RESUMO
Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs)1-4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. A quantitative analysis of hundreds of cell lines reveals that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship. Mathematical modelling suggests that nonlinearity might arise from transient enhancer-promoter interactions being translated into slower promoter bursting dynamics in individual cells, therefore uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism of how distal enhancers act from large genomic distances, and of how topologically associating domain boundaries block distal enhancers. Finally, we show that enhancer strength also determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation. Our measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.
Assuntos
Cromossomos , Elementos Facilitadores Genéticos , Animais , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Genômica , Mamíferos/genética , Regiões Promotoras Genéticas/genéticaRESUMO
The factors that sequester transcriptionally repressed heterochromatin at the nuclear periphery are currently unknown. In a genome-wide RNAi screen, we found that depletion of S-adenosylmethionine (SAM) synthetase reduces histone methylation globally and causes derepression and release of heterochromatin from the nuclear periphery in Caenorhabditis elegans embryos. Analysis of histone methyltransferases (HMTs) showed that elimination of two HMTs, MET-2 and SET-25, mimics the loss of SAM synthetase, abrogating the perinuclear attachment of heterochromatic transgenes and of native chromosomal arms rich in histone H3 lysine 9 methylation. The two HMTs target H3K9 in a consecutive fashion: MET-2, a SETDB1 homolog, mediates mono- and dimethylation, and SET-25, a previously uncharacterized HMT, deposits H3K9me3. SET-25 colocalizes with its own product in perinuclear foci, in a manner dependent on H3K9me3, but not on its catalytic domain. This colocalization suggests an autonomous, self-reinforcing mechanism for the establishment and propagation of repeat-rich heterochromatin.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Núcleo Celular/química , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/genética , Cromossomos/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Genoma Helmíntico , Histona-Lisina N-Metiltransferase/análise , Histona-Lisina N-Metiltransferase/genética , Laminas/metabolismo , Metionina Adenosiltransferase/metabolismo , Metilação , MutaçãoRESUMO
Bicaudal D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. Given that BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize these components to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles and the tandem spindles during female meiosis II. Furthermore, BicD is required to localize clathrin and the microtubule-stabilizing factors transforming acidic coiled-coil protein (D-TACC/Tacc) and Mini spindles (Msps) correctly to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, immediately after the establishment of the female pronucleus, D-TACC and Caenorhabditis elegans BicD, tacc and Chc are also needed for pronuclear fusion, suggesting that the underlying mechanism might be more widely used across species.
Assuntos
Fator D do Complemento , Proteínas de Drosophila , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Fator D do Complemento/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Meiose , Microtúbulos/metabolismo , Fuso Acromático/metabolismoRESUMO
SUMMARY: Recent studies suggest that the loop extrusion activity of Structural Maintenance of Chromosomes complexes is central to proper organization of genomes in vivo. Polymer physics-based modeling of chromosome structure has been instrumental to assess which structures such extrusion can create. Only few laboratories however have the technical and computational expertise to create in silico models combining dynamic features of chromatin and loop extruders. Here, we present 3DPolyS-LE, a self-contained, easy to use modeling and simulation framework allowing non-specialists to ask how specific properties of loop extruders and boundary elements impact on 3D chromosome structure. 3DPolyS-LE also provides algorithms to compare predictions with experimental Hi-C data. AVAILABILITY AND IMPLEMENTATION: Software available at https://gitlab.com/togop/3DPolyS-LE; implemented in Python and Fortran 2003 and supported on any Unix-based operating system (Linux and Mac OS). SUPPLEMENTARY INFORMATION: Supplementary information are available at Bioinformatics online.
Assuntos
Cromatina , Cromossomos , Simulação por Computador , Software , GenomaRESUMO
The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-wide gene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while in hermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domains in males, while the DCC prevents this in hermaphrodites.
Assuntos
Caenorhabditis elegans/genética , Mecanismo Genético de Compensação de Dose/genética , Cromossomo X/química , Cromossomo X/genética , Animais , Regulação da Expressão Gênica , Organismos Hermafroditas/genética , Masculino , Modelos GenéticosRESUMO
The existence of different domains within the nucleus has been clear from the time, in the late 1920s, that heterochromatin and euchromatin were discovered. The observation that heterochromatin is less transcribed than euchromatin suggested that microscopically identifiable structures might correspond to functionally different domains of the nucleus. Until 15 years ago, studies linking gene expression and subnuclear localization were limited to a few genes. As we discuss in this Review, new genome-wide techniques have now radically changed the way nuclear organization is analyzed. These have provided a much more detailed view of functional nuclear architecture, leading to the emergence of a number of new paradigms of chromatin folding and how this folding evolves during development.
Assuntos
Núcleo Celular/genética , Genes , Genoma , Animais , Linhagem da Célula/genética , Núcleo Celular/ultraestrutura , Heterocromatina/metabolismo , Humanos , MicroscopiaRESUMO
To understand whether the spatial organization of the genome reflects the cell's differentiated state, we examined whether genes assume specific subnuclear positions during Caenorhabditis elegans development. Monitoring the radial position of developmentally controlled promoters in embryos and larval tissues, we found that small integrated arrays bearing three different tissue-specific promoters have no preferential position in nuclei of undifferentiated embryos. However, in differentiated cells, they shifted stably toward the nuclear lumen when activated, or to the nuclear envelope when silent. In contrast, large integrated arrays bearing the same promoters became heterochromatic and nuclear envelope-bound in embryos. Tissue-specific activation of promoters in these large arrays in larvae overrode the perinuclear anchorage. For transgenes that carry both active and inactive promoters, the inward shift of the active promoter was dominant. Finally, induction of master regulator HLH-1 prematurely induced internalization of a muscle-specific promoter array in embryos. Fluorescence in situ hybridization confirmed analogous results for the endogenous endoderm-determining gene pha-4. We propose that, in differentiated cells, subnuclear organization arises from the selective positioning of active and inactive developmentally regulated promoters. We characterize two forces that lead to tissue-specific subnuclear organization of the worm genome: large repeat-induced heterochromatin, which associates with the nuclear envelope like repressed genes in differentiated cells, and tissue-specific promoters that shift inward in a dominant fashion over silent promoters, when they are activated.
Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular , Colágeno/genética , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/genética , Modelos Genéticos , Células Musculares/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Transativadores/genética , Transgenes/genéticaRESUMO
Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteínas de Caenorhabditis elegans/biossíntese , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Estresse Oxidativo/genética , Doença de Parkinson/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Neurônios Dopaminérgicos/metabolismo , Drosophila melanogaster , Regulação da Expressão Gênica , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
C. elegans has recently emerged as a valuable model to understand the link between nuclear organization and cell fate, by combining microscopy approaches, genome-wide mapping techniques with advanced genetics. Crucial to these analyses are techniques to determine the genome-wide interaction pattern of proteins with DNA. Chromatin immunoprecipitation has proven valuable but it requires considerable amounts of starting material. This is sometimes difficult to achieve, in particular for specific genotypes (balanced strains, different sexes, severe phenotypes ). As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Based upon this pipeline, we provide a comparative analysis of libraries generated with different starting material and discuss important library features. Moreover, we introduce an adaptation of an imaging based tool to visualize in vivo the cell-specific tridimensional binding pattern of any protein of interest.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , DNA de Helmintos/metabolismo , Análise de Sequência de DNA/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Animais , Sítios de Ligação , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Metilação de DNA , Análise de Célula Única , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismoRESUMO
The structural maintenance of chromosome (SMC) complexes-cohesin and condensins-are crucial for chromosome separation and compaction during cell division. During the interphase, mammalian cohesins additionally fold the genome into loops and domains. Here we show that, in Caenorhabditis elegans, a species with holocentric chromosomes, condensin I is the primary, long-range loop extruder. The loss of condensin I and its X-specific variant, condensin IDC, leads to genome-wide decompaction, chromosome mixing and disappearance of X-specific topologically associating domains, while reinforcing fine-scale epigenomic compartments. In addition, condensin I/IDC inactivation led to the upregulation of X-linked genes and unveiled nuclear bodies grouping together binding sites for the X-targeting loading complex of condensin IDC. C. elegans condensin I/IDC thus uniquely organizes holocentric interphase chromosomes, akin to cohesin in mammals, as well as regulates X-chromosome gene expression.
Assuntos
Adenosina Trifosfatases , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas de Ligação a DNA , Complexos Multiproteicos , Cromossomo X , Animais , Caenorhabditis elegans/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromossomo X/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Coesinas , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Interfase/genética , Genoma Helmíntico , Genes Ligados ao Cromossomo X , Cromossomos/genéticaRESUMO
AIMS: This study aimed to explore rural Australians' perceptions of social and cultural factors influencing alcohol use in their communities. METHODS: Semi-structured interviews exploring rural community key informants' (n = 46) perceptions of social and cultural factors influencing alcohol consumption in their community were conducted. A narrative analysis identified cultural capital as a salient concept for explaining how rural community life is created and sustained via drinking practices. RESULTS: Themes relating to participants' accounts of learning to drink, normal drinking; exclusion because of not drinking and problematic drinkers are described. CONCLUSION: In rural communities, beliefs and values about drinking as a positive social practice are transmitted, rewarded and reproduced across multiple groups and settings, reinforcing that drinking is an integral part of Australian rural culture. Drinking is so important that engaging in drinking practices creates and sustains cultural capital. As a result, alcohol-related harm is of little concern to rural dwellers.
Assuntos
Consumo de Bebidas Alcoólicas/psicologia , Transtornos Relacionados ao Uso de Álcool/psicologia , Cultura , Consumo de Bebidas Alcoólicas/etnologia , Austrália , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , População Rural , Valores Sociais , Temperança/psicologiaRESUMO
DNA adenine methylation identification (DamID) is a powerful method to determine DNA binding profiles of proteins at a genomic scale. The method leverages the fusion between a protein of interest and the Dam methyltransferase of E. coli, which methylates proximal DNA in vivo. Here, we present an optimized procedure, which was developed for tissue-specific analyses in Caenorhabditis elegans and successfully used to footprint genes actively transcribed by RNA polymerases and to map transcription factor binding in gene regulatory regions. The present protocol details C. elegans-specific steps involved in the preparation of transgenic lines and genomic DNA samples, as well as broadly applicable steps for the DamID procedure, including the isolation of methylated DNA fragments, the preparation of multiplexed libraries, Nanopore sequencing, and data analysis. Two distinctive features of the approach are (i) the use of an efficient recombination-based strategy to selectively analyze rare cell types and (ii) the use of Nanopore sequencing, which streamlines the process. The method allows researchers to go from genomic DNA samples to sequencing results in less than a week, while being sensitive enough to report reliable DNA footprints in cell types as rare as 2 cells per animal.
RESUMO
The gpa-4 promoter-driven expression is described as specific for ASIL and ASIR chemosensory neurons in the nematode Caenorhabditis elegans, yet this was mostly examined in adult animals. Here we used a recombination-mediated reporter to test the previously used gpa-4 promoter expression. This reporter highlights all cells in which the gpa-4 promoter has been active at one point or another during development. We show that the gpa-4 promoter is indeed active in ASI, yet to our surprise, thispromoter drives also expression in many other cell types, including the somatic gonad, the seam cells, a subset of tail and head neurons, and muscle cells, demonstrating a widespread activity of this transgenic gpa-4 promoter during embryonic and post-embryonic development.
RESUMO
In response to DNA damage and replication pausing, eukaryotes activate checkpoint pathways that prevent genomic instability by coordinating cell cycle progression with DNA repair. The intra-S-phase checkpoint has been proposed to protect stalled replication forks from pathological rearrangements that could result from unscheduled recombination. On the other hand, recombination may be needed to cope with either stalled forks or double-strand breaks resulting from hydroxyurea treatment. We have exploited fission yeast to elucidate the relationship between replication fork stalling, loading of replication and recombination proteins onto DNA, and the intra-S checkpoint. Here, we show that a functional recombination machinery is not essential for recovery from replication fork arrest and instead can lead to nonfunctional fork structures. We find that Rad22-containing foci are rare in S-phase cells, but peak in G2 phase cells after a perturbed S phase. Importantly, we find that the intra-S checkpoint is necessary to avoid aberrant strand-exchange events during a hydroxyurea block.
Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética/genética , Fase S/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Eletroforese em Gel Bidimensional , Fase G2/efeitos dos fármacos , Fase G2/genética , Fase G2/fisiologia , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Instabilidade Genômica/fisiologia , Hidroxiureia/toxicidade , Recombinação Genética/efeitos dos fármacos , Fase S/efeitos dos fármacos , Fase S/genética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
DNA-FISH remains the method of choice to visualize genomic regions in situ ranging from a single locus to entire chromosomes. Current methods to generate probes rely on expensive kits that vary in labeling efficiency and are limited by the size and/or amount of starting material and by the choice of fluorophores. Here we describe a protocol to prepare inexpensive ($20) DNA-FISH probes using an isothermal polymerase, incorporating labeled nucleotides while amplifying minute amounts of any template (PCR fragments/BAC/YAC/fosmids). For complete details on the use and execution of this protocol, please refer to Grosmaire et al. (2019) and Sharma et al. (2014).
Assuntos
Hibridização in Situ Fluorescente/métodos , Sondas Moleculares , Reação em Cadeia da Polimerase/métodos , Animais , Humanos , Camundongos , Sondas Moleculares/química , Sondas Moleculares/metabolismoRESUMO
Differential gene expression across cell types underlies development and cell physiology in multicellular organisms. Caenorhabditis elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNA Polymerase DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, in sorted embryonic blastomeres and in different tissues from intact young adults by driving tissue-specific Dam fusion expression. We obtained meaningful transcriptional footprints in line with RNA-sequencing (RNA-seq) studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 3901 candidate genes with putatively active transcription in XXX cells, including the few previously known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX cells and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of RNA polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.
Assuntos
Proteínas de Caenorhabditis elegans/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Pegadas de Proteínas/métodos , RNA-Seq/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Animais , Blastômeros/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Perfilação da Expressão Gênica/normas , Células Neuroendócrinas/metabolismo , Especificidade de Órgãos , Pegadas de Proteínas/normas , RNA-Seq/normas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genéticaRESUMO
Stable cell fate is an essential feature for multicellular organisms in which individual cells achieve specialized functions. Caenorhabditis elegans is a great model to analyze the determinants of cell fate stability because of its invariant lineage. We present a tractable cell fate challenge system that uses the induction of fate-specifying transcription factors. We show that wild-type differentiated animals are highly resistant to fate challenge. Removal of heterochromatin marks showed marked differences: the absence of histone 3 lysine 9 methylation (H3K9) has no effect on fate stability, whereas Polycomb homolog mes-2 mutants lacking H3K27 methylation terminally arrest larval development upon fate challenge. Unexpectedly, the arrest correlated with widespread cell proliferation rather than transdifferentiation. Using a candidate RNAi larval arrest-rescue screen, we show that the LIN-12Notch pathway is essential for hyperplasia induction. Moreover, Notch signaling appears downstream of food-sensing pathways, as dauers and first larval stage diapause animals are resistant to fate challenge. Our results demonstrate an equilibrium between proliferation and differentiation regulated by Polycomb and Notch signaling in the soma during the nematode life cycle.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Proliferação de Células/fisiologia , Larva/fisiologia , Proteínas do Grupo Polycomb/metabolismo , Receptores Notch/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Plasticidade Celular/fisiologia , Técnicas de Silenciamento de Genes , Hiperplasia/metabolismo , Proteínas de Membrana/metabolismo , Metilação , Fenótipo , Interferência de RNA , Receptores Notch/genética , Inanição/fisiopatologiaRESUMO
We report the reproductive strategy of the nematode Mesorhabditis belari This species produces only 9% males, whose sperm is necessary to fertilize and activate the eggs. However, most of the fertilized eggs develop without using the sperm DNA and produce female individuals. Only in 9% of eggs is the male DNA utilized, producing sons. We found that mixing of parental genomes only gives rise to males because the Y-bearing sperm of males are much more competent than the X-bearing sperm for penetrating the eggs. In this previously unrecognized strategy, asexual females produce few sexual males whose genes never reenter the female pool. Here, production of males is of interest only if sons are more likely to mate with their sisters. Using game theory, we show that in this context, the production of 9% males by M. belari females is an evolutionary stable strategy.
Assuntos
Óvulo/fisiologia , Partenogênese , Rhabditoidea/fisiologia , Razão de Masculinidade , Animais , Evolução Biológica , Feminino , Teoria dos Jogos , Genes Ligados ao Cromossomo X/fisiologia , Genes Ligados ao Cromossomo Y/fisiologia , Masculino , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologiaRESUMO
We describe a straightforward two-step PCR-based method to insert arrays of lac or tet operators (lacO or tetO) at specific loci in the budding yeast genome. The method entails insertion of a marker generated by PCR with classical long primers recognizing the locus of interest, followed by the replacement of this marker by a linearized plasmid bearing an array of lacI- or tetR-binding motifs. Using this technique, loci located either in the yeast genome or on yeast artificial chromosomes can be efficiently tagged. We provide a set of plasmids with different markers for cloning-free integration of lacO or tetO repeats into the yeast genome.
Assuntos
Cromatina/genética , Vetores Genéticos , Saccharomyces cerevisiae/genética , Sitios de Sequências Rotuladas , DNA Fúngico/genética , Marcação de Genes , Engenharia Genética , Óperon Lac , Regiões Operadoras Genéticas , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
Over the past decade, advances in molecular methods have strikingly improved the resolution at which nuclear genome folding can be analyzed. This revealed a wealth of conserved features organizing the one dimensional DNA molecule into tridimensional nuclear domains. In this review, we briefly summarize the main findings and highlight how models based on polymer physics shed light on the principles underlying the formation of these domains. Finally, we discuss the mechanistic similarities allowing self-organization of these structures and the functional importance of these in the maintenance of transcriptional programs.