RESUMO
Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/µg of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.
Assuntos
DNA Fúngico/genética , Genoma Fúngico , Proteínas Luminescentes/genética , Mutação , Penicillium/genética , Transformação Genética/genética , Proteínas Luminescentes/análise , Microscopia de Fluorescência , Penicillium/enzimologia , Plasmídeos/genética , Poligalacturonase/genética , Protoplastos/enzimologiaRESUMO
The yeast two-hybrid system is a powerful tool for screening protein-protein interactions and has also been used for large-scale studies. We evaluated two protein-coding sequences as reporter genes for the yeast two-hybrid system, to determine if it was suitable as an alternative screening strategy. Aspergillus awamori glucoamylase activity results in clear haloes around colonies producing this enzyme after growth on starch plates and staining with iodine vapors. However, transcription activation by Gal4 on Gal-regulated promoters was insufficient for this type of phenotypic visualization. A modified gene of Aequoria victoria enhanced green fluorescent protein (EGFP) was tested to determine its suitability for interaction screenings with flow cytometry. When the EGFP reporter gene system was incorporated into the cells, Gal4 transcriptional activation produced sufficient fluorescence for detection with the flow cytometer, especially when there were strong interactions
Assuntos
Genes Reporter , Leveduras/genética , Técnicas do Sistema de Duplo-Híbrido , Sequência de Bases , Clonagem Molecular , Citometria de Fluxo , Dados de Sequência Molecular , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Alinhamento de SequênciaRESUMO
The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.
Assuntos
Animais , Feminino , Camundongos , Adenovírus Humanos/isolamento & purificação , Barreira Hematoencefálica , Encéfalo/virologia , Cerebelo/citologia , Cerebelo/virologia , Estudo Comparativo , Genes Reporter , Vetores Genéticos/administração & dosagem , Vetores Genéticos/isolamento & purificação , Vetores Genéticos/farmacocinética , Hipocampo/virologia , Colículos Inferiores/virologia , Injeções Intravenosas , Proteínas Luminescentes/análise , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos Endogâmicos BALB C , Neuroglia/virologia , Neurônios/virologia , Células de Purkinje/virologia , Células Piramidais/virologia , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Cauda/irrigação sanguínea , Distribuição TecidualRESUMO
We made reporter HIV-1 DNA constructs carrying green fluorescent protein (GFP) gene and exchangeable env of subtype E. The recombinant constructs were used to produce infectious reporter viruses, which induced infected cells to emit green fluorescent light and rendered them easily detectable at single cell level. Because the env in this construct can be easily exchanged, viruses with different antigenic epitopes can be made. We used these reporter viruses to set up a neutralizing antibody assay based on fluorescence reduction by flow cytometric measurement. The result of this new assay correlated with the standard infectivity reduction assay using primary isolates. Because this new assay is faster and much less costly than the standard assay using a p24 endpoint and can be performed in peripheral blood mononuclear cells (PBMC), it provides a useful tool for analysis of HIV-1 immune responses.
Assuntos
Determinação de Ponto Final/métodos , Imunofluorescência/métodos , Genes Reporter/fisiologia , Genes Virais/fisiologia , Proteínas de Fluorescência Verde , HIV-1/genética , Humanos , Indicadores e Reagentes/análise , Proteínas Luminescentes/análise , Testes de Neutralização/métodos , Sensibilidade e Especificidade , Fatores de Tempo , Latência Viral/imunologiaRESUMO
Con el microscopio de fondo oscuro los eritrocitos de larvas de Telmatobius laticeps, Telmatobius pisanoi, Hyla pulchella andina y Bufo arenarum, presentan cuadros de luminiscencia dque difieren según la especie y el estadio; el fenómeno disminuye en los estadios cercanos a la metamorfosis hasta desaparecer en el adulto. La luminiscencia, visible en granulaciones ubicadas tanto en el núcleo como en el citoplasma, se vincularía con los diferentes tipos de hemoglobina dependientes, a su vez, del sitio eritopoyético