Aditi Singh

Max Planck Institute for Developmental Biology

One of the most extensive forms of natural genome editing occurs in ciliates, a group of microbial eukaryotes. Ciliate germline and somatic genomes are contained in distinct nuclei within the same cell. During the massive reorganization process of somatic genome development, ciliates eliminate tens of thousands of DNA sequences from a germline geno...

Most eukaryotes have one nucleus and nuclear genome per cell. Ciliates have instead evolved distinct nuclei that coexist in each cell: a silent germline vs. transcriptionally active somatic nuclei. In the best-studied model species, both nuclei can divide asexually, but only germline nuclei undergo meiosis and karyogamy during sex. Thereafter, thou...

The unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sex, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique internal eliminated sequences (IESs) distributed throughout the genome require precise exc...

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition 1 . All non-standard ciliate genetic codes involve stop codon reassignments 1,2,3 . Two of these codes are ambiguous 1–3 , with “stop” codons either translated or terminating translation depending...

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition 1 . All non-standard ciliate genetic codes involve stop codon reassignments 1,2,3 . Two of these codes are ambiguous 1–3 , with “stop” codons either translated or terminating translation depending...

The unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sexual reproduction, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique Internal Eliminated Sequences (IESs) distributed throughout the genome req...

Ciliates are unicellular eukaryotes with two distinct kinds of nuclei in each cell: transcriptionally active somatic macronuclei (MAC) and silent germline micronuclei (MIC). In the best-studied model species, both nuclei can divide asexually, but only germline MICs participate in meiosis, karyogamy, and development into new MACs. During MIC-to-MAC...

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition ¹ . All non-standard ciliate genetic codes involve stop codon reassignments 1,2,3 . Two of these codes are ambiguous ¹⁻³ , with "stop" codons either translated or terminating translation depending...

Chromatin remodeling is required for essential cellular processes, including DNA replication, DNA repair, and transcription regulation. The ciliate germline and soma are partitioned into two distinct nuclei within the same cell. During a massive editing process that forms a somatic genome, ciliates eliminate thousands of DNA sequences from a germli...

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout the ciliate germline genome are deleted during the development of the streamlined somatic genome. The genus Blepharisma represents one...

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 pers...

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 macronu...

Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we...

In ambiguous stop/sense genetic codes, the stop codon(s) not only terminate translation but can also encode amino acids. Such codes have evolved at least four times in eukaryotes, twice among ciliates (Condylostoma magnum and Parduczia sp.). These have appeared to be isolated cases whose next closest relatives use conventional stop codons. However,...

Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we...

During a sophisticated developmental process, ciliates excise numerous internally eliminated sequences (IESs) from a germline genome copy, producing a functional somatic genome. Most IESs ultimately originate from transposons but homology is obscured by sequence decay. To obtain more representative perspectives on ciliate genome editing, we assembl...

A bstract In ambiguous stop/sense genetic codes, the stop codon(s) not only terminate translation but can also encode amino acids. Such codes have evolved at least four times in eukaryotes, twice among ciliates ( Condylostoma magnum and Parduczia sp.). These have appeared to be isolated cases whose next closest relatives use conventional stop codon...

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the ger...

The germ-soma distinction is a defining feature of multicellular eukaryotes. Analogous to this, ciliates, a ubiquitous microbial eukaryote lineage, have morphologically and functionally distinct nuclei, but within single cells: the germline micronucleus (MIC) and somatic macronucleus (MAC). The origins and mechanisms of the MIC to MAC transformatio...

The past two decades have seen extensive research done to pinpoint the role of microRNAs (miRNAs) that have led to discovering thousands of miRNAs in humans. It is not, therefore, surprising to see many of them implicated in a number of common as well as rare human diseases. In this review article, we summarize the progress in our understanding of...

[This corrects the article DOI: 10.1371/journal.pone.0206667.].

5-methylcytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have n...

Effect of inhibitor treatment on cell survival and IES retention. Immunofluorescence using; (a) only Alexa Fluor 488 secondary antibody on a population of Paramecium where majority of cells have fragmented parental macronucleus,(b, left panel) C.elegans embryo stained against 5- mCytosine as a negative control for immunofluorescence, (c, right pane...

Full length gels on IES retention PCRs corresponding to S2E Fig. (PDF)

Absence of evidence of C methylation in Paramecium mac genome. Left panel, percent calculated using C/(C + T) for each C in Paramecium genome. There was no bias in C conversion among the three different nucleotide contexts (right panel). (PDF)

Effect of inhibitor treatment on cell survival and IES retention. a) Immunocytochemistry with antibody against 5-methylcytosine after Azacytidine and Decitabine treatment for three consecutive days. Scale bar: 5μm. (b) & (d) Survival test on cells treated with Azacytidine/ Decitabine; sick; cells did not undergo normal vegetative division rate, dea...

List of primers. List of primers to check IES retention PCRs. (PDF)

Full length gels on IES retention PCRs corresponding to S2C Fig. (PDF)

Percentage of methylation calculated after mass spectrometry done on total genomic DNA samples from different Paramecium during autogamy when new macronuclei are observed in the cell. Drosophila, E. coli, Human MCF7 DNA and Human T47D DNA (provided by Storm Therapeutics Limited) were used as a positive control for the detection of methylated cytosi...

The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including...

Table of Pgm and PgmL proteins encoded by published Paramecium genomes and their ParameciumDB accession numbers

Sequences of the transposase core domains used for the alignement shown in Supplementary file 1.

DNA-seq datasets from ENA project PRJEB24171 (this study)

Analysis of IES excision reads in PGM and PGML knockdowns

MUSCLE alignment of the transposase core domains of ciliate domesticated PB transposases and other PB transposases

Sequences of the cysteine-rich domains used for the alignment shown in Figure 1—figure supplement 1

Sequence of the synthetic PGML genes used for protein production in insect cells

Analysis of post-autogamous progeny in small-scale PGML knockdowns

Analysis of post-autogamous progeny in middle- and large-scale PGML knockdowns

During development in the ciliate Paramecium, excess DNA interspersed throughout the germline genome is deleted to generate a new somatic genome. In this process, most of the intervening DNA is excised by a Piggybac-derived transposase, assisted by small RNAs (scnRNAs and iesRNAs) and chromatin remodelling. As the list of genes involved in DNA elim...

In vitro study was carried out in an important medicinal plant Tinospora cordifolia (Willd.) Miers belonging to the family Menispermaceae. Vegetative parts such as stem, leaf and nodal explants were excised from an elite in vivo grown mature plant and thereafter cultured on Murashige-Skoog (MS) medium supplemented with different hormonal concentrat...

In vitro study was carried out in an important medicinal plant Tinospora cordifolia (Willd.) Miers belonging to the family Menispermaceae. Vegetative parts such as stem, leaf and nodal explants were excised from an elite in vivo grown mature plant and thereafter cultured on Murashige-Skoog (MS) medium supplemented with different hormonal concentrat...

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a var...

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The...

In vitro study was carried out in an important medicinal plant Tinospora cordifolia (Willd.) Miers belonging to the family Menispermaceae.Vegetative parts such as stem, leaf and nodal explants were excised from an elite in vivo grown mature plant and thereaftercultured on Murashige-Skoog (MS) medium supplemented with different hormonal concentra...