RESUMEN
This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.
Asunto(s)
Roturas del ADN de Doble Cadena , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Programas Informáticos , Meiosis/genética , Genoma Fúngico , Mapeo Cromosómico/métodos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Biología Computacional/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismoRESUMEN
DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3' UTR, a 22 bp 5' UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86-88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles' Dmc1 and close to the reptiles', but far away from the teleost's. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells' meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells' development and differentiation in Chinese soft-shell turtles, even in the reptiles.
Asunto(s)
Gametogénesis , Filogenia , Tortugas , Animales , Tortugas/genética , Tortugas/metabolismo , Masculino , Gametogénesis/genética , Femenino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Testículo/metabolismo , Clonación Molecular , Secuencia de Aminoácidos , Meiosis/genética , Ovario/metabolismo , Espermatocitos/metabolismo , Pueblos del Este de AsiaRESUMEN
In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.
Asunto(s)
ADN Mitocondrial , Inestabilidad Genómica , Retroelementos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Retroelementos/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Reparación del ADN por Unión de Extremidades , Roturas del ADN de Doble Cadena , Meiosis/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genéticaRESUMEN
Supernumerary B chromosomes contribute to intraspecific karyotypic variation. B chromosomes have been detected in more than 2000 organisms; they possess unique and diverse features, including non-Mendelian inheritance. Here, we report one or more B chromosomes in the gynodioecious plant Atractylodes lancea. Among 54 A. lancea lines, 0-2 B chromosomes were detected in both hermaphroditic and female plants, with the B chromosomes appearing as DAPI-bright regions within the nuclei. Genomic in situ hybridization revealed that the B chromosomes had no conserved A chromosome DNA sequences, confirmed by fluorescence in situ hybridization probed with independently dissected B chromosomes. In male meiosis, the B chromosome did not pair with an A chromosome and was therefore eliminated; accordingly, only 20.1% and 18.6% of these univalent B chromosomes remained at the end of meiosis for the 1B lines of KY17-148 and KY17-118, respectively. However, we also found that B chromosomes were transmitted from male parents in 40.8%-44.2% and 47.2% of the next generation; although these transmission rates from male parents were not essentially different from Mendelian inheritance (0.5), the transmission of gametes carrying B chromosomes increased through fertilization or seed development. B chromosomes were transmitted from three of four 1B female parents to 64.3%-92.6% of the next generation, suggesting B chromosome accumulation. We propose that the B chromosome of A. lancea has a specific sequence and persists via non-Mendelian inheritance from female parents. Overall, A. lancea, with its unique characteristics, is a promising model for understanding the structure, evolution, and mechanism of non-Mendelian inheritance of B chromosomes.
Asunto(s)
Atractylodes , Cromosomas de las Plantas , Hibridación Fluorescente in Situ , Meiosis , Cromosomas de las Plantas/genética , Atractylodes/genética , Meiosis/genéticaRESUMEN
Multiple sex chromosomes usually arise from chromosomal rearrangements which involve ancestral sex chromosomes. There is a fundamental condition to be met for their long-term fixation: the meiosis must function, leading to the stability of the emerged system, mainly concerning the segregation of the sex multivalent. Here, we sought to analyze the degree of differentiation and meiotic pairing properties in the selected fish multiple sex chromosome system present in the wolf-fish Hoplias malabaricus (HMA). This species complex encompasses seven known karyotype forms (karyomorphs) where the karyomorph C (HMA-C) exhibits a nascent XY sex chromosomes from which the multiple X1X2Y system evolved in karyomorph HMA-D via a Y-autosome fusion. We combined genomic and cytogenetic approaches to analyze the satellite DNA (satDNA) content in the genome of HMA-D karyomorph and to investigate its potential contribution to X1X2Y sex chromosome differentiation. We revealed 56 satDNA monomers of which the majority was AT-rich and with repeat units longer than 100 bp. Seven out of 18 satDNA families chosen for chromosomal mapping by fluorescence in situ hybridization (FISH) formed detectable accumulation in at least one of the three sex chromosomes (X1, X2 and neo-Y). Nine satDNA monomers showed only two hybridization signals limited to HMA-D autosomes, and the two remaining ones provided no visible FISH signals. Out of seven satDNAs located on the HMA-D sex chromosomes, five mapped also to XY chromosomes of HMA-C. We showed that after the autosome-Y fusion event, the neo-Y chromosome has not substantially accumulated or eliminated satDNA sequences except for minor changes in the centromere-proximal region. Finally, based on the obtained FISHpatterns, we speculate on the possible contribution of satDNA to sex trivalent pairing and segregation.
Asunto(s)
Characiformes , ADN Satélite , Hibridación Fluorescente in Situ , Cromosomas Sexuales , Animales , ADN Satélite/genética , Cromosomas Sexuales/genética , Masculino , Characiformes/genética , Femenino , Evolución Molecular , Meiosis/genética , Cariotipo , Cromosoma Y/genéticaRESUMEN
Azoospermia (the complete absence of spermatozoa in the semen) is a common cause of male infertility. The etiology of azoospermia is poorly understood. Whole-genome analysis of azoospermic men has identified a number of candidate genes, such as the X-linked testis-expressed 11 (TEX11) gene. Using a comparative genomic hybridization array, an exonic deletion (exons 10-12) of TEX11 had previously been identified in two non-apparent azoospermic patients. However, the putative impact of this genetic alteration on spermatogenesis and the azoospermia phenotype had not been validated functionally. We therefore used a CRISPR/Cas9 system to generate a mouse model (Tex11Ex9-11del/Y) with a partial TEX11 deletion that mimicked the human mutation. Surprisingly, the mutant male Tex11Ex9-11del/Y mice were fertile. The sperm concentration, motility, and morphology were normal. Similarly, the mutant mouse line's testis transcriptome was normal, and the expression of spermatogenesis genes was not altered. These results suggest that the mouse equivalent of the partial deletion observed in two infertile male with azoospermia has no impact on spermatogenesis or fertility in mice, at least of a FVB/N genetic background and until 10 months of age. Mimicking a human mutation does not necessarily lead to the same human phenotype in mice, highlighting significant differences species.
Asunto(s)
Azoospermia , Meiosis , Espermatogénesis , Animales , Masculino , Ratones , Espermatogénesis/genética , Meiosis/genética , Azoospermia/genética , Azoospermia/patología , Infertilidad Masculina/genética , Eliminación de Secuencia , Humanos , Testículo/metabolismo , Testículo/patología , Sistemas CRISPR-CasRESUMEN
Male infertility can be caused by chromosomal abnormalities, mutations and epigenetic defects. Epigenetic modifiers pre-program hundreds of spermatogenic genes in spermatogonial stem cells (SSCs) for expression later in spermatids, but it remains mostly unclear whether and how those genes are involved in fertility. Here, we report that Wfdc15a, a WFDC family protease inhibitor pre-programmed by KMT2B, is essential for spermatogenesis. We found that Wfdc15a is a non-canonical bivalent gene carrying both H3K4me3 and facultative H3K9me3 in SSCs, but is later activated along with the loss of H3K9me3 and acquisition of H3K27ac during meiosis. We show that WFDC15A deficiency causes defective spermiogenesis at the beginning of spermatid elongation. Notably, depletion of WFDC15A causes substantial disturbance of the testicular protease-antiprotease network and leads to an orchitis-like inflammatory response associated with TNFα expression in round spermatids. Together, our results reveal a unique epigenetic program regulating innate immunity crucial for fertility.
Asunto(s)
Homeostasis , Espermátides , Espermatogénesis , Masculino , Animales , Espermatogénesis/genética , Ratones , Espermátides/metabolismo , Testículo/metabolismo , Histonas/metabolismo , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/genética , Epigénesis Genética , Infertilidad Masculina/genética , Ratones Endogámicos C57BL , Meiosis/genética , Células Madre Germinales Adultas/metabolismo , Ratones Noqueados , Inmunidad Innata/genética , Espermatogonias/metabolismoRESUMEN
Meiosis is a critical process in sexual reproduction, and errors during this cell division can significantly impact fertility. Successful meiosis relies on the coordinated action of numerous genes involved in DNA replication, strand breaks, and subsequent rejoining. DNA topoisomerase enzymes play a vital role by regulating DNA topology, alleviating tension during replication and transcription. To elucidate the specific function of DNA topoisomerase 1α ( A t T O P 1 α ) in male reproductive development of Arabidopsis thaliana, we investigated meiotic cell division in Arabidopsis flower buds. Combining cytological and biochemical techniques, we aimed to reveal the novel contribution of A t T O P 1 α to meiosis. Our results demonstrate that the absence of A t T O P 1 α leads to aberrant chromatin behavior during meiotic division. Specifically, the top1α1 mutant displayed altered heterochromatin distribution and clustered centromere signals at early meiotic stages. Additionally, this mutant exhibited disruptions in the distribution of 45s rDNA signals and a reduced frequency of chiasma formation during metaphase I, a crucial stage for genetic exchange. Furthermore, the atm-2×top1α1 double mutant displayed even more severe meiotic defects, including incomplete synapsis, DNA fragmentation, and the presence of polyads. These observations collectively suggest that A t T O P 1 α plays a critical role in ensuring accurate meiotic progression, promoting homologous chromosome crossover formation, and potentially functioning in a shared DNA repair pathway with ATAXIA TELANGIECTASIA MUTATED (ATM) in Arabidopsis microspore mother cells.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Segregación Cromosómica , ADN-Topoisomerasas de Tipo I , Meiosis , Arabidopsis/genética , Arabidopsis/enzimología , Meiosis/fisiología , Meiosis/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Recombinación Genética , MutaciónRESUMEN
Recombination hotspot-activating DNA sites (e.g., M26, CCAAT, Oligo-C) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe. We used a fission yeast-optimized CRISPR-Cas9 system (SpEDIT) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.
Asunto(s)
Sistemas CRISPR-Cas , Meiosis , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Meiosis/genética , Sistemas CRISPR-Cas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Recombinación Genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Edición Génica/métodosRESUMEN
The non-Mendelian transmission of sex chromosomes during gametogenesis carries significant implications, influencing sex ratios and shaping evolutionary dynamics. Here we focus on known mechanisms that drive non-Mendelian inheritance of X chromosomes during spermatogenesis and their impact on population dynamics in species with different breeding systems. In Drosophila and mice, X-linked drivers targeting Y-bearing sperm for elimination or limiting their fitness, tend to confer unfavourable effects, prompting the evolution of suppressors to mitigate their impact. This leads to a complex ongoing evolutionary arms race to maintain an equal balance of males and females. However, in certain insects and nematodes with XX/X0 sex determination, the preferential production of X-bearing sperm through atypical meiosis yields wild-type populations with highly skewed sex ratios, suggesting non-Mendelian transmission of the X may offer selective advantages in these species. Indeed, models suggest X-meiotic drivers could bolster population size and persistence under certain conditions, challenging the conventional view of their detrimental effects. Furthering our understanding of the diverse mechanisms and evolutionary consequences of non-Mendelian transmission of X chromosomes will provide insights into genetic inheritance, sex determination, and population dynamics, with implications for fundamental research and practical applications.
Asunto(s)
Dinámica Poblacional , Razón de Masculinidad , Cromosoma X , Animales , Cromosoma X/genética , Masculino , Femenino , Procesos de Determinación del Sexo , Espermatogénesis/genética , Cruzamiento , Ratones , Meiosis/genética , Drosophila/genética , Humanos , Evolución BiológicaRESUMEN
Whole-genome duplication (WGD) occurs in all kingdoms and impacts speciation, domestication, and cancer outcome. However, doubled DNA management can be challenging for nascent polyploids. The study of within-species polyploidy (autopolyploidy) permits focus on this DNA management aspect, decoupling it from the confounding effects of hybridization (in allopolyploid hybrids). How is autopolyploidy tolerated, and how do young polyploids stabilize? Here, we introduce a powerful model to address this: the genus Cochlearia, which has experienced many polyploidization events. We assess meiosis and other polyploid-relevant phenotypes, generate a chromosome-scale genome, and sequence 113 individuals from 33 ploidy-contrasting populations. We detect an obvious autopolyploidy-associated selection signal at kinetochore components and ion transporters. Modeling the selected alleles, we detail evidence of the kinetochore complex mediating adaptation to polyploidy. We compare candidates in independent autopolyploids across three genera separated by 40 million years, highlighting a common function at the process and gene levels, indicating evolutionary flexibility in response to polyploidy.
Asunto(s)
Evolución Molecular , Genoma de Planta , Cinetocoros , Poliploidía , Cinetocoros/metabolismo , Duplicación de Gen , Adaptación Fisiológica/genética , Meiosis/genéticaRESUMEN
Polyploids are essential in plant evolution and species formation, providing a rich genetic reservoir and increasing species diversity. Complex polyploids with higher ploidy levels often have a dosage effect on the phenotype, which can be highly detrimental to gametes, making them rare. In this study, offspring plants resulting from an autoallotetraploid (RRRC) derived from the interspecific hybridization between allotetraploid Raphanobrassica (RRCC, 2n = 36) and diploid radish (RR, 2n = 18) were obtained. Fluorescence in situ hybridization (FISH) using C-genome-specific repeats as probes revealed two main genome configurations in these offspring plants: RRRCC (2n = 43, 44, 45) and RRRRCC (2n = 54, 55), showing more complex genome configurations and higher ploidy levels compared to the parental plants. These offspring plants exhibited extensive variation in phenotypic characteristics, including leaf type and flower type and color, as well as seed and pollen fertility. Analysis of chromosome behavior showed that homoeologous chromosome pairing events are widely observed at the diakinesis stage in the pollen mother cells (PMCs) of these allopolyploids, with a range of 58.73% to 78.33%. Moreover, the unreduced C subgenome at meiosis anaphase II in PMCs was observed, which provides compelling evidence for the formation of complex allopolyploid offspring. These complex allopolyploids serve as valuable genetic resources for further analysis and contribute to our understanding of the mechanisms underlying the formation of complex allopolyploids.
Asunto(s)
Aneuploidia , Cromosomas de las Plantas , Poliploidía , Raphanus , Raphanus/genética , Cromosomas de las Plantas/genética , Hibridación Fluorescente in Situ , Brassica/genética , Hibridación Genética , Meiosis/genética , Genoma de Planta , Polen/genética , FenotipoRESUMEN
Hi-C, a genome-wide chromosome conformation capture assay, is a powerful tool used to study three-dimensional genome organization by converting physical pairwise interactions into counts of pairwise interactions. To study the many temporally regulated facets of meiotic recombination in S. cerevisiae, the Hi-C assay must be robust such that fine- and wide-scale comparisons between genetic datasets can be made. Here we describe an updated protocol for Hi-C (Hi-C2B) that generates reproducible libraries of interaction data with low noise and for a relatively low cost.
Asunto(s)
Cromosomas Fúngicos , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Meiosis/genética , Cromosomas Fúngicos/genética , Recombinación Genética , Genoma FúngicoRESUMEN
Homologous recombination plays pivotal roles in physical attachments and genetic diversity. In the past, it was studied among individuals from different populations. However, only few gametes from individual could generate offspring, which limits its exploration in nature selection. In the last few years, preimplantation blastocysts based on trio SNP-chip data were available in individuals for preimplantation genetic testing (PGT). In this protocol, we demonstrate how to detect meiotic recombination events and construct the genetic map based on trio SNP-chip data, obtained from biopsied blastocysts and their related individuals in PGT cycles, which may allow better understanding of recombination events in nature selection.
Asunto(s)
Blastocisto , Meiosis , Polimorfismo de Nucleótido Simple , Humanos , Meiosis/genética , Blastocisto/metabolismo , Blastocisto/citología , Femenino , Diagnóstico Preimplantación/métodos , Mapeo Cromosómico/métodos , Recombinación Homóloga , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Recombinación GenéticaRESUMEN
In recent years, targeted genome editing has emerged as an indispensable tool for creating animal models, facilitating a comprehensive exploration of the molecular mechanisms governing a myriad of biological processes. Within this scientific landscape, the investigation of meiosis in mice has attracted considerable attention across numerous research laboratories. The precision and versatility of the CRISPR/Cas9 genome editing system have revolutionized our ability to generate mice with tailored genetic alterations, including point mutations and null mutations. These genetic modifications have provided invaluable insights into the intricate functionality of various meiotic genes and their associated variants. In this context, we present a detailed state of the art protocol for the creation of novel mouse models, each bearing specific genetic modifications within key meiotic genes, through the application of CRISPR/Cas9 technology. Furthermore, we showcase two distinct genetic modifications, accomplished within our laboratory, that can serve as valuable reference points for researchers seeking to elucidate the molecular intricacies of meiosis in mammals.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Meiosis , Animales , Meiosis/genética , Ratones , Edición Génica/métodos , Masculino , Modelos Animales , Femenino , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies we adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin-binding sites by ChIP-seq. High-quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin-binding sites.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Cromatina , Espermatocitos , Animales , Espermatocitos/metabolismo , Espermatocitos/citología , Ratones , Masculino , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Meiosis/genética , Inmunoprecipitación de Cromatina/métodos , Sitios de UniónRESUMEN
During meiosis, homologous chromosomes reciprocally exchange segments of DNA via the formation of crossovers. However, the frequency and position of crossover events along chromosomes are not random. Each chromosome must receive at least one crossover, and the formation of a crossover at one location inhibits the formation of additional crossovers nearby. These crossover patterning phenomena are referred to as "crossover assurance" and "crossover interference," respectively. One key method for quantifying meiotic crossover patterning is to immunocytologically measure the position and intensity of crossover-associated protein foci along the length of meiotic prophase I chromosomes. This approach was recently used to map the position of a conserved E3 ligase, HEI10, along Arabidopsis pachytene chromosomes, providing experimental support for a novel mechanistic "coarsening model" for crossover patterning. Here we describe a user-friendly method for automatically measuring the position and intensity of recombination-associated foci along meiotic prophase I chromosomes that is broadly applicable to studies in different eukaryotic species.
Asunto(s)
Intercambio Genético , Meiosis , Meiosis/genética , Arabidopsis/genética , Cromosomas de las Plantas/genética , Profase Meiótica I , Recombinación GenéticaRESUMEN
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 excision to guarantee a functional new MAC genome. Here, we characterize a pair of paralogous PHD finger proteins involved in DNA elimination. DevPF1, the early-expressed paralog, is present in only some of the gametic and post-zygotic nuclei during meiosis. Both DevPF1 and DevPF2 localize in the new developing MACs, where IES excision occurs. Upon DevPF2 knockdown (KD), long IESs are preferentially retained and late-expressed small RNAs decrease; no length preference for retained IESs was observed in DevPF1-KD and development-specific small RNAs were abolished. The expression of at least two genes from the new MAC with roles in genome reorganization seems to be influenced by DevPF1- and DevPF2-KD. Thus, both PHD fingers are crucial for new MAC genome development, with distinct functions, potentially via regulation of non-coding and coding transcription in the MICs and new MACs.
Asunto(s)
Edición Génica , Paramecium tetraurelia , Proteínas Protozoarias , Paramecium tetraurelia/genética , Paramecium tetraurelia/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Macronúcleo/genética , Macronúcleo/metabolismo , Genoma de Protozoos , Micronúcleo Germinal/metabolismo , Micronúcleo Germinal/genética , Meiosis/genéticaRESUMEN
Our understanding of the molecular pathways that regulate oogenesis and define cellular identity in the Arthropod female reproductive system and the extent of their conservation is currently very limited. This is due to the focus on model systems, including Drosophila and Daphnia, which do not reflect the observed diversity of morphologies, reproductive modes, and sex chromosome systems. We use single-nucleus RNA and ATAC sequencing to produce a comprehensive single nucleus atlas of the adult Artemia franciscana female reproductive system. We map our data to the Fly Cell Atlas single-nucleus dataset of the Drosophila melanogaster ovary, shedding light on the conserved regulatory programs between the two distantly related Arthropod species. We identify the major cell types known to be present in the Artemia ovary, including germ cells, follicle cells, and ovarian muscle cells. Additionally, we use the germ cells to explore gene regulation and expression of the Z chromosome during meiosis, highlighting its unique regulatory dynamics and allowing us to explore the presence of meiotic sex chromosome silencing in this group.
Asunto(s)
Artemia , Drosophila melanogaster , Células Germinativas , Meiosis , Oogénesis , Ovario , Cromosomas Sexuales , Animales , Femenino , Cromosomas Sexuales/genética , Células Germinativas/metabolismo , Artemia/genética , Meiosis/genética , Ovario/metabolismo , Oogénesis/genética , Drosophila melanogaster/genética , Núcleo Celular/genética , Reproducción/genéticaRESUMEN
Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications.