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 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 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.

scholarly journals Single-cell multiomics reveals increased plasticity, resistant populations and stem-cell-like blasts in KMT2A-rearranged leukemia

Blood ◽  
2021 ◽  
Author(s):  
Changya Chen ◽  
Wenbao Yu ◽  
Fatemeh Alikarami ◽  
Qi Qiu ◽  
Chia-hui Chen ◽  
...  
Keyword(s):  
Single Cell ◽  
Young Patients ◽  
Cytotoxic Lymphocytes ◽  
Extrinsic Factors ◽  
Hematopoietic Stem ◽  
Younger Patients ◽  
Factors Affecting ◽  
Immune Mediated ◽  
Younger Age ◽  
Increased Risk

KMT2A-rearranged (KMT2A-r) infant ALL is a devastating malignancy with a dismal outcome, and younger age at diagnosis is associated with increased risk of relapse. To discover age-specific differences and critical drivers that mediate poor outcome in KMT2A-r ALL, we subjected KMT2A-r leukemias and normal hematopoietic cells from patients of different ages to single cell multi-omics analyses. We uncovered the following critical new insights: leukemia cells from patients younger than 6 months have significantly increased lineage plasticity. Steroid response pathways are downregulated in the most immature blasts from younger patients. We identify a hematopoietic stem and progenitor-like (HSPC-like) population in the blood of younger patients that contains leukemic blasts and form an immunosuppressive signaling circuit with cytotoxic lymphocytes. These observations offer a compelling explanation for the ability of leukemias in young patients to evade chemotherapy and immune mediated control. Our analysis also revealed pre-existing lymphomyeloid primed progenitors and myeloid blasts at initial diagnosis of B-ALL. Tracking of leukemic clones in two patients whose leukemia underwent a lineage switch documented the evolution of such clones into frank AML. These findings provide critical insights into KMT2A-r ALL and have clinical implications for molecularly targeted and immunotherapy approaches. Beyond infant ALL, our study demonstrates the power of single cell multi-omics to detect tumor intrinsic and extrinsic factors affecting rare but critical subpopulations within a malignant population that ultimately determines patient outcome.

scholarly journals Comprehensive benchmarking of computational deconvolution of transcriptomics data

2020 ◽  
Author(s):  
Francisco Avila Cobos ◽  
José Alquicira-Hernandez ◽  
Joseph Powell ◽  
Pieter Mestdagh ◽  
Katleen De Preter
Keyword(s):  
Single Cell ◽  
Cell Types ◽  
Cell Type ◽  
Factors Affecting ◽  
Marker Selection ◽  
Cell Type Composition ◽  
Type Composition ◽  
Comparable Performance ◽  
Transcriptomics Data ◽  
Combined Impact

AbstractMany computational methods to infer cell type proportions from bulk transcriptomics data have been developed. Attempts comparing these methods revealed that the choice of reference marker signatures is far more important than the method itself. However, a thorough evaluation of the combined impact of data transformation, pre-processing, marker selection, cell type composition and choice of methodology on the results is still lacking.Using different single-cell RNA-sequencing (scRNA-seq) datasets, we generated hundreds of pseudo-bulk mixtures to evaluate the combined impact of these factors on the deconvolution results. Along with methods to perform deconvolution of bulk RNA-seq data we also included five methods specifically designed to infer the cell type composition of bulk data using scRNA-seq data as reference.Both bulk and single-cell deconvolution methods perform best when applied to data in linear scale and the choice of normalization can have a dramatic impact on the performance of some, but not all methods. Overall, single-cell methods have comparable performance to the best performing bulk methods and bulk methods based on semi-supervised approaches showed higher error and lower correlation values between the computed and the expected proportions. Moreover, failure to include cell types in the reference that are present in a mixture always led to substantially worse results, regardless of any of the previous choices. Taken together, we provide a thorough evaluation of the combined impact of the different factors affecting the computational deconvolution task across different datasets and propose general guidelines to maximize its performance.

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 AGE-CORRELATED CHANGES IN EXPRESSION OF MICRONUCLEAR DAMAGE AND REPAIR IN PARAMECIUM TETRAURELIA

Genetics ◽  
1977 ◽  
Vol 87 (2) ◽  
pp. 259-274
Author(s):  
Steven R Rodermel ◽  
Joan Smith-Sonneborn
Keyword(s):  
Life Span ◽  
High Mortality ◽  
Ultraviolet Irradiation ◽  
Paramecium Tetraurelia ◽  
Dark Repair ◽  
Abrupt Increase ◽  
Self Fertilization ◽  
Cross Fertilization

ABSTRACT In Paramecium, age is defined as the number of mitotic divisions which have elapsed since the previous cross-fertilization (conjugation) or self-fertilization (autogamy). As the mitotic interval between fertilizations increases, the percentage of nonviable progeny clones increases. In the current study, resolution of conflicting previous reports on the pattern of increase of death and reduced viability in progeny from aging parent cells is found. Some exautogamous clones exhibit a high mortality at young clonal ages, others show no mortality throughout their life span, but most (73%) show an abrupt increase in the percent death and reduced viability in progeny from cells 50-80 fissions old. Ultraviolet-irradiation-induced micronuclear mutations, repairable by photoreactivation, increased with increased clonal age when monitored by percent death and reduced viability of exautogamous progeny of irradiated cells. Loss of dark repair is considered a contributor to the increased expression of micronuclear mutations with increased clonal age.

scholarly journals Machine Learning-Assisted Identification of Factors Contributing to the Technical Variability Between Bulk and Single-Cell RNA-Seq Experiments

2022 ◽  
Author(s):  
Sofya Lipnitskaya ◽  
Yang Shen ◽  
Stefan Legewie ◽  
Holger Klein ◽  
Kolja Becker
Keyword(s):  
Gene Expression ◽  
Machine Learning ◽  
Single Cell ◽  
Rna Sequencing ◽  
Quantitative Difference ◽  
Rna Seq ◽  
Sequencing Data ◽  
Factors Affecting ◽  
Expression Variability ◽  
Technical Variability

Abstract Background: Recent studies in the area of transcriptomics performed on single-cell and population levels reveal noticeable variability in gene expression measurements provided by different RNA sequencing technologies. Due to increased noise and complexity of single-cell RNA-Seq (scRNA-Seq) data over the bulk experiment, there is a substantial number of variably-expressed genes and so-called dropouts, challenging the subsequent computational analysis and potentially leading to false positive discoveries. In order to investigate factors affecting technical variability between RNA sequencing experiments of different technologies, we performed a systematic assessment of single-cell and bulk RNA-Seq data, which have undergone the same pre-processing and sample preparation procedures. Results: Our analysis indicates that variability between gene expression measurements as well as dropout events are not exclusively caused by biological variability, low expression levels, or random variation. Furthermore, we propose FAVSeq, a machine learning-assisted pipeline for detection of factors contributing to gene expression variability in matched RNA-Seq data provided by two technologies. Based on the analysis of the matched bulk and single-cell dataset, we found the 3'-UTR and transcript lengths as the most relevant effectors of the observed variation between RNA-Seq experiments, while the same factors together with cellular compartments were shown to be associated with dropouts. Conclusions: Here, we investigated the sources of variation in RNA-Seq profiles of matched single-cell and bulk experiments. In addition, we proposed the FAVSeq pipeline for analyzing multimodal RNA sequencing data, which allowed to identify factors affecting quantitative difference in gene expression measurements as well as the presence of dropouts. Hereby, the derived knowledge can be employed further in order to improve the interpretation of RNA-Seq data and identify genes that can be affected by assay-based deviations. Source code is available under the MIT license at https://github.com/slipnitskaya/FAVSeq.

scholarly journals Sex enhances survival in Paramecium

2019 ◽  
Author(s):  
Amarinder Singh Thind ◽  
Valerio Vitali ◽  
Mario R. Guarracino ◽  
Francesco Catania
Keyword(s):  
Genetic Variation ◽  
Stress Response ◽  
Sexual Reproduction ◽  
Stress Resistance ◽  
Evolutionary Biology ◽  
Molecular Mechanisms ◽  
Adaptive Significance ◽  
Paramecium Tetraurelia ◽  
Self Fertilization

AbstractThe pervasiveness of sex despite its well-known costs is a long-standing puzzle in evolutionary biology. Current explanations for the success of sex in nature largely rely on the adaptive significance of the new or rare genotypes that sex may generate. Less explored is the possibility that sex-underlying molecular mechanisms can enhance fitness and convey benefits to the individuals that bear the immediate costs of sex. Here we show that self-fertilization can increase stress resistance in the ciliate Paramecium tetraurelia. This advantage is independent of new genetic variation, coupled with a reduced nutritional input, and offers fresh insights into the mechanistic origin of sex. In addition to providing evidence that the molecular underpinnings of sexual reproduction and the stress response are linked in P. tetraurelia, these findings supply an explanation for the persistence of self-fertilization in this ciliate.

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