RESUMO
The intranuclear position of a set of genes was analyzed with respect to the territories occupied by the whole chromosomes in which these genes are localized. Genes and their respective chromosome territories were simultaneously visualized in three-dimensionally preserved nuclei applying dual color fluorescence in situ hybridization. Three coding (DMD, MYH7, and HBB) and two noncoding sequences (D1Z2 and an anonymous sequence) were analyzed in four different cell types, including cells where DMD and MYH7 are actively transcribed. Spatial analysis by confocal laser scanning microscopy revealed that the genes are preferentially located in the periphery of chromosome territories. This positioning was independent from the activity of the genes. In contrast, the non-expressed anonymous fragment was found randomly distributed or localized preferentially in the interior of the corresponding chromosome territory. Furthermore, the distribution of the analyzed genes within the territorial peripheries was found to be highly characteristic for each gene, and, again, independent from its expression. The impact of these findings with regard to the three-dimensional arrangement of the linear DNA string within chromosome territories, as well as with respect to a putative nuclear subcompartment confining gene expression, are discussed.
Assuntos
Cromossomos Humanos/genética , Genes , Células Cultivadas , Cromossomos Humanos/ultraestrutura , Distrofina/genética , Fluoresceína-5-Isotiocianato , Expressão Gênica , Globinas/genética , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Microscopia Confocal , Cadeias Pesadas de Miosina/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
RNA polymerase II transcripts accumulate within mammalian nuclei at distinct sites and exhibit varying morphology. Certain RNA species are organized in elongated structures, whereas others appear as dot-like concentrations. To analyze the status of the RNA within these accumulations, we investigated the composition of accumulations derived from Epstein-Barr virus (EBV) genes, human papilloma virus 18 (HPV18) open reading frames E6 and E7, as well as heat shock protein 89a (hsp89alpha) and 89beta (hsp89beta) genes. No differential distribution of exon and intron sequences within concentrations of EBV RNA could be observed. Whereas accumulations of hsp89alpha and hsp89beta always coincided with Sm antigen foci, the RNA of EBV and HPV18 never co-localized with these foci. This excludes Sm antigen foci as the only sites of splicing and suggests gene-specific variation in the nuclear localization of transcripts. Two sets of experiments were performed to assess whether transcripts in the RNA accumulations are in statu nascendi or products released from a discrete gene locus. Because RNA transcripts derived from EBV genes, which are located on both ends of the genome, were all distributed along the entire length of the RNA signals, they cannot be derived from a highly decondensed genomic DNA extending throughout elongated RNA accumulations. Furthermore, removal of labeled RNA sequences and subsequent visualization of DNA confirmed the confinement of the genomic sequences to a small subregion of the area occupied by accumulated RNA. Therefore, this study supports the view of RNA accumulations as a stream of molecules that delineate a path from a dot-like gene locus toward the nuclear envelope for export into the cytoplasm.
Assuntos
Autoantígenos/análise , Núcleo Celular/química , Proteínas de Ligação a DNA , RNA Mensageiro/análise , RNA Nuclear/análise , Ribonucleoproteínas Nucleares Pequenas , Linfoma de Burkitt , Fracionamento Celular , Citoplasma/química , DNA Viral/análise , Éxons/genética , Genes Virais/genética , Células HeLa , Proteínas de Choque Térmico/genética , Herpesvirus Humano 4/genética , Humanos , Íntrons/genética , Membrana Nuclear/química , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Precursores de RNA/análise , Splicing de RNA , RNA Viral/análise , Transcrição Gênica , Células Tumorais Cultivadas , Proteínas Centrais de snRNPRESUMO
The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescence in situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs.