RESUMO
Transgenic strains of Drosophila melanogaster capable of expressing a cDNA fragment corresponding to open reading frame (ORF) of the gene for the German cockroach densonucleosis virus capsid protein VP2 (ORF VP2) in specific tissues and at a certain stage of development depending on the type of chosen driver strains (GAL-UAS system) were obtained. The ORF VP2 transcription was examined at the imago stage after crossing the obtained transgenic Drosophila with the driver line expressing the inducer protein (GAL4) under control of actin promoter (the ORF VP2 expression is induced in all tissues of the first-generation Drosophila). It was demonstrated that the greater part of transcribed foreign RNA was represented by three spliced variants in which RNA fragments either between nucleotides 137 and 353 or between nucleotides 609 and 1925 were excised; the third spliced variant was represented by RNA lacking both introns. Using the next-generation sequencing (NGS) technique, the proportion of unspliced form relative to spliced variants of the analyzed RNA was assessed. It was shown that the ratio of unspliced form to the identified spliced variants of the analyzed RNA was approximately 1:6. It is suggested that splicing of viral RNA foreign to Drosophila can be a sort of defense mechanism preventing the large-scale production of the capsid protein, potentially hazardous to the host organism.
Assuntos
Blattellidae/virologia , Proteínas do Capsídeo/biossíntese , DNA Complementar/biossíntese , Animais , Animais Geneticamente Modificados , Proteínas do Capsídeo/genética , Densovirus/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Fases de Leitura Aberta/genética , Splicing de RNA/genéticaRESUMO
The intracellular localization of the fusion protein composed of green fluorescent protein (GFP) and one of the capsid proteins (namely VP1) of the German cockroach densovirus BgDV1 was investigated using the HeLa human cell culture. The intracellular localization of GFP was analyzed in a series of control experiments. Histochemical analysis with GFP antibodies showed that the fusion protein is localized exclusively inside the nucleus of cells because of the transitory expression of the corresponding vector constructions, whereas the GFP is located both in the nucleus and the cytoplasm. We can conclude that the signal of the nuclear localization of the capsid protein of the German cockroach densovirus is functionally active, not only within the cells of this insect but within the human cell culture as well. This observation extends the experimental possibilities for studying the genetic control of intracellular traffic of densovirus proteins.
Assuntos
Proteínas do Capsídeo/genética , Densovirus/genética , Transporte Proteico/genética , Proteínas Virais/genética , Animais , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Técnicas In VitroRESUMO
In recent decades, novel microscopic methods commonly referred to as super- resolution microscopy have been developed. These methods enable the visualization of a cell with a resolution of up to 10 nm. The application of these methods is of great interest in studying the structure and function of the cell nucleus. The review describes the main achievements in this field.