraciliature of Opisthotrichum janus, Epidinium ecaudatum, and Ophryoscolex purkynjei, belonging to the family Ophryoscolecidae, from pyridinated silver carbonate impregnated specimens. The adoral polybrachykinety is C-shaped in Opi. janus, whereas it is spiral-shaped in Epi. ecaudatum and Oph. purkynjei. The vestibular polybrachykinety is slender and short in Opi. janus, whereas slender and long in Epi. ecaudatum and Oph. purkynjei. The paralabial kineties are composed of several short kineties along the adoral polybrachykinety. The dorsal polybrachykinety is displaced posteriorly. This study provides information on the diagnostic features of the subfamily Ophryoscolecinae to which Opi. janus, Epi. ecaudatum, and Oph. purkynjei belong. A C-shaped or spiral-shaped adoral polybrachykinety and slender vestibular polybrachykinety are the diagnostic features of the subfamily Ophryoscolecinae in addition to their general features of a cylindrical body, tubular vestibulum, dorsal ciliary zone considerably displaced posteriorly, well developed skeletal plates, and macronucleus beneath the right body surface. Opi. janus, Epi. ecaudatum, and Oph. purkynjei show the basic form of morphogenesis of the ciliature, similar to that in the subfamily Diplodiniinae, with four primordia developing into the oral and dorsal ciliature.
Topics: Cilia; Ciliophora; Species Specificity
PubMed: 29128561
DOI: 10.1016/j.ejop.2017.10.001