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1.
Results Probl Cell Differ ; 68: 321-353, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31598863

RESUMO

When shifting research focus from model to non-model species, many differences in the working approach should be taken into account and usually methodological modifications are required because of the lack of genetics/genomics and developmental information for the vast majority of organisms. This lack of data accounts for the largely incomplete understanding of how the two components-genes and developmental programs-are intermingled in the process of evolution. A deeper level of knowledge was reached for a few model animals, making it possible to understand some of the processes that guide developmental changes during evolutionary time. However, it is often difficult to transfer the obtained information to other, even closely related, animals. In this chapter, we present and discuss some examples, such as the choice of molecular markers to be used to characterize differentiation and developmental processes. The chosen examples pertain to the study of germline in molluscs, reptiles, and other non-model animals.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Células Germinativas/citologia , Células Germinativas/metabolismo , Moluscos/citologia , Répteis , Animais , Biomarcadores/análise , Répteis/embriologia
2.
Genome Biol ; 20(1): 151, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370870

RESUMO

BACKGROUND: In multicellular organisms, alternative splicing is central to tissue differentiation and identity. Unicellular protists lack multicellular tissue but differentiate into variable cell types during their life cycles. The role of alternative splicing in transitions between cell types and establishing cellular identity is currently unknown in any unicellular organism. RESULTS: To test whether alternative splicing in unicellular protists plays a role in cellular differentiation, we conduct RNA-seq to compare splicing in female and male sexual stages to asexual intraerythrocytic stages in the rodent malaria parasite Plasmodium berghei. We find extensive changes in alternative splicing between stages and a role for alternative splicing in sexual differentiation. Previously, general gametocyte differentiation was shown to be modulated by specific transcription factors. Here, we show that alternative splicing establishes a subsequent layer of regulation, controlling genes relating to consequent sex-specific differentiation of gametocytes. CONCLUSIONS: We demonstrate that alternative splicing is reprogrammed during cellular differentiation of a unicellular protist. Disruption of an alternative splicing factor, PbSR-MG, perturbs sex-specific alternative splicing and decreases the ability of the parasites to differentiate into male gametes and oocysts, thereby reducing transmission between vertebrate and insect hosts. Our results reveal alternative splicing as an integral, stage-specific phenomenon in these protists and as a regulator of cellular differentiation that arose early in eukaryotic evolution.


Assuntos
Processamento Alternativo , Plasmodium berghei/genética , Animais , Células Germinativas/metabolismo , Estágios do Ciclo de Vida/genética , Camundongos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Transcrição Genética
3.
BMC Bioinformatics ; 20(1): 391, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307385

RESUMO

BACKGROUND: Asymmetry during cellular division, both in the uneven partitioning of damaged cellular components and of cell volume, is a cell biological phenomenon experienced by many unicellular organisms. Previous work based on a deterministic model claimed that such asymmetry in the partitioning of cell volume and of aging-associated damage confers a fitness benefit in avoiding clonal senescence, primarily by diversifying the cellular population. However, clonal populations of unicellular organisms are already naturally diversified due to the inherent stochasticity of biological processes. RESULTS: Applying a model of aging cells that accounts for natural cell-to-cell variations across a broad range of parameter values, here we show that the parameters directly controlling the accumulation and repair of damage are the most important factors affecting fitness and clonal senescence, while the effects of both segregation of damaged components and division asymmetry are frequently minimal and generally context-dependent. CONCLUSIONS: We conclude that damage segregation and division asymmetry, perhaps counterintuitively, are not necessarily beneficial from an evolutionary perspective.


Assuntos
Envelhecimento , Modelos Biológicos , Animais , Divisão Celular , Dano ao DNA , Reparo do DNA , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Processos Estocásticos
4.
Artigo em Inglês | MEDLINE | ID: mdl-31176867

RESUMO

In rice field eel (Monopterus albus), germ cell development in the developing gonad has been revealed in detail. However, it is unclear how primordial germ cells (PGCs) migrate to the somatic part of the gonad (genital ridge). This study visualized PGC migration by injecting a chimeric mRNA containing a fluorescent protein fused to the 3' untranslated region (3'UTR) of three different genes, nanos3 of zebrafish (Danio rerio) and dead end (dnd) and vasa of rice field eel. The mRNAs were injected either alone or in pairs into embryos at the one-cell stage. The results showed that mRNAs containing nanos3 and dnd 3'UTRs labeled PGCs over a wider time frame than those containing vasa 3'UTR, suggesting that nanos3 and dnd 3'UTRs are suitable for visualizing PGCs in rice field eel. Using this direct visualization method, the normal migration route of PGCs was observed from the 50%-epiboly stage to hatching stage for the first time, and the ectopic PGCs were also visualized during this period in rice field eel. These findings extend our knowledge of germ cell development, and lay a foundation for further research on the relationship between PGCs and sex differentiation, and on incubation conditions for embryos in rice field eel.


Assuntos
Células Germinativas/metabolismo , Smegmamorpha/embriologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células Germinativas/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Smegmamorpha/genética , Smegmamorpha/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
5.
Nat Commun ; 10(1): 2529, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175278

RESUMO

Substitution of lysine 27 with methionine in histone H3.3 is a recently discovered driver mutation of pediatric high-grade gliomas. Mutant cells show decreased levels and altered distribution of H3K27 trimethylation (H3K27me3). How these chromatin changes are established genome-wide and lead to tumorigenesis remains unclear. Here we show that H3.3K27M-mediated alterations in H3K27me3 distribution result in ectopic DNA replication and cell cycle progression of germ cells in Caenorhabditis elegans. By genetically inducing changes in the H3.3 distribution, we demonstrate that both H3.3K27M and pre-existing H3K27me3 act locally and antagonistically on Polycomb Repressive Complex 2 (PRC2) in a concentration-dependent manner. The heterochromatin changes result in extensive gene misregulation, and genetic screening identified upregulation of JNK as an underlying cause of the germcell aberrations. Moreover, JNK inhibition suppresses the replicative fate in human tumor-derived H3.3K27M cells, thus establishing C. elegans as a powerful model for the identification of potential drug targets for treatment of H3.3K27M tumors.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular , Replicação do DNA , Regulação da Expressão Gênica , Histonas/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Neoplasias Encefálicas , Caenorhabditis elegans , Carcinogênese , Cromatina , Regulação Neoplásica da Expressão Gênica , Células Germinativas/metabolismo , Glioma , Heterocromatina , Código das Histonas , Metilação , Complexo Repressor Polycomb 2/metabolismo
6.
Mol Cell ; 74(5): 982-995.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31076285

RESUMO

PIWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Mitocôndrias/genética , Fatores de Iniciação de Peptídeos/genética , RNA Helicases/genética , RNA Interferente Pequeno/genética , Animais , Drosophila melanogaster/genética , Endorribonucleases/genética , Feminino , Fertilidade/genética , Células Germinativas/metabolismo , Mitocôndrias/metabolismo , Mutação , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Proteínas de Ligação a RNA/genética
7.
Theriogenology ; 131: 61-71, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30947076

RESUMO

The analysis of early gonadogenesis during larval development requires a molecular marker that is specifically expressed in the germ cell lineage, such as the vasa gene. In this study, we cloned and characterized vasa in the striped catfish (Pangasianodon hypophthalmus), and designated this as Phy-vasa. Phy-vasa contained all of the predicted consensus motifs that are shared among the vasa genes in other fish species, including RG and RGG repeats, ATPase motifs, and a DEAD-box, and phylogenetic analysis using various DEAD-box family proteins demonstrated that the Phy-vasa protein clustered within the Vasa family. Reverse transcription polymerase chain reaction (RT-PCR) indicated that Phy-vasa mRNA only occurred in the testis and ovary, and in situ hybridization showed that the gene was expressed only in the germ cells, with strong expression in the spermatogonia and oogonia. To investigate early gonadogenesis in catfish larvae, we undertook histological characterization and in situ hybridization using a Phy-vasa probe, which showed that migration of the primordial germ cells (PGCs) most commonly occurred in larvae at 2-10 days post-fertilization (dpf), the PGCs started to be surrounded by gonadal somatic cells at around 10-20 dpf, and rapid proliferation of the PGCs had begun by 30 dpf. These findings provide a valuable insight into early gonadal development in the striped catfish.


Assuntos
Peixes-Gato/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Peixes/metabolismo , Células Germinativas/metabolismo , Gônadas/metabolismo , Animais , Peixes-Gato/genética , RNA Helicases DEAD-box/química , Proteínas de Peixes/química , Filogenia , RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína
8.
Genes Cells ; 24(5): 377-389, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30929290

RESUMO

In Caenorhabditis elegans, germline cells remain transcriptionally silenced during embryogenesis. The transcriptional silencing is achieved by two different mechanisms: One is the inhibition of RNA polymerase II in P2-P4 cells at the establishment stage, and another is chromatin-based silencing in two primordial germ cells (PGCs) at the maintenance stage; however, the molecular mechanism underlying chromatin-based silencing is less understood. We investigated the role of the chromodomain protein MRG-1, which is an essential maternal factor for germline development, in transcriptional silencing in PGCs. PGCs lacking maternal MRG-1 showed increased levels of two histone modifications (H3K4me2 and H4K16ac), which are epigenetic markers for active transcription, and precocious activation of germline promoters. Loss of MES-4, a H3K36 methyltransferase, also caused similar derepression of the germline genes in PGCs, suggesting that both MRG-1 and MES-4 function in chromatin-based silencing in PGCs. In addition, the mrg-1 null mutant showed abnormal chromosome structures and a decrease in homologous recombinase RAD-51 foci in PGCs, but the mes-4 null mutant did not show such phenotypes. Taken together, we propose that MRG-1 has two distinct functions: chromatin-based transcriptional silencing and preserving genomic integrity at the maintenance stage of PGCs.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Células Germinativas/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Cromatina/metabolismo , Instabilidade Genômica , Células Germinativas/citologia , Código das Histonas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
9.
Methods Cell Biol ; 151: 473-486, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948027

RESUMO

In many species, sperm must locate the female gamete to achieve fertilization. Molecules diffusing from the egg envelope, or the female genital tract, guide the sperm toward the oocyte through a process called chemotaxis. Sperm chemotaxis has been studied for more than 100 years being a widespread phenomenon present from lower plants to mammals. This process has been mostly studied in external fertilizers where gametes undergo a significant dilution, as compared to internal fertilizers where the encounter is more defined by the topology of the female tract and only a small fraction of sperm appear to chemotactically respond. Here, we summarize the main methods to measure sperm swimming responses to a chemoattractant, both in populations and in individual sperm. We discuss a novel chemotactic index (CI) to score sperm chemotaxis in external fertilizers having circular trajectories. This CI is based on the sperm progressive displacement and its orientation angle to the chemoattractant source.


Assuntos
Quimiotaxia/genética , Fertilização/genética , Motilidade Espermática/genética , Animais , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Mamíferos/genética , Mamíferos/crescimento & desenvolvimento , Desenvolvimento Vegetal
10.
PLoS Genet ; 15(3): e1008004, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30921322

RESUMO

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.


Assuntos
Células Germinativas/metabolismo , Proteína Fosfatase 1/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Genoma , Células Germinativas/fisiologia , Meiose/fisiologia , Prófase Meiótica I/fisiologia , Metilação , Monoéster Fosfórico Hidrolases , Proteína Fosfatase 1/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno
11.
Gen Comp Endocrinol ; 277: 56-65, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30878349

RESUMO

Unlike its paralog Foxl2, which is well known for its role in ovarian development in vertebrates, the function of Foxl3 is still unclear. Foxl3 is an ancient duplicated copy of Foxl2. It is present as a single copy in ray-finned fish. But, due to repeated losses, it is absent in most tetrapods. Our transcriptomic data, however, show that two Foxl3s (Foxl3a and its paralog Foxl3b) are present in Japanese eel. Foxl3a is predominantly expressed in the pituitary, and Foxl3b is predominantly expressed in the gills. Both Foxl3s show a sex-dimorphic expression, being higher expression in testes than in ovaries. Moreover, Foxl3a and Foxl3b were exclusively expressed during gonadal differentiation in control eels (100% male). Conversely, Foxl3a and Foxl3b significantly decreased after gonadal differentiation in E2-treated eels (100% female). Furthermore, in accordance the difference in adhesive ability between somatic cells and germline cells in testes, Foxl3s showed a high expression in suspension cells (putative germline cells) and low expression in adhesive cells (putative somatic cells). In situ hybridization further showed that Foxl3a and Foxl3b were expressed in the testicular germline cells. In addition, Foxl3s expression was not changed by sex steroids in in vitro testes culture. Taken together, our results suggest that the teleost-specific Foxl3 paralog was repeatedly lost in most fish after the third round of whole genome duplication. The two germline-expressed Foxl3s had higher expression levels in males than in females during gonadal differentiation in Japanese eel. These results demonstrated that Foxl3s might play an important role in germline sexual fate determination from ancient fish to modern fish.


Assuntos
Anguilla/genética , Anguilla/fisiologia , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Gônadas/fisiologia , Diferenciação Sexual/fisiologia , Sequência de Aminoácidos , Animais , Tamanho Corporal/efeitos dos fármacos , Estradiol/farmacologia , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , Gônadas/efeitos dos fármacos , Masculino , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Diferenciação Sexual/efeitos dos fármacos , Diferenciação Sexual/genética , Esteroides/farmacologia , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/metabolismo
12.
Differentiation ; 106: 23-34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30852470

RESUMO

The extracellular matrix (ECM) proteins play an important role in the establishment of the sex-dependent structure of developing gonads. The matrix metalloproteinases (MMPs) are the major players in the regulation of ECM. Our hypothesis was that the MMPs-dependent regulation of EMC is crucial for the establishment of the correct, either testis or ovary, structure of developing gonad. We cultured developing mouse gonads in vitro in the presence of the MMPs inhibitors (α-2-macroglobulin, leupeptin, phosphoramidon) or the MMPs activator, APMA (4-aminophenylmercuric acetate). These inhibitors and activator inhibit/activate, to a different degree, matrix metalloproteinases, but the exact mechanism of inhibition/activation remains unknown. We found that the MMP inhibitors increased accumulation of ECM in the developing gonads. The α-2-macroglobulin had the weakest, and the phosphoramidon the strongest effect on the ECM and the structure of the gonads. The α-2-macroglobulin caused a slight increase of ECM and did not disrupt the gonad structure. Leupeptin led to the strong accumulation of ECM, resulted in the formation of the structures resembling testis cords in both testes and ovaries, and caused increase of apoptosis and complete loss of germ cells. Phosphoramidon caused the strongest accumulation of ECM, which separated individual cells and completely prevented intercellular adhesion both in the testes and in the ovaries. As a result of aberrant morphology, the sex of the phosphoramidon-treated gonads was morphologically unrecognizable. The APMA - the activator of MMP caused ECM loss, which led to the loss of cell adhesion, cell dispersion and an aberrant morphology of the gonads. These results indicate that the ECM accumulation is MMPs-dependent and that the correct amount and distribution of ECM during gonad development plays a key role in the formation of the gonad structure.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Células Germinativas/citologia , Gônadas/citologia , Metaloproteinases da Matriz/metabolismo , Diferenciação Sexual , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Gônadas/metabolismo , Masculino , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL
13.
J Fish Biol ; 94(5): 772-780, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30873617

RESUMO

In this study, a 2198 bp full-length cDNA of spinyhead croaker Collichthys lucidus vasa gene encoding 616 amino-acid residues was obtained. Multiple alignment revealed that C. lucidus vasa has eight conserved characteristic motifs of the DEAD box protein family and has the highest identity to large yellow croaker Larimichthys croceas. Reverse-transcription (RT)-PCR and Western blot analyses indicated that the vasa messenger (m)RNA and Vasa protein are specifically expressed in the gonads in both sexes. In situ hybridisation (ISH) demonstrated that vasa RNA is exclusively detected in the germ cells in C. lucidus gonads and its temporospatial expression reveals a dynamic pattern during oogenesis. Surprisingly, C. lucidus vasa 3'UTR can direct stable and specific GFP expression in the primordial germ cells (PGC) of medaka Oryzias latipes embryos. Taken together, these results suggest that because C. lucidus vasa expression delineates critical stages of oogenesis, it may be a useful molecular marker for the identification of gonadal germ cells, facilitating the isolation and utilization of germ cells in future study.


Assuntos
Evolução Biológica , Proteínas de Peixes/genética , Células Germinativas/citologia , Perciformes/genética , Animais , Clonagem Molecular , DNA Complementar/genética , Feminino , Proteínas de Peixes/química , Células Germinativas/metabolismo , Gônadas/metabolismo , Hibridização In Situ , Masculino , Oogênese , Oryzias/genética , Perciformes/embriologia , Filogenia
14.
Sci Data ; 6(1): 8, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30918261

RESUMO

Germline stem cells are germ cells at an early developmental stage, so their development is key to ensuring human reproduction. There is increasing evidence that long noncoding RNA (lncRNA) and circular RNA (circRNA) play important roles in the development of germ cells. This data descriptor provides unique lncRNA and circRNA transcriptomic information for mouse germline stem cells. Using the Illumina HiSeqx 2000 system, a total of 511,836,732 raw reads were generated. High-quality transcripts, lncRNAs, and circRNAs were identificated and quantified using the reads, and more precise annotations of lncRNAs (especially 9357 novel lncRNAs) and circRNAs were performed in the germline stem cells. We then analyzed the transcript structures, genetic variants, and the interaction between circRNA and microRNA to provide the basis for subsequent functional experiments. This comprehensive dataset will help advance data sharing and deepen our understanding of mouse germline stem cells, providing a theoretical foundation for research on germ cell development and human reproduction, among others.


Assuntos
Células Germinativas Embrionárias , Células Germinativas , RNA Longo não Codificante , RNA , Animais , Células Germinativas Embrionárias/citologia , Células Germinativas Embrionárias/metabolismo , Genoma , Células Germinativas/citologia , Células Germinativas/metabolismo , Camundongos
15.
Nat Commun ; 10(1): 1407, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926776

RESUMO

RAD51 assembly on single-stranded (ss)DNAs is a crucial step in the homology-dependent repair of DNA damage for genomic stability. The formation of the RAD51 filament is promoted by various RAD51-interacting proteins including RAD51 paralogues. However, the mechanisms underlying the differential control of RAD51-filament dynamics by these factors remain largely unknown. Here, we report a role for the human RAD51 paralogue, SWSAP1, as a novel regulator of RAD51 assembly. Swsap1-deficient cells show defects in DNA damage-induced RAD51 assembly during both mitosis and meiosis. Defective RAD51 assembly in SWSAP1-depleted cells is suppressed by the depletion of FIGNL1, which binds to RAD51 as well as SWSAP1. Purified FIGNL1 promotes the dissociation of RAD51 from ssDNAs. The dismantling activity of FIGNL1 does not require its ATPase but depends on RAD51-binding. Purified SWSAP1 inhibits the RAD51-dismantling activity of FIGNL1. Taken together, our data suggest that SWSAP1 protects RAD51 filaments by antagonizing the anti-recombinase, FIGNL1.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Recombinases Rec A/fisiologia , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromossomos Humanos/metabolismo , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Células Germinativas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Modelos Biológicos , Ligação Proteica , Recombinases Rec A/genética
16.
Genes Genet Syst ; 94(1): 3-12, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30905890

RESUMO

Next-generation sequencing (NGS) has been used to determine the reference sequences of model organisms. This allows us to identify mutations by the chromosome number and sequence position where the base sequence has been altered, independent of any phenotypic alteration. Because the re-sequencing method by NGS covers all of the genome, it enables detection of the small number of spontaneous de novo germline mutations that occur in the reproductive lineage. The spontaneous mutation rate varies depending on the environment; for example, it increases when 8-oxoguanine accumulates. If the mutation rate (per replication) is greater than 1/genome size (2n), at least one mutation would generally occur in each cell division on average, producing cells with a different genome from the parent cell. Organisms with larger genomes and more divisions by cells in the reproductive lineage are expected to show higher mutation rates per generation, if the mutation rate per replication is constant among species. The accumulation of mutations that arose in the genome of ancestor cells has resulted in heterogeneity and diversity among extant species. In this sense, the ability to produce mutations in cells of the reproductive lineage can be considered as a key feature of organisms, even if mutations also present an unavoidable risk.


Assuntos
Linhagem da Célula , Mutação em Linhagem Germinativa , Animais , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Taxa de Mutação
17.
Nat Commun ; 10(1): 1433, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926893

RESUMO

Malaria infections occurring below the limit of detection of standard diagnostics are common in all endemic settings. However, key questions remain surrounding their contribution to sustaining transmission and whether they need to be detected and targeted to achieve malaria elimination. In this study we analyse a range of malaria datasets to quantify the density, detectability, course of infection and infectiousness of subpatent infections. Asymptomatically infected individuals have lower parasite densities on average in low transmission settings compared to individuals in higher transmission settings. In cohort studies, subpatent infections are found to be predictive of future periods of patent infection and in membrane feeding studies, individuals infected with subpatent asexual parasite densities are found to be approximately a third as infectious to mosquitoes as individuals with patent (asexual parasite) infection. These results indicate that subpatent infections contribute to the infectious reservoir, may be long lasting, and require more sensitive diagnostics to detect them in lower transmission settings.


Assuntos
Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Parasitos/fisiologia , Plasmodium falciparum/fisiologia , Animais , Células Germinativas/metabolismo , Humanos , Parasitemia/parasitologia , Probabilidade , Fatores de Tempo
18.
Cancer Immunol Immunother ; 68(6): 897-905, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30863922

RESUMO

Immune-checkpoint inhibition (ICI) treatments improve outcomes for metastatic melanoma; however, > 60% of treated patients do not respond to ICI. Current biomarkers do not reliably explain ICI resistance. Given the link between ICI and autoimmunity, we investigated if genetic susceptibility to autoimmunity modulates ICI efficacy. In 436 patients with metastatic melanoma receiving single line ICI or combination treatment, we tested 25 SNPs, associated with > 2 autoimmune diseases in recent genome-wide association studies, for modulation of ICI efficacy. We found that rs17388568-a risk variant for allergy, colitis and type 1 diabetes-was associated with increased anti-PD-1 response, with significance surpassing multiple testing adjustments (OR 0.26; 95% CI 0.12-0.53; p = 0.0002). This variant maps to a locus of established immune-related genes: IL2 and IL21. Our study provides first evidence that autoimmune genetic susceptibility may modulate ICI efficacy, suggesting that systematic testing of autoimmune risk loci could reveal personalized biomarkers of ICI response.


Assuntos
Doenças Autoimunes/terapia , Biomarcadores Tumorais/genética , Predisposição Genética para Doença/genética , Imunoterapia/métodos , Melanoma/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Biomarcadores Tumorais/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Feminino , Células Germinativas/imunologia , Células Germinativas/metabolismo , Humanos , Interleucina-2/genética , Interleucinas/genética , Masculino , Melanoma/genética , Melanoma/imunologia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Fatores de Risco
19.
DNA Repair (Amst) ; 75: 18-28, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30710866

RESUMO

A missense mutation in C. elegans RAD-54, a homolog of RAD54 that operates in the homologous recombination (HR) pathway, was found to decrease ATPase activity in vitro. The hypomorphic mutation caused hypersensitivity of C. elegans germ cells to double-strand DNA breaks (DSBs). Although the formation of RAD-51 foci at DSBs was normal in both the mutant and knockdown worms, their subsequent dissipation was slow. The rad-54-deficient phenotypes were greatly aggravated when combined with an xpf-1 mutation, suggesting a conservative role of single-strand annealing (SSA) for DSB repair in HR-defective worms. The phenotypes of doubly-deficient rad-54;xpf-1 worms were partially suppressed by a mutation of lig-4, a nonhomologous end-joining (NHEJ) factor. In summary, RAD-54 is required for the dissociation of RAD-51 from DSB sites in C. elegans germ cells. Also, NHEJ and SSA exert negative and positive effects, respectively, on genome stability when HR is defective.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Células Germinativas/metabolismo , Recombinação Homóloga , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , DNA de Cadeia Simples/genética , Mutação
20.
Dev Cell ; 48(6): 827-839.e9, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30799227

RESUMO

The recent work of Besseling and Bringmann (2016) identified a molecular intervention for C. elegans in which premature segregation of maternal and paternal chromosomes in the fertilized oocyte can produce viable animals exhibiting a non-Mendelian inheritance pattern. Overexpression in embryos of a single protein regulating chromosome segregation (GPR-1) provides a germline derived clonally from a single parental gamete. We present a collection of strains and cytological assays to consistently generate and track non-Mendelian inheritance. These tools allow reproducible and high-frequency (>80%) production of non-Mendelian inheritance, the facile and simultaneous homozygosis for all nuclear chromosomes in a single generation, the precise exchange of nuclear and mitochondrial genomes between strains, and the assessments of non-canonical mitosis events. We show the utility of these strains by demonstrating a rapid assessment of cell lineage requirements (AB versus P1) for a set of genes (lin-2, lin-3, lin-12, and lin-31) with roles in C. elegans vulval development.


Assuntos
Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Padrões de Herança/genética , Animais , Biomarcadores/metabolismo , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromossomos/genética , Cruzamentos Genéticos , Feminino , Fluorescência , Masculino , Microtúbulos/metabolismo , Mosaicismo , Mutação/genética , Faringe/metabolismo , Fenótipo , Vulva/embriologia , Vulva/metabolismo , Zigoto/metabolismo
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