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BackgroundDNA 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3713-7) contains supplementary material, which is available to authorized users.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ

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The influence of a 2-T static magnetic field on the cardiac rhythm was studied with 24-hour electrocardiographic monitoring in 12 healthy volunteers for 1 hour before exposure, 1 hour during exposure, and 22 hours after exposure. Four other subjects were exposed to 1 T, and nine control subjects were exposed to 0 T. In the 2-T group, the mean cardiac cycle length (CCL) was 912 msec +/- 83 before exposure. A significant 17% increase in CCL was observed after 10 minutes of exposure. No further significant variation was observed during exposure, and the CCL was back to preexposure values 10 minutes after exposure. No other arrhythmogenic effect was noted during the 24-hour monitoring. No statistically significant change was observed at either 0 or 1 T. The magnetically induced blood-flow potentials superimposed on the T wave were observed. The CCL increase during exposure could reflect a direct or indirect effect of magnetic fields on the sinus node, which is probably harmless in healthy subjects. However, its safety in dysrhythmic patients remains to be determined.

A hallmark of active centromeres is the presence of the histone H3 variant CenH3 in the centromeric chromatin, which ensures faithful genome distribution at each cell division. A functional centromere can be inactivated, but the molecular mechanisms underlying the process of centromere inactivation remain largely unknown. Here, we describe the loss of CenH3 protein as part of a developmental program leading to the formation of the somatic nucleus in the eukaryote Paramecium. We identify two proteins whose depletion prevents developmental loss of CenH3: the domesticated transposase Pgm involved in the formation of DNA double strand cleavages and the Polycomb-like lysine methyltransferase Ezl1 necessary for trimethylation of histone H3 on lysine 9 and lysine 27. Taken together, our data support a model in which developmentally programmed centromere loss is caused by the elimination of DNA sequences associated with CenH3.

Whole-Genome Duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages.The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods and the relative contributions of different selective pressures to their maintenance is still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the P. aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and one additional outgroup, allowing us to track post-WGD evolution in 13 species that share a common ancestral WGD. We found similar biases in gene retention compatible with dosage constraints playing a major role opposing post-WGD gene loss across all 13 species.Interestingly we found that post-WGD gene loss was slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. We also report a lack of recent segmental duplications in Paramecium, which we interpret as additional evidence for strong selective pressures against individual genes dosage changes. Finally, we hope that this exceptional dataset of 13 species sharing an ancestral WGD and two closely related outgroup species will be a useful resource for future studies and will help establish Paramecium as a major model organism in the study of post-WGD evolution.