scholarly journals Fifty generations of amitosis: tracing asymmetric allele segregation in polyploid cells with single-cell DNA sequencing

2021 ◽  
Author(s):  
Valerio Vitali ◽  
Rebecca Rothering ◽  
Francesco Catania
Keyword(s):  
Single Cell ◽  
Evolutionary Significance ◽  
Unicellular Eukaryote ◽  
Paramecium Tetraurelia ◽  
Somatic Variation ◽  
Somatic Nucleus ◽  
Phenotypic Assortment ◽  
Polyploid Cells ◽  
Random Segregation ◽  
Allele Segregation

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis are acquired by assessing the rate of phenotypic assortment. The phenotypic diversification of heterozygous clones during successive cell divisions reveals the random segregation of alleles to daughter nuclei. Though powerful, this experimental approach relies on the availability of phenotypic markers. Here, we present an approach that overcomes the requirement for phenotypic assortment. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dimorphism and a highly polyploid somatic nucleus, we use single-cell whole-genome sequencing to track the assortment of developmentally acquired somatic DNA variants. Accounting for genome representation biases, we measure the effect of amitosis on allele segregation across the first ~50 amitotic divisions post self-fertilization and compare our empirical findings with theoretical predictions estimated via mathematical modeling. In line with our simulations, we show that amitosis in P. tetraurelia produces measurable but modest levels of somatic assortment. In forgoing the requirement for phenotypic assortment and employing developmental, environmentally induced somatic variation as molecular markers, our work provides a new powerful approach to investigate the consequences of amitosis in polyploid cells.

scholarly journals Fifty Generations of Amitosis: Tracing Asymmetric Allele Segregation in Polyploid Cells with Single-Cell DNA Sequencing

2021 ◽  
Vol 9 (9) ◽  
pp. 1979
Author(s):  
Valerio Vitali ◽  
Rebecca Rothering ◽  
Francesco Catania
Keyword(s):  
Single Cell ◽  
Evolutionary Significance ◽  
Unicellular Eukaryote ◽  
Paramecium Tetraurelia ◽  
Somatic Nucleus ◽  
Factors Affecting ◽  
Phenotypic Assortment ◽  
Empirical Estimates ◽  
Self Fertilization ◽  
Allele Segregation

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis have been gleaned by assessing the rate of phenotypic assortment. Though powerful, this experimental approach relies on the availability of phenotypic markers. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dualism and a highly polyploid somatic nucleus, we probe the limits of single-cell whole-genome sequencing to study the consequences of amitosis. To this end, we first evaluate the suitability of single-cell sequencing to study the AT-rich genome of P. tetraurelia, focusing on common sources of genome representation bias. We then asked: can alternative rearrangements of a given locus eventually assort after a number of amitotic divisions? To address this question, we track somatic assortment of developmentally acquired Internal Eliminated Sequences (IESs) up to 50 amitotic divisions post self-fertilization. To further strengthen our observations, we contrast empirical estimates of IES retention levels with in silico predictions obtained through mathematical modeling. In agreement with theoretical expectations, our empirical findings are consistent with a mild increase in variation of IES retention levels across successive amitotic divisions of the macronucleus. The modest levels of somatic assortment in P. tetraurelia suggest that IESs retention levels are largely sculpted at the time of macronuclear development, and remain fairly stable during vegetative growth. In forgoing the requirement for phenotypic assortment, our approach can be applied to a wide variety of amitotic species and could facilitate the identification of environmental and genetic factors affecting amitosis.

scholarly journals Exploration of the Germline Genome of the Ciliate Chilodonella uncinata through Single-Cell Omics (Transcriptomics and Genomics)

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Xyrus X. Maurer-Alcalá ◽  
Rob Knight ◽  
Laura A. Katz
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Gc Content ◽  
Gene Families ◽  
Genome Rearrangements ◽  
Paramecium Tetraurelia ◽  
Protein Coding ◽  
Genome Data ◽  
Large Gene ◽  
Disproportionate Number

ABSTRACTSeparate germline and somatic genomes are found in numerous lineages across the eukaryotic tree of life, often separated into distinct tissues (e.g., in plants, animals, and fungi) or distinct nuclei sharing a common cytoplasm (e.g., in ciliates and some foraminifera). In ciliates, germline-limited (i.e., micronuclear-specific) DNA is eliminated during the development of a new somatic (i.e., macronuclear) genome in a process that is tightly linked to large-scale genome rearrangements, such as deletions and reordering of protein-coding sequences. Most studies of germline genome architecture in ciliates have focused on the model ciliatesOxytricha trifallax,Paramecium tetraurelia, andTetrahymena thermophila, for which the complete germline genome sequences are known. Outside of these model taxa, only a few dozen germline loci have been characterized from a limited number of cultivable species, which is likely due to difficulties in obtaining sufficient quantities of “purified” germline DNA in these taxa. Combining single-cell transcriptomics and genomics, we have overcome these limitations and provide the first insights into the structure of the germline genome of the ciliateChilodonella uncinata, a member of the understudied classPhyllopharyngea. Our analyses reveal the following: (i) large gene families contain a disproportionate number of genes from scrambled germline loci; (ii) germline-soma boundaries in the germline genome are demarcated by substantial shifts in GC content; (iii) single-cell omics techniques provide large-scale quality germline genome data with limited effort, at least for ciliates with extensively fragmented somatic genomes. Our approach provides an efficient means to understand better the evolution of genome rearrangements between germline and soma in ciliates.IMPORTANCEOur understanding of the distinctions between germline and somatic genomes in ciliates has largely relied on studies of a few model genera (e.g.,Oxytricha,Paramecium,Tetrahymena). We have used single-cell omics to explore germline-soma distinctions in the ciliateChilodonella uncinata, which likely diverged from the better-studied ciliates ~700 million years ago. The analyses presented here indicate that developmentally regulated genome rearrangements between germline and soma are demarcated by rapid transitions in local GC composition and lead to diversification of protein families. The approaches used here provide the basis for future work aimed at discerning the evolutionary impacts of germline-soma distinctions among diverse ciliates.

scholarly journals Hyperosmotic Stress Response Memory is Modulated by Gene Positioning in Yeast

Cells ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 582 ◽  
Author(s):  
Zacchari Ben Meriem ◽  
Yasmine Khalil ◽  
Pascal Hersen ◽  
Emmanuelle Fabre
Keyword(s):  
Single Cell ◽  
Quantitative Description ◽  
Time Lapse ◽  
Chromatin Domain ◽  
Unicellular Eukaryote ◽  
Cellular Memory ◽  
Fluorescent Reporter ◽  
Activation Patterns ◽  
Stress Memory ◽  
Gene Positioning

Cellular memory is a critical ability that allows microorganisms to adapt to potentially detrimental environmental fluctuations. In the unicellular eukaryote Saccharomyces cerevisiae, cellular memory can take the form of faster or slower responses within the cell population to repeated stresses. Using microfluidics and fluorescence time-lapse microscopy, we studied how yeast responds to short, pulsed hyperosmotic stresses at the single-cell level by analyzing the dynamic behavior of the stress-responsive STL1 promoter (pSTL1) fused to a fluorescent reporter. We established that pSTL1 exhibits variable successive activation patterns following two repeated short stresses. Despite this variability, most cells exhibited a memory of the first stress as decreased pSTL1 activity in response to the second stress. Notably, we showed that genomic location is important for the memory effect, since displacement of the promoter to a pericentromeric chromatin domain decreased the transcriptional strength of pSTL1 and led to a loss of memory. This study provides a quantitative description of a cellular memory that includes single-cell variability and highlights the contribution of chromatin structure to stress memory.

5-Azacytidine affects the programming of expression of the somatic nucleus of Paramecium

Development ◽  
1989 ◽  
Vol 105 (3) ◽  
pp. 559-568 ◽  
Author(s):  
F.W. Kwok ◽  
S.F. Ng
Keyword(s):  
Sexual Reproduction ◽  
Cell Lines ◽  
Compensatory Mechanism ◽  
Paramecium Tetraurelia ◽  
Growth Depression ◽  
Somatic Nucleus ◽  
Asexual Propagation ◽  
Oral Development ◽  
Asexual Cycle

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomatogenic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5′ position is discussed.

scholarly journals Construction of thousands of single cell genome sequencing libraries using combinatorial indexing

2016 ◽  
Author(s):  
Sarah A. Vitak ◽  
Kristof A. Torkenczy ◽  
Jimi L. Rosenkrantz ◽  
Andrew J. Fields ◽  
Lena Christiansen ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Sequencing ◽  
Copy Number ◽  
Low Cost ◽  
Single Cells ◽  
Copy Number Variant ◽  
Tumor Evolution ◽  
Somatic Variation ◽  
Cell Genome ◽  
Single Cell Genome

AbstractSingle cell genome sequencing has proven to be a valuable tool for the detection of somatic variation, particularly in the context of tumor evolution and neuronal heterogeneity. Current technologies suffer from high per-cell library construction costs which restrict the number of cells that can be assessed, thus imposing limitations on the ability to quantitatively measure genomic heterogeneity within a tissue. Here, we present Single cell Combinatorial Indexed Sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single cell libraries for the purpose of somatic copy number variant detection. In total, we constructed libraries for 16,698 single cells from a combination of cultured cell lines, frontal cortex tissue from Macaca mulatta, and two human adenocarcinomas. This novel technology provides the opportunity for low-cost, deep characterization of somatic copy number variation in single cells, providing a foundational knowledge across both healthy and diseased tissues.

scholarly journals The Conjugation-Specific Die5 Protein Is Required for Development of the Somatic Nucleus in both Paramecium and Tetrahymena

2010 ◽  
Vol 9 (7) ◽  
pp. 1087-1099 ◽  
Author(s):  
Atsushi Matsuda ◽  
Annie Wan-Yi Shieh ◽  
Douglas L. Chalker ◽  
James D. Forney
Keyword(s):  
Gene Disruption ◽  
Tetrahymena Thermophila ◽  
Paramecium Tetraurelia ◽  
Somatic Nucleus ◽  
Genome Amplification ◽  
Content Type ◽  
Chromosome Fragmentation ◽  
Genome Reorganization ◽  
Somatic Genome ◽  
Nuclear Differentiation

ABSTRACTDevelopment in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specificDefective inIESExcision 5 (DIE5) genes ofParamecium tetraurelia(PtDIE5) andTetrahymena thermophila(TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei.TetrahymenaΔDIE5cells arrested late in development and failed to complete genome amplification, whereas RNAi-treatedParameciumcells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. InTetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p inParameciumstill allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis ofDIE5establishes the action of a conserved mechanism within the genome reorganization pathway.

Limits of imagination: the 150th Anniversary of Mendel’s Laws, and why Mendel failed to see the importance of his discovery for Darwin’s theory of evolution

Genome ◽  
2015 ◽  
Vol 58 (9) ◽  
pp. 415-421 ◽  
Author(s):  
Rama S. Singh
Keyword(s):  
Genetic Variation ◽  
Quantum Mechanics ◽  
Niels Bohr ◽  
One Dimension ◽  
Evolutionary Significance ◽  
Theory Of Evolution ◽  
Determining Factors ◽  
Random Segregation ◽  
Hybrid Development ◽  
Mendel’S Laws

Mendel is credited for discovering Laws of Heredity, but his work has come under criticism on three grounds: for possible falsification of data to fit his expectations, for getting undue credit for the laws of heredity without having ideas of segregation and independent assortment, and for being interested in the development of hybrids rather than in the laws of heredity. I present a brief review of these criticisms and conclude that Mendel deserved to be called the father of genetics even if he may not, and most likely did not, have clear ideas of segregation and particulate determiners as we know them now. I argue that neither Mendel understood the evolutionary significance of his findings for the problem of genetic variation, nor would Darwin have understood their significance had he read Mendel’s paper. I argue that the limits to imagination, in both cases, came from their mental framework being shaped by existing paradigms—blending inheritance in the case of Darwin, hybrid development in the case of Mendel. Like Einstein, Darwin’s natural selection was deterministic; like Niels Bohr, Mendel’s Laws were probabilistic—based on random segregation of trait-determining “factors”. Unlike Einstein who understood quantum mechanics, Darwin would have been at a loss with Mendel’s paper with no guide to turn to. Geniuses in their imaginations are like heat-seeking missiles locked-in with their targets of deep interests and they generally see things in one dimension only. Imagination has limits; unaided imagination is like a bird without wings — it goes nowhere.

scholarly journals Quantitative predictions of random segregation models of the ciliate macronucleus

1976 ◽  
Vol 27 (2) ◽  
pp. 227-238 ◽  
Author(s):  
John R. Preer
Keyword(s):  
Ploidy Level ◽  
Paramecium Tetraurelia ◽  
Regular Distribution ◽  
Random Segregation ◽  
Chromosome Fragments

SUMMARYModels of the macronucleus in Paramecium tetraurelia which assume known levels of ploidy and random segregation of subunits smaller than a haploid set at both fission and macronuclear regeneration (MR) are not consistent with the hypothesis that senescence is due to aneuploid imbalance. Either senescence has some other basis or there is a mechanism for regular distribution of subunits at MR. Random segregation models for fission and MR are consistent with most data on survival and heterozygote stability, but if the ploidy level is 860 they fail to account for the data of Nyberg (this volume). Since the ploidy level may be higher than 860, models of random segregation cannot be ruled out for Paramecium. Models of the macronucleus in hypotrichs which assume randomly segregating chromosome fragments are consistent with data on survival and on stability of heterozygous genotypes at fission.

scholarly journals Homoacetogenesis in Deep-SeaChloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in TerrestrialDehalococcoidetes

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Holly L. Sewell ◽  
Anne-Kristin Kaster ◽  
Alfred M. Spormann
Keyword(s):  
Single Cell ◽  
Deep Sea ◽  
Formate Dehydrogenase ◽  
Single Cells ◽  
Reductive Dehalogenation ◽  
Evolutionary Significance ◽  
Rrna Gene ◽  
Deep Sea Sediment ◽  
Deep Sea Sediments ◽  
Anaerobic Benzoate Oxidation

ABSTRACTThe deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylumChloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in suchChloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum IChloroflexiwhich were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, aRhodobacternitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marineChloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylumChloroflexi(DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrialDehalococcoides/Dehalogenimonasspp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity inDehalococcoides/Dehalogenimonasspp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrialDehalococcoides/Dehalogenimonasspp. and of reductive dehalogenation, as well as the biology of abundant deep-seaChloroflexi.IMPORTANCEThe deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylumChloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marineChloroflexi. Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenatingDehalococcoides.

scholarly journals The memory of hyperosmotic stress response in yeast is modulated by gene-positioning

2019 ◽  
Author(s):  
Zacchari Ben Meriem ◽  
Yasmine Khalil ◽  
Pascal Hersen ◽  
Emmanuelle Fabre
Keyword(s):  
Single Cell ◽  
Dynamical Behavior ◽  
Quantitative Description ◽  
Time Lapse ◽  
Chromatin Domain ◽  
Unicellular Eukaryote ◽  
Cellular Memory ◽  
Fluorescent Reporter ◽  
Stress Memory ◽  
Gene Positioning

AbstractCellular memory is a critical ability displayed by microorganisms in order to adapt to potentially detrimental environmental fluctuations. In the unicellular eukaryoteS. cerevisiaecellular memory can take the form of a faster or a decreased response following repeated stresses in cell population. Using microfluidics and fluorescence time-lapse microscopy, we studied how yeasts respond to short-pulsed hyperosmotic stresses at the single-cell level by analyzing the dynamical behavior of the stress responsive STL1 promoter fused to a fluorescent reporter. We established that pSTL1 shows variability in its successive activations following two repeated short stresses. Despite this variability, most cells displayed a memory of past stresses through a decreased activity of pSTL1 upon repeated stress. Notably, we showed that genomic location is important for the memory effect since promoter displacement to a pericentromeric chromatin domain leads to a decreased transcriptional strength of pSTL1 and to the loss of memory. This study provides a quantitative description of a cellular memory that includes single-cell variability and points towards the contribution of the chromatin structure in stress memory.

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