RESUMO
BACKGROUND: Soybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of the GmWRKY TFs in resistance to SMV are largely unclear. RESULTS: Here, 185 GmWRKYs were characterized in soybean (Glycine max), among which 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. The transcriptome data and RT-qPCR results showed that the expression of GmWRKY164 decreased after imidazole treatment and had higher expression levels in the incompatible combination between soybean cultivar variety Jidou 7 and SMV strain N3. Remarkably, the silencing of GmWRKY164 reduced callose deposition and enhanced virus spread during SMV infection. In addition, the transcript levels of the GmGSL7c were dramatically lower upon the silencing of GmWRKY164. Furthermore, EMSA and ChIP-qPCR revealed that GmWRKY164 can directly bind to the promoter of GmGSL7c, which contains the W-box element. CONCLUSION: Our findings suggest that GmWRKY164 plays a positive role in resistance to SMV infection by regulating the expression of GmGSL7c, resulting in the deposition of callose and the inhibition of viral movement, which provides guidance for future studies in understanding virus-resistance mechanisms in soybean.
Assuntos
Resistência à Doença , Regulação da Expressão Gênica de Plantas , Glycine max , Doenças das Plantas , Proteínas de Plantas , Potyvirus , Fatores de Transcrição , Glycine max/virologia , Glycine max/genética , Resistência à Doença/genética , Doenças das Plantas/virologia , Doenças das Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Potyvirus/fisiologia , Potyvirus/patogenicidade , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras GenéticasRESUMO
Specific recognition of centromere-specific histone variant CENP-A-containing chromatin by CENP-N is an essential process in the assembly of the kinetochore complex at centromeres prior to mammalian cell division. However, the mechanisms of CENP-N recruitment to centromeres/kinetochores remain unknown. Here, we show that a CENP-A-specific RG loop (Arg80/Gly81) plays an essential and dual regulatory role in this process. The RG loop assists the formation of a compact "ladder-like" structure of CENP-A chromatin, concealing the loop and thus impairing its role in recruiting CENP-N. Upon G1/S-phase transition, however, centromeric chromatin switches from the compact to an open state, enabling the now exposed RG loop to recruit CENP-N prior to cell division. Our results provide the first insights into the mechanisms by which the recruitment of CENP-N is regulated by the structural transitions between compaction and relaxation of centromeric chromatin during the cell cycle.
Assuntos
Ciclo Celular/fisiologia , Centrômero/química , Centrômero/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Linhagem Celular , Proliferação de Células , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Cromossomos/metabolismo , Células HeLa , Humanos , Cinetocoros/química , Cinetocoros/metabolismo , Ligação Proteica , Transporte Proteico , Fase S/fisiologiaRESUMO
During meiotic prophase, chromosomes pair and synapse with their homologs and undergo programmed DNA double-strand break (DSB) formation to initiate meiotic recombination. These DSBs are processed to generate a limited number of crossover recombination products on each chromosome, which are essential to ensure faithful segregation of homologous chromosomes. The nematode Caenorhabditis elegans has served as an excellent model organism to investigate the mechanisms that drive and coordinate these chromosome dynamics during meiosis. Here we focus on our current understanding of the regulation of DSB induction in C. elegans. We also review evidence that feedback regulation of crossover formation prolongs the early stages of meiotic prophase, and discuss evidence that this can alter the recombination pattern, most likely by shifting the genome-wide distribution of DSBs.
Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Pontos de Checagem do Ciclo Celular/genética , Troca Genética , Meiose/genética , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Quebras de DNA de Cadeia DuplaRESUMO
Applying machine learning to combinatorial optimization problems has the potential to improve both efficiency and accuracy. However, existing learning-based solvers often struggle with generalization when faced with changes in problem distributions and scales. In this paper, we propose a new approach called ASP: Adaptive Staircase Policy Space Response Oracle to address these generalization issues and learn a universal neural solver. ASP consists of two components: Distributional Exploration, which enhances the solver's ability to handle unknown distributions using Policy Space Response Oracles, and Persistent Scale Adaption, which improves scalability through curriculum learning. We have tested ASP on several challenging COPs, including the traveling salesman problem, the vehicle routing problem, and the prize collecting TSP, as well as the real-world instances from TSPLib and CVRPLib. Our results show that even with the same model size and weak training signal, ASP can help neural solvers explore and adapt to unseen distributions and varying scales, achieving superior performance. In particular, compared with the same neural solvers under a standard training pipeline, ASP produces a remarkable decrease in terms of the optimality gap with 90.9% and 47.43% on generated instances and real-world instances for TSP, and a decrease of 19% and 45.57% for CVRP.
RESUMO
Cohesins are ancient and ubiquitous regulators of chromosome architecture and function, but their diverse roles and regulation remain poorly understood. During meiosis, chromosomes are reorganized as linear arrays of chromatin loops around a cohesin axis. This unique organization underlies homolog pairing, synapsis, double-stranded break induction, and recombination. We report that axis assembly in Caenorhabditis elegans is promoted by DNA-damage response (DDR) kinases that are activated at meiotic entry, even in the absence of DNA breaks. Downregulation of the cohesin-destabilizing factor WAPL-1 by ATM-1 promotes axis association of cohesins containing the meiotic kleisins COH-3 and COH-4. ECO-1 and PDS-5 also contribute to stabilizing axis-associated meiotic cohesins. Further, our data suggest that cohesin-enriched domains that promote DNA repair in mammalian cells also depend on WAPL inhibition by ATM. Thus, DDR and Wapl seem to play conserved roles in cohesin regulation in meiotic prophase and proliferating cells.
Assuntos
Proteínas Cromossômicas não Histona , Meiose , Animais , Proteínas Cromossômicas não Histona/genética , Cromossomos , Proteínas de Ciclo Celular/genética , Pareamento Cromossômico , Caenorhabditis elegans/genética , Mamíferos/genética , CoesinasRESUMO
Meiotic chromosome segregation relies on synapsis and crossover (CO) recombination between homologous chromosomes. These processes require multiple steps that are coordinated by the meiotic cell cycle and monitored by surveillance mechanisms. In diverse species, failures in chromosome synapsis can trigger a cell cycle delay and/or lead to apoptosis. How this key step in 'homolog engagement' is sensed and transduced by meiotic cells is unknown. Here we report that in C. elegans, recruitment of the Polo-like kinase PLK-2 to the synaptonemal complex triggers phosphorylation and inactivation of CHK-2, an early meiotic kinase required for pairing, synapsis, and double-strand break (DSB) induction. Inactivation of CHK-2 terminates DSB formation and enables CO designation and cell cycle progression. These findings illuminate how meiotic cells ensure CO formation and accurate chromosome segregation.
Most animals, plants, and fungi reproduce sexually, meaning that the genetic information from two parents combines during fertilization to produce offspring. This parental genetic information is carried within the reproductive cells in the form of chromosomes. Reproductive cells in the ovaries or testes first multiply through normal cell division, but then go through a unique type of cell division called meiosis. During meiosis, pairs of chromosomes the two copies inherited from each parent must find each other and physically line up from one end to the other. As they align side-by-side with their partners, chromosomes also go through a mixing process called recombination, during which regions of one chromosome cross over to the paired chromosome to exchange information. Scientists are still working to understand how this process of chromosome alignment and crossing-over is controlled. If chromosomes fail to line up or cross over during meiosis, eggs or sperm can end up with too many or too few chromosomes. If these faulty reproductive cells combine during fertilization this can lead to birth defects and developmental problems. To minimize this problem, reproductive cells have a quality control mechanism during meiosis called "crossover assurance", which limits how often mistakes occur. Zhang et al. have investigated how cells can tell if their chromosomes have accomplished this as they undergo meiosis. They looked at egg cells of the roundworm C. elegans, whose meiotic processes are similar to those in humans. In C. elegans, a protein called CHK-2 regulates many of the early events during meiosis. During successful meiosis, CHK-2 is active for only a short amount of time. But if there are problems during recombination, CHK-2 stays active for longer and prevents the cell division from proceeding. Zhang et al. uncovered another protein that affects for how long CHK-2 stays switched on. When chromosomes align with their partners, a protein called PLK-2 sticks to other proteins at the interface between the aligned chromosomes. A combination of microscopy and test tube experiments showed that when PLK-2 is bound to this specific location, it can turn off CHK-2. However, if the chromosome alignment fails, PLK-2 is not activated to switch off CHK-2. Therefore, CHK-2 is only switched off when the chromosomes are properly aligned and move on to the next step in crossing-over, which then allows meiosis to proceed. Thus, PLK-2 and CHK-2 work together to detect errors and to slow down meiosis if necessary. Further experiments in mammalian reproductive cells will reveal how similar the crossover assurance mechanism is in different organisms. In the future, improved understanding of quality control during meiosis may eventually lead to improvements in assisted reproduction.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Pareamento Cromossômico , Meiose , Complexo Sinaptonêmico/metabolismoRESUMO
Centromere identity is defined by nucleosomes containing CENP-A, a histone H3 variant. The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner. We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68. However, a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading. Here, we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability. We also find that mice harboring the corresponding mutations are lethal. Together, these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.
Assuntos
Centrômero , Nucleossomos , Animais , Autoantígenos/metabolismo , Ciclo Celular , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Histonas/genética , Histonas/metabolismo , CamundongosRESUMO
CENP-A (centromeric protein A), a histone H3 variant, specifies centromere identity and is essential to centromere maintenance. Little is known about how protein levels of CENP-A are controlled in mammalian cells. Here, we report that the phosphorylation of CENP-A Ser68 primes the ubiquitin-proteasome-mediated proteolysis of CENP-A during mitotic phase in human cultured cells. We identify two major polyubiquitination sites that are responsible for this phosphorylation-dependent degradation. Substituting the two residues, Lys49 and Lys124, with arginines abrogates proper CENP-A degradation and results in CENP-A mislocalization to non-centromeric regions. Furthermore, we find that DCAF11 (DDB1 and CUL4 associated factor 11/WDR23) is the E3 ligase that specifically mediates the observed polyubiquitination. Deletion of DCAF11 hampers CENP-A degradation and causes its mislocalization. We conclude that the Ser68 phosphorylation plays an important role in regulating cellular CENP-A homeostasis via DCAF11-mediated degradation to prevent ectopic localization of CENP-A during the cell cycle.
Assuntos
Ciclo Celular , Proteína Centromérica A/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Serina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação , Animais , Centrômero , Proteína Centromérica A/química , Proteína Centromérica A/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Culina/genética , Proteínas de Ligação a DNA/genética , Feminino , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nucleossomos , Fosforilação , Proteólise , Serina/química , Serina/genética , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
The centromere regulates proper chromosome segregation, and its dysfunction is implicated in chromosomal instability (CIN). However, relatively little is known about how centromere dysfunction occurs in cancer. Here, we define the consequences of phosphorylation by cyclin E1/CDK2 on a conserved Ser18 residue of centromere-associated protein CENP-A, an essential histone H3 variant that specifies centromere identity. Ser18 hyperphosphorylation in cells occurred upon loss of FBW7, a tumor suppressor whose inactivation leads to CIN. This event on CENP-A reduced its centromeric localization, increased CIN, and promoted anchorage-independent growth and xenograft tumor formation. Overall, our results revealed a pathway that cyclin E1/CDK2 activation coupled with FBW7 loss promotes CIN and tumor progression via CENP-A-mediated centromere dysfunction. Cancer Res; 77(18); 4881-93. ©2017 AACR.
Assuntos
Autoantígenos/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/patologia , Instabilidade Cromossômica , Proteínas Cromossômicas não Histona/metabolismo , Neoplasias do Colo/patologia , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas F-Box/metabolismo , Proteínas Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Apoptose , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Centrômero , Proteína Centromérica A , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteína 7 com Repetições F-Box-WD , Feminino , Histonas/metabolismo , Humanos , Fosforilação , Células Tumorais CultivadasRESUMO
The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.
Assuntos
Autoantígenos/metabolismo , Ciclo Celular/fisiologia , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Fosfatase 1/metabolismo , Serina/metabolismo , Western Blotting , Proteína Quinase CDC2 , Proteína Centromérica A , Cromatina/genética , Imunofluorescência , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Mitose/fisiologia , Nucleossomos , FosforilaçãoRESUMO
Mammalian histone H3.3 is a variant of the canonical H3.1 essential for genome reprogramming in fertilized eggs and maintenance of chromatin structure in neuronal cells. An H3.3-specific histone chaperone, DAXX, directs the deposition of H3.3 onto pericentric and telomeric heterochromatin. H3.3 differs from H3.1 by only five amino acids, yet DAXX can distinguish the two with high precision. By a combination of structural, biochemical and cell-based targeting analyses, we show that Ala87 and Gly90 are the principal determinants of human H3.3 specificity. DAXX uses a shallow hydrophobic pocket to accommodate the small hydrophobic Ala87 of H3.3, whereas a polar binding environment in DAXX prefers Gly90 in H3.3 over the hydrophobic Met90 in H3.1. An H3.3-H4 heterodimer is bound by the histone-binding domain of DAXX, which makes extensive contacts with both H3.3 and H4.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Histonas/química , Chaperonas Moleculares/química , Proteínas Nucleares/química , Proteínas Correpressoras , Dimerização , Humanos , Modelos MolecularesRESUMO
A novel phenylacetic acid (PAA)-induced CoA-ligase-encoding gene, designated as phlC, has been cloned from penicillin-producing fungus Penicillium chrysogenum. The open reading frame of phlC cDNA was 1671 bp and encoded a 556 amino acid residues protein with the consensus AMP binding site and a peroxisomal targeting signal 1 on its C terminus. The deduced amino acid sequence showed 37% and 38% identity with characterized P. chrysogenum Phl and PhlB protein, respectively. Functional recombinant PhlC protein was overexpressed in Escherichia coli. The purified recombinant enzyme was capable to convert PAA into its corresponding CoA ester with a specific activity of 129.5 ± 3.026 pmol/min per mg protein. Similar to Phl and PhlB, PhlC displayed broad substrate spectrum and showed higher activities to medium- and long-chain fatty acids. The catalytic properties of PhlC have been determined and compared to those of Phl and PhlB.
Assuntos
Acetilcoenzima A/biossíntese , Coenzima A Ligases/genética , Penicillium chrysogenum/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Penicillium chrysogenum/genética , Fenilacetatos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
According to the GenBank sequences (GenBank Accession No. AF539467), one pair of primers was designed to amplify hly gene of Aeromonas hydrophila by PCR. After sequencing, homology analysis indicated that a DNA fragment of 1485 bp was amplified from isolated DNA from Aeromonas hydrophila, and it shared more than 99% homology in nucleotide sequence compared with other reference strains in GenBank. The gene was cloned in pET-28a vector to construct a recombinant plasmid pET-28a-hly, which was transformed into Escherichia coli BL21 (DE3), and the recombinant strain BL21(DE3)(pET-28a-hly) was obtained. The hemolysin was highly expressed when the recombinant strain BL21 (DE3) (pET-28a-hly) was induced by IPTG. The expressed protein was 56 kD as estimated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the expressed Hly protein was confirmed by Western blotting. Mice were immunized with inactivated whole bacteria vaccine and the genetic engineering vaccines showing promise that all these vaccines have a high protective ability. The results showed that the recombinant strain BL21 (DE3)(pET-28a-hly) could be candidate of hemolysin toxoid vaccine to provide protective immunity against diseases caused by Aeromonas hydrophila.
Assuntos
Aeromonas hydrophila/genética , Proteínas Hemolisinas/metabolismo , Proteínas Recombinantes/imunologia , Toxoides/imunologia , Animais , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Proteínas Hemolisinas/genética , Imunização , Camundongos , Dados de Sequência Molecular , Distribuição Aleatória , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência , Vacinas Sintéticas/imunologiaRESUMO
In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.