RESUMO
During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Meiose/fisiologia , Complexo Sinaptonêmico/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Cromátides/ultraestrutura , Pareamento Cromossômico/fisiologia , DNA/química , DNA/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Complexo Sinaptonêmico/fisiologia , Complexo Sinaptonêmico/ultraestruturaRESUMO
More than 50% of cases of primary ovarian insufficiency (POI) and nonobstructive azoospermia in humans are classified as idiopathic infertility. Meiotic defects may relate to at least some of these cases. Mutations in genes coding for synaptonemal complex (SC) components have been identified in humans, and hypothesized to be causative for the observed infertile phenotype. Mutation SYCE1 c.721C>T (former c.613C>T)-a familial mutation reported in two sisters with primary amenorrhea-was the first such mutation found in an SC central element component-coding gene. Most fundamental mammalian oogenesis events occur during the embryonic phase, and eventual defects are identified many years later, thus leaving few possibilities to study the condition's etiology and pathogenesis. Aiming to validate an approach to circumvent this difficulty, we have used the CRISPR/Cas9 technology to generate a mouse model with an SYCE1 c.721C>T equivalent genome alteration. We hereby present the characterization of the homozygous mutant mice phenotype, compared to their wild type and heterozygous littermates. Our results strongly support a causative role of this mutation for the POI phenotype in human patients, and the mechanisms involved would relate to defects in homologous chromosome synapsis. No SYCE1 protein was detected in homozygous mutants and Syce1 transcript level was highly diminished, suggesting transcript degradation as the basis of the infertility mechanism. This is the first report on the generation of a humanized mouse model line for the study of an infertility-related human mutation in an SC component-coding gene, thus representing a proof of principle.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Mutação Puntual/genética , Insuficiência Ovariana Primária/genética , Animais , Pareamento Cromossômico/genética , Pareamento Cromossômico/fisiologia , Proteínas de Ligação a DNA/genética , Feminino , Citometria de Fluxo , Predisposição Genética para Doença/genética , Homozigoto , Humanos , Imuno-Histoquímica , Meiose/genética , Meiose/fisiologia , Camundongos , Mutação/genéticaRESUMO
The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.
Assuntos
Proteínas de Membrana Transportadoras/genética , Transportadores de Ácidos Monocarboxílicos/genética , Neuroglia/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Linhagem Celular , Sobrevivência Celular , Pareamento Cromossômico , Drosophila , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios/metabolismoRESUMO
The XX/XY system is the rule among mammals. However, many exceptions from this general pattern have been discovered since the last decades. One of these non-conventional sex chromosome mechanisms is the multiple sex chromosome system, which is evolutionary fixed among many bat species of the family Phyllostomidae, and has arisen by a translocation between one original gonosome (X or Y chromosome), and an autosome, giving rise to a "neo-XY body." The aim of this work is to study the synaptic behavior and the chromatin remodeling of multiple sex chromosomes in different species of phyllostomid bats using electron microscopy and molecular markers. Testicular tissues from adult males of the species Artibeus lituratus, Artibeus planirostris, Uroderma bilobatum, and Vampyrodes caraccioli from the eastern Amazonia were analyzed by optical/electron microscopy and immunofluorescence of meiotic proteins involved in synapsis (SYCP3 and SYCE3), sister-chromatid cohesion (SMC3), and chromatin silencing (BRCA1, γ-H2AX, and RNApol 2). The presence of asynaptic axes-labeled by BRCA1 and γ-H2AX-at meiotic prophase in testes that have a normal development of spermatogenesis, suggests that the basic mechanism that arrests spreading of transcriptional silencing (meiotic sex chromosome inactivation (MSCI)) to the autosomal segments may be per se the formation of a functional synaptonemal complex between homologous or non-homologous regions, and thus, this SC barrier might be probably related to the preservation of fertility in these systems.
Assuntos
Quirópteros/genética , Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Processos de Determinação Sexual/genética , Cromossomo X/genética , Cromossomo Y/genética , Animais , Pareamento Cromossômico/genética , Masculino , Estágio Paquíteno/fisiologia , Espermatócitos/metabolismo , Espermatogênese/fisiologiaRESUMO
In the zebra finch, 2 alternative morphs regarding centromere position were described for chromosome 6. This polymorphism was interpreted to be the result of a pericentric inversion, but other causes of the centromere repositioning were not ruled out. We used immunofluorescence localization to examine the distribution of MLH1 foci on synaptonemal complexes to test the prediction that pericentric inversions cause synaptic irregularities and/or crossover suppression in heterozygotes. We found complete suppression of crossing over in the region involved in the rearrangement in male and female heterozygotes. In contrast, the same region showed high levels of crossing over in homozygotes for the acrocentric form of this chromosome. No inversion loops or synaptic irregularities were detected along bivalent 6 in heterozygotes suggesting that heterologous pairing is achieved during zygotene or early pachytene. Altogether these findings strongly indicate that the polymorphic chromosome 6 originated by a pericentric inversion. Since inversions are common rearrangements in karyotypic evolution in birds, it seems likely that early heterologous pairing could help to fix these rearrangements, preventing crossing overs in heterozygotes and their deleterious effects on fertility.
Assuntos
Inversão Cromossômica , Pareamento Cromossômico/genética , Cromossomos/genética , Troca Genética/genética , Tentilhões/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Aviárias/genética , Centrômero/genética , Mapeamento Cromossômico , Feminino , Heterozigoto , Homozigoto , Masculino , Meiose , Proteínas Nucleares/genética , Complexo Sinaptonêmico/genéticaRESUMO
Background Currently, the technology called Clearfield® is used in the development of crops resistant to herbicides that inhibit the enzyme acetohydroxy acid synthase (AHAS, EC 2.2.1.6). AHAS is the first enzyme of the biosynthetic pathway that produces the branched-chain of the essential amino acids valine, leucine, and isoleucine. Therefore, multiple copies of the AHAS gene might be of interest for breeding programs targeting herbicide resistance. In this work, the characterization of the AHAS gene was accomplished for the Chenopodium quinoa Regalona-Baer cultivar. Cloning, sequencing, and Southern blotting were conducted to determine the number of gene copies. Results The presence of multiple copies of the AHAS gene as has been shown previously in several other species is described. Six copies of the AHAS gene were confirmed with Southern blot analyses. CqHAS1 and CqAHAS2 variants showed the highest homology with AHAS mRNA sequences found in the NR Database. A third copy, CqAHAS3, shared similar fragments with both CqAHAS1 and CqAHAS2, suggesting duplication through homeologous chromosomes pairing. Conclusions The presence of multiple copies of the gene AHAS shows that gene duplication is a common feature in polyploid species during evolution. In addition, to our knowledge, this is the first report of the interaction of sub-genomes in quinoa.
Assuntos
Acetolactato Sintase/genética , Duplicação Gênica , Chenopodium quinoa/enzimologia , Chenopodium quinoa/genética , Pareamento Cromossômico , Resistência a HerbicidasRESUMO
Artificial Neural Network (ANN) design is a complex task because its performance depends on the architecture, the selected transfer function, and the learning algorithm used to train the set of synaptic weights. In this paper we present a methodology that automatically designs an ANN using particle swarm optimization algorithms such as Basic Particle Swarm Optimization (PSO), Second Generation of Particle Swarm Optimization (SGPSO), and a New Model of PSO called NMPSO. The aim of these algorithms is to evolve, at the same time, the three principal components of an ANN: the set of synaptic weights, the connections or architecture, and the transfer functions for each neuron. Eight different fitness functions were proposed to evaluate the fitness of each solution and find the best design. These functions are based on the mean square error (MSE) and the classification error (CER) and implement a strategy to avoid overtraining and to reduce the number of connections in the ANN. In addition, the ANN designed with the proposed methodology is compared with those designed manually using the well-known Back-Propagation and Levenberg-Marquardt Learning Algorithms. Finally, the accuracy of the method is tested with different nonlinear pattern classification problems.
Assuntos
Algoritmos , Redes Neurais de Computação , Reconhecimento Automatizado de Padrão/métodos , Pareamento Cromossômico/fisiologia , Neurônios/fisiologiaRESUMO
φC31 integrase encoded by Streptomyces phage can mediate site-specific recombination between phage and host genomes. The recombination direction is generally considered to be accurately regulated, but the regulatory mechanisms involved are still unclear. Recently, some hyperactive mutants of φC31 integrase that can bypass the regulatory steps have been isolated and extensively studied. A putative coiled-coil region is found to play a critical role in controlling recombination direction. Further analysis led us to the speculation that at least two regions in the N-terminal domain of φC31 integrase are involved in the tetrameric interfaces and that the putative coiled coil interacts with one of the regions to regulate the recombination direction.
Assuntos
Bacteriófagos/enzimologia , Integrases/metabolismo , Recombinação Genética , Sequência de Aminoácidos , Pareamento Cromossômico , Integrases/química , Modelos Genéticos , Dados de Sequência Molecular , Mutação/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Cytological investigation revealed complete asynapsis during microsporogenesis in 2 wild accessions of Paspalum jesuiticum collected in distinct Brazilian regions. Both accessions were hexaploid (2n = 6x = 60) and 60 univalents could be counted at diakinesis. In this phase, the majority of meiocytes exhibited univalents with both chromatids. After alignment at the metaphase plate, the chromatids segregated to the poles. Only 1 meiotic division (equational) occurred, and after cytokinesis, a dyad with 2n microspores was formed. The genetic constitution of the 2n gametes was equivalent to that of first division restitution (FDR). Since recombination did not occur, 100% transmission of parental heterozygosity could be expected from the FDR 2n gametes. The meiotic behavior of both accessions suggested that they resulted from a recent natural hybridization event. The potential use of the 2n gametes in Paspalum breeding programs has been discussed.
Assuntos
Pareamento Cromossômico , Diploide , Paspalum/genética , Pólen/genética , Cromátides/genética , Citocinese , Gametogênese Vegetal , Paspalum/citologia , Paspalum/fisiologia , Pólen/citologia , Pólen/fisiologia , Recombinação GenéticaRESUMO
The Xy(p) sex determination mechanism is the system most frequent and ancestral to Coleoptera. Moreover, the presence of argyrophilous material associated with the sex bivalent is described as being responsible for the maintenance and association of these chromosomes. There are no karyotype data available regarding the genus Lagria and no consensus in the literature regarding the argyrophilous material present in the lumen of sex bivalent. Therefore, the aim of this work was to investigate the mechanism of sex chromosome bivalent association in Lagria villosa by analyzing the argyrophilous nature of the material present in the Xy(p) lumen. It was also intended to characterize L. villosa cytogenetically. The analysis of meiotic cells showed 2n = 18 = 16+Xy(p) for males and 2n = 18 = 16+XX in females and the meiotic formula was 2n = 8(II)+Xy(p). The C-banding showed blocks of pericentromeric heterochromatin in all chromosomes except in the y(p) chromosome. In these regions, the use of fluorochromes revealed the presence of heterochromatin containing GC rich DNA sequences. The study of synaptonemal complex showed a gradual increase in the electron-density of the axial elements of the sex chromosomes and their association with strongly electron-dense material. The pepsin pretreatment revealed that the material impregnated by silver is protein.