Z-DNA, a new in situ marker for transcription.
Z-DNA forms transiently behind the active RNA polymerases, because of the mechanical torsional stress produced during transcription. In this paper, we explore the possibility that the distribution of Z-DNA stretches signals the sites related to nuclear transcription. To localize transcription, the in situ assay for active RNA polymerases, that allows the elongation of the already initiated transcripts but no initiation of new ones (run-on experiments), was carried out in isolated nuclei of Allium cepa L. root meristems. Both nucleolar and non-nucleolar sites appeared labelled. Nucleoli were most active in transcription than the multiple non-nucleolar foci altogether. In situ immunodetection of Z-DNA provided images that were comparable to those obtained after the run-on assay, with one exception: while Z-DNA and transcription sites were scattered throughout the whole nucleus, Z-DNA also accumulated in the nuclear periphery, where no transcription foci were detected in run-on assays. The peripheral Z-conformation signals might correspond to dsRNA segments present in the pre-mRNA in the process of their export to cytoplasm. The Z-containing structures nearly disappeared when non-nucleolar RNA polymerase II-dependent transcription had been previously abolished by the adenosine analogue DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). This inhibition selectively decreased the amount of all nucleoplasmic Z structures. On the other hand, the inhibition of the nucleolar RNA polymerase I by cordycepin (3'-deoxyadenosine) prevented the presence of Z-DNA in nucleoli. We propose to use the in situ immunodetection of Z-DNA as a marker of the transcription level in both nucleolus and non-peripheral nucleoplasmic regions of nuclei. Co-detection of Z-DNA and of intermediate filament (IF) proteins, the major components of the nuclear matrix, was also carried out. The IFA antibody recognizes a conserved epitope essential for dimerization of the multiple IF proteins. They co-localized with most nucleolar Z-DNA, but not with the nucleoplasmic ones. In the nuclear periphery, the Z-positive signals were adjacent to the IF proteins constituting the lamina, though both signals did not often co-localize.
Cerná
A
A
Institute of Experimental Botany, ASCR, Prague, Czech Republic.
Cuadrado
A
A
Jouve
N
N
Díaz de la Espina
S M
SM
De la Torre
C
C
D000490:Allium / Q000235:genetics; D015415:Biomarkers; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D043542:DNA, Z-Form / Q000187:drug effects / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D003839:Deoxyadenosines / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
D015415:Biomarkers; D043542:DNA, Z-Form; D003839:Deoxyadenosines; D004004:Dichlororibofuranosylbenzimidazole; D012321:DNA-Directed RNA Polymerases; C058120:cordycepin