RESUMO
The fate of future generations depends on a high-quality germ line. For a female to successfully produce offspring, her oocytes must be successfully specified and their contents meticulously organized. Germ cells are specified by two general mechanisms: inductive and inherited. In the inductive mechanism, the primordial germ cells (PGCs) are induced by signals from the surrounding cells. In the inherited mechanism, PGCs are specified by passing localized germ plasm material from the oocyte to the future germ cells. The Balbiani body, a conserved oocyte aggregate, facilitates the organization of the oocyte into a polarized cell with discrete cytoplasmic domains, including localizing the germ plasm. In the mouse, the Balbiani body is implicated in oocyte survival, while in frogs and zebrafish the Balbiani body carries specific mRNAs to the vegetal pole. These asymmetric mRNAs form the foundation of the functionally polarized oocyte and play important roles in axial patterning and germ plasm formation of the embryo.
Assuntos
Polaridade Celular/genética , Puffs Cromossômicos/genética , Oócitos/citologia , Vertebrados/genética , Animais , Humanos , Padrões de Herança/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.
Assuntos
Chironomidae/citologia , Chironomidae/genética , Puffs Cromossômicos/metabolismo , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Puffs Cromossômicos/química , Puffs Cromossômicos/genética , Genes de Insetos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/química , Ribonucleoproteínas/genéticaRESUMO
The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate the molecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification.
Assuntos
Puffs Cromossômicos/genética , Dípteros/genética , Receptores de Esteroides/metabolismo , Animais , Sítios de Ligação/genética , Replicação do DNA , Dípteros/classificação , Ecdisona/administração & dosagem , Loci Gênicos , Larva/genética , Técnicas de Amplificação de Ácido Nucleico , Receptores de Esteroides/genética , Glândulas Salivares/metabolismoRESUMO
We have developed a new molecular beacon design that requires an additional UV pulse for fluorescence activation. This improves the signal-to-noise ratio tremendously compared to previous approaches and allows for a precise control of the time point and location of RNA labelling.
Assuntos
Corantes Fluorescentes/química , RNA Mensageiro/análise , Animais , Chironomidae , Puffs Cromossômicos/genética , Fluorescência , Microscopia Confocal , Glândulas Salivares/citologia , Raios UltravioletaRESUMO
The eco- and genotoxicity of silver nanoparticles (AgNPs) was investigated in the fourth instar larvae of the aquatic midge, Chironomus riparius. AgNPs did not have acute toxicity in C. riparius, but did exhibited chronic toxicity on development (pupation and emergence failure) and reproduction. Genotoxicity also occurred in AgNPs exposed C. riparius. Differential Display PCR (DD-PCR), based on the Annealing Control Primer (ACP) technique, was conducted to investigate the underlying toxic mechanism, which identified altered gene expression in C. riparius after treatment with AgNPs. The possible toxicity mechanism of AgNPs in C. riparius involves the down regulation of the ribosomal protein gene (CrL15) affecting the ribosomal assembly and consequently, protein synthesis. Up regulation of the gonadotrophin releasing hormone gene (CrGnRH1) might lead to the activation of gonadotrophin releasing hormone mediated signal transduction pathways and reproductive failure. Up regulation of the Balbiani ring protein gene (CrBR2.2) may be an indication of the organism's protection mechanism against the AgNPs. The overall results suggest that the toxicity of AgNPs towards aquatic organisms should be thoroughly investigated to allow for their safe use, as they seem to exhibit important toxicity towards C. riparius.
