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Frontiers in Cell and Developmental... 2019In the ciliate , a 631-amino acid -MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key...
In the ciliate , a 631-amino acid -MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key component of an autocrine, cell-growth promoting self-signaling mechanism. While its 283-amino acid N-terminal domain includes all the structural specificities of the mitogen-activated protein kinases required for a catalytic function, the 348-amino acid C-terminal domain is structurally unique with undetermined functions. By expressing the two -MAPK1 domains tagged with the green fluorescent protein in mammalian fibroblasts, the yeast and the ciliate , evidence was obtained that the C-terminal domain contains all the sequence information responsible for the -MAPK1 subcellular localization. However, in fibroblasts and this information determined a nucleolar localization of the GFP-tagged C-terminal domain, and a ciliary localization in . In the light of these findings, the -MAPK1 localization in was re-examined via immunoreactions and shown to be ciliary besides that nuclear, as is the case for the mammalian intestinal cell kinase with which the -MAPK1 N-terminal domain shares a strong sequence identity and a catalytic function.
PubMed: 31681773
DOI: 10.3389/fcell.2019.00244 -
Cell Cycle (Georgetown, Tex.) Feb 2019Ciliates represent a morphologically and genetically distinct group of single-celled eukaryotes that segregate germline and somatic functions into two types of nuclei...
Ciliates represent a morphologically and genetically distinct group of single-celled eukaryotes that segregate germline and somatic functions into two types of nuclei and exhibit complex cytogenetic events during the sexual process of conjugation, which is under the control of the so-called "mating type systems". Studying conjugation in ciliates may provide insight into our understanding of the origins and evolution of sex and fertilization. In the present work, we studied in detail the sexual process of conjugation using the model species Euplotes vannus, and compared these nuclear events with those occurring in other ciliates. Our results indicate that in E. vannus: 1) conjugation requires about 75 hours to complete: the longest step is the development of the new macronucleus (ca. 64h), followed by the nuclear division of meiosis I (5h); the mitotic divisions usually take only 2h; 2) there are three prezygotic divisions (mitosis and meiosis I and II), and two of the eight resulting nuclei become pronuclei; 3) after the exchange and fusion of the pronuclei, two postzygotic divisions occur; two of the four products differentiate into the new micronucleus and macronucleus, respectively, and the parental macronucleus degenerates completely; 4) comparison of the nuclear events during conjugation in different ciliates reveals that there are generally three prezygotic divisions while the number of postzygotic divisions is highly variable. These results can serve as reference to investigate the mating type system operating in this species and to analyze genes involved in the different steps of the sexual process.
Topics: Cell Nucleus; Ciliophora; Conjugation, Genetic; Euplotes; Kinetics; Macronucleus; Meiosis; Mitosis
PubMed: 30563432
DOI: 10.1080/15384101.2018.1558871 -
The Journal of Eukaryotic Microbiology 2015Ciliate nuclear architecture, in particular the sequestration of a transcriptionally silent germline genome, allows for the accumulation of mutations that are "hidden"...
Ciliate nuclear architecture, in particular the sequestration of a transcriptionally silent germline genome, allows for the accumulation of mutations that are "hidden" from selection during many rounds of asexual reproduction. After sexual conjugation, these mutations are expressed, potentially resulting in highly variable phenotypes. Morphological traits are widely used in ciliate taxonomy, however, the extent to which the values of these traits are robust to change in the face of mutation remains largely unknown. In this study, we examine the effects of mutations accumulated in the germline genome to test the mutational robustness of four traits commonly used in ciliate morphological taxonomy (number of somatic kineties, number of postoral kineties, macronuclear size, and cell size). We found that the number of postoral kineties is robust to mutation, confirming that it should be preferentially used in taxonomy. By contrast, we found that, as in other unicellular and multicellular species, cell and macronucleus sizes change in response to mutation. Thus, we argue that cell and macronucleus sizes, which are widely used in taxonomy, should be treated cautiously for species identification. Finally, we found evidence of correlations between cell and macronucleus sizes and fitness, suggesting possible mutational pleiotropy. This study demonstrates the importance of, and methods for, determining mutational robustness to guide morphological taxonomy in ciliates.
Topics: Genetic Pleiotropy; Genome, Protozoan; Mutation; Tetrahymena thermophila
PubMed: 25227613
DOI: 10.1111/jeu.12174 -
Microorganisms Feb 2023Locus-specific gene amplification and genome-wide endoreplication generate the elevated copy number of ribosomal DNA (rDNA, 9000 C) and non-rDNA (90 C) chromosomes in...
Locus-specific gene amplification and genome-wide endoreplication generate the elevated copy number of ribosomal DNA (rDNA, 9000 C) and non-rDNA (90 C) chromosomes in the developing macronucleus of . Subsequently, all macronuclear chromosomes replicate once per cell cycle during vegetative growth. Here, we describe an unanticipated, programmed switch in the regulation of replication initiation in the rDNA minichromosome. Early in development, the 21 kb rDNA minichromosome is preferentially amplified from 2 C to ~800 C from well-defined origins, concurrent with genome-wide endoreplication (2 C to 8-16 C) in starved mating (endoreplication (ER) Phase 1). Upon refeeding, rDNA and non-rDNA chromosomes achieve their final copy number through resumption of just the endoreplication program (ER Phase 2). Unconventional rDNA replication intermediates are generated primarily during ER phase 2, consistent with delocalized replication initiation and possible formation of persistent RNA-DNA hybrids. Origin usage and replication fork elongation are affected in non-rDNA chromosomes as well. Despite the developmentally programmed 10-fold reduction in the ubiquitous eukaryotic initiator, the Origin Recognition Complex (ORC), active initiation sites are more closely spaced in ER phases 1 and 2 compared to vegetative growing cells. We propose that initiation site selection is relaxed in endoreplicating macronuclear chromosomes and may be less dependent on ORC.
PubMed: 36838456
DOI: 10.3390/microorganisms11020491 -
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 -
BMC Genomics Apr 2017DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and...
BACKGROUND
DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences.
RESULTS
We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome.
CONCLUSIONS
We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
Topics: DNA Transposable Elements; DNA, Protozoan; Flow Cytometry; Genomics; Paramecium
PubMed: 28446146
DOI: 10.1186/s12864-017-3713-7 -
RNA (New York, N.Y.) Apr 2017Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of...
Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as have a phenotypically silent germline micronucleus and an expressed somatic macronucleus, which is differentiated anew from a germline genome copy in sexual reproduction. We show that the nuclear-localized Piwi protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.
Topics: Argonaute Proteins; Chromosomes; DNA, Protozoan; Gene Rearrangement; Genome, Protozoan; Macronucleus; Micronucleus, Germline; Protozoan Proteins; RNA, Protozoan; RNA, Small Interfering; Reproduction, Asexual; Tetrahymena
PubMed: 28053272
DOI: 10.1261/rna.060012.116 -
Eukaryotic Cell Feb 2015The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and...
The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle.
Topics: Gene Expression Regulation, Developmental; Life Cycle Stages; Protozoan Proteins; SUMO-1 Protein; Sumoylation; Tetrahymena thermophila; Ubiquitin-Activating Enzymes
PubMed: 25527524
DOI: 10.1128/EC.00252-14 -
BMC Microbiology Dec 2020The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated...
Three redescriptions in Tintinnopsis (Protista: Ciliophora: Tintinnina) from coastal waters of China, with cytology and phylogenetic analyses based on ribosomal RNA genes.
BACKGROUND
The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated cytological and molecular information. Tintinnopsis, the most speciose tintinnine genus, is also the most problematic: it is known to be non-monophyletic, but it cannot be revised until more of its species are studied with modern methods.
RESULTS
Here, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, from coastal waters of China, were studied. Lorica and cell features were morphometrically investigated in living and protargol-stained specimens, and sequences of three ribosomal RNA (rRNA) loci were phylogenetically analyzed. The three species show a complex ciliary pattern (with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields), but differ in lorica morphology, details of the somatic ciliature and rRNA gene sequences. Tintinnopsis hemispiralis is further distinguished by a ciliary tuft (a ribbon of very long cilia originated from the middle portion of the ventral kinety and extending out of the lorica) and multiple macronuclear nodules. Both T. kiaochowensis and T. uruguayensis have two macronuclear nodules, but differ in the number of somatic kineties and the position of the posterior kinety. Two neotypes are fixed for T. hemispiralis and T. kiaochowensis to stabilize the species names objectively, mainly because of the previous unavailability of type materials. By phylogenetic analysis and comparison with closely-related species, we infer that the ciliary tuft and details such as the commencement of the rightmost kinety in the lateral ciliary field are synapomorphies that may help clarify the systematics of Tintinnopsis-like taxa.
CONCLUSION
The redescriptions of three poorly known Tintinnopsis species, namely T. hemispiralis, T. kiaochowensis, and T. uruguayensis firstly revealed their ciliary patterns and rRNA sequences. This study expands knowledge and database of tintinnines and helps in identifying potential synapomorphies for future taxonomic rearrangements.
Topics: China; Cilia; Ciliophora; DNA, Protozoan; Genes, rRNA; Macronucleus; Phylogeny; RNA, Ribosomal; Seawater; Species Specificity
PubMed: 33317450
DOI: 10.1186/s12866-020-02057-2 -
Cell Reports Mar 2017RNAi-mediated positive feedback loops are pivotal for the maintenance of heterochromatin, but how they are downregulated at heterochromatin-euchromatin borders is not...
RNAi-mediated positive feedback loops are pivotal for the maintenance of heterochromatin, but how they are downregulated at heterochromatin-euchromatin borders is not well understood. In the ciliated protozoan Tetrahymena, heterochromatin is formed exclusively on the sequences that are removed from the somatic genome by programmed DNA elimination, and an RNAi-mediated feedback loop is important for assembling heterochromatin on the eliminated sequences. In this study, we show that the heterochromatin protein 1 (HP1)-like protein Coi6p, its interaction partners Coi7p and Lia5p, and the histone demethylase Jmj1p are crucial for confining the production of small RNAs and the formation of heterochromatin to the eliminated sequences. The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. The results provide direct evidence for the existence of a dedicated mechanism that counteracts a positive feedback loop between RNAi and heterochromatin at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome.
Topics: Base Sequence; DNA, Protozoan; Feedback; Genome, Protozoan; Heterochromatin; Macronucleus; Protein Binding; Protozoan Proteins; RNA Interference; Tetrahymena
PubMed: 28273462
DOI: 10.1016/j.celrep.2017.02.024