RESUMO
A specific form of gene silencing that was observed visually as a mosaic distribution of fluorescent and non-fluorescent cells apparently dispersed at random within tissues was found in a few green fluorescent protein (GFP)-transformed tobacco lines. To characterize this event quantitatively, we studied flow cytometric measurements in GFP-expressing and -silenced cells in T1 and T2 progeny of four selected plants. The proportion of silenced cells varied considerably among the T1 lines but with notable genotype differences. Mosaic expression was inherited into the T2 generation in which the majority of progenies tested exhibited a level of silencing similar to that of their T1 parental plants. However, in some T2 progenies segregation, evident as a decrease or increase in the proportion of fluorescent cells, was observed. We discuss several factors, such as copy number, promoter activity or polyploidy, that may be the possible causes of the gene silencing, but none sufficiently explain the appearance of the mosaic distribution.
Assuntos
Proteínas Luminescentes/genética , Mosaicismo/genética , Nicotiana/genética , Sequência de Bases , Primers do DNA , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Nicotiana/citologia , Nicotiana/metabolismoRESUMO
For precision of bacteriological diagnosis of Listeria monocytogenes (L. m.) the immune fluorescent technique (IFT) was assessed. On using the described immunization pattern high titres of specific antibodies were obtained from the immunized rabbits. The obtained hyperimmune sera yielded very good results when used in IFT, and they were suitable for conjugate production. By means of IFT L.M. was demonstrated from cultures, organ replicas and tissue sections of experimentally infected rabbits. The most brilliant fluorescence was achieved with conjugate of the corresponding serotype, the polytype conjugate yielded demonstrable fluorescence with all serotypes. Listerias showed intensive fluorescence, and in contrast staining of replicas and tissue sections with Evans blue they could be well differentiated from the background.