Protocol of electron microscope in situ nucleic acid hybridization for the exclusive detection of double-stranded DNA sequences in cells containing large amounts of homologous single-stranded DNA and RNA sequences: application to adenovirus type 5 infected HeLa cells.
In order to gain a further insight into the relationships of the complex process of replication of adenovirus genomes to the substructures which occur in the nuclei of adenovirus type 5 (Ad5) infected HeLa cells, we have visualized directly, at the electron microscopic level, viral double-stranded DNA (dsDNA) in late infected nuclei by the use of a post-embedding in situ hybridization technique with a biotinylated specific DNA probe. The procedure is based on the removal of single-stranded (ss) nucleic acids by S1 nuclease. The highest levels of signal density for viral dsDNA were detected over the fibrils of the large, centrally located viral genome storage site and over the viral nucleoids of both clustered and isolated viruses. Lower but significant signals were observed over the fibrillo-granular network of the peripheral replicative zones, where both transcription and replication of viral DNA occur. On the other hand, the labeling of the enclosed viral ssDNA accumulation sites, also involved in viral replication but not transcription, was negligible, which suggests that, in the latter, the newly synthesized viral dsDNA immediately extends into the adjacent peripheral replicative zone to be transcribed and/or replicated.
Puvion-Dutilleul
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Laboratoire de Biologie et Ultrastructure du Noyau de l'UPR272 CNRS, Villejuif, France.
D000260:Adenoviruses, Human / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure*; D001710:Biotin; D002478:Cells, Cultured; D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000235:genetics / Q000648:ultrastructure*; D004279:DNA, Viral / Q000235:genetics / Q000648:ultrastructure*; D005814:Genes, Viral / Q000235:genetics; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008854:Microscopy, Electron / Q000379:methods; D012367:RNA, Viral / Q000235:genetics / Q000648:ultrastructure*; D014779:Virus Replication
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral; D001710:Biotin