RerRNA transcripts are detected throughout the nucleolus by RNA-FISH (fluorescent in
RerRNA transcripts are detected throughout the nucleolus by RNA-FISH (fluorescent in situ hybridization) (Fig. 1C, bottom row). Nevertheless, essentially the most prominent 45S rRNA gene DNA-FISH signals are external for the nucleolus (Fig. 1B [red signals], C [top row, green signals]; note that NOR associations can lead to fewer than four signals). rRNA genes inside the nucleolus are decondensed and more hard to detect by DNA-FISH, according to the extent of their dispersal (e.g., cf. the two nuclei in Fig. 1B). HISTONE DEACETYLASE six (HDA6) is required for uniparental rRNA gene silencing in hybrid Arabidopsis suecica and for developmentally regulated silencing of variant 1 rRNA genes in nonhybrid A. thaliana (Earley et al. 2006, 2010). HDA6 localizes throughout nuclei, including the nucleolus (Fig. 1C). In hda6 mutants, NORs decondense (Probst et al. 2004; Earley et al. 2006), and rRNA gene FISH signals inside the nucleolus increase (Fig. 1D). In leaves of wild-type plants (ecotype Col-0), variant two and three rRNA gene Akt2 review subtypes are expressed,In eukaryotes, 45S ribosomal RNA (rRNA) genes are tandemly arrayed at nucleolus organizer regions (NORs) (see[Keywords: transcription; gene silencing; DNA methylation; histone deacetylation; chromatin assembly; RNA polymerase I; ribosomal RNA gene] 8 These authors contributed equally to this work. Present addresses: 9Laboratoire Genome et Developpement des Plantes, UMR 5096 CNRS-University of Perpignan by means of Domitia, Perpignan, France; 10 Division of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Linnean Center for Plant Biology, Uppsala, Sweden; 11 Monsanto Company, St. Louis, MO 63107, USA 12 Corresponding authors E-mail [email protected] E-mail [email protected] Report is on the web at genesdev.org/cgi/doi/10.1101/gad.221648.113. Freely readily available on the internet by way of the Genes Improvement Open Access solution.GENES Improvement 27:1545550 2013 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/13; genesdev.orgpontvianne et al.Figure 1. Partitioning of active and silent rRNA genes between the nucleolus and nucleoplasm. (A) Relationships involving the nucleolus, NORs, and 45S rRNA gene repeats. The drawing at the major depicts a metaphase chromosome with all the remnants of a nucleolus associated with the secondary constriction, the place of active rRNA genes in the preceding interphase. The black oval represents a chromomere in which rRNA genes are assembled into dense heterochromatin. In a. thaliana, insertions/deletions inside the 39 external transcribed region define rRNA gene variant kinds. (B) Localization of rRNA genes (rDNA) and Pol I. DNA-FISH using an rRNA gene probe (red signals) and immunolocalization of Flag-tagged Pol I using an anti-Flag antibody (green signals) have been performed inside a. thaliana mAChR1 medchemexpress interphase nuclei. DNA was counterstained with DAPI (gray signals). (C) Subnuclear localization of rRNA genes, pre-rRNA transcripts, and Flag-tagged HDA6. rRNA gene or transcript FISH signals are shown in green, immunolocalized HDA6 is in red, and DAPI-stained DNA is in blue. Merged signals are shown in the appropriate column. (D) DNA-FISH detection of rRNA genes in wild-type (Col-0) and hda6 nuclei. rRNA gene FISH signals are shown in red and are merged with all the DAPI (blue) image in the appropriate column. (E) Detection of rRNA gene variant forms and their transcripts by PCR working with genomic DNA or reversetranscribed (RT) cDNA of wild-type (Col-0) or hda6 plants. The amplified area is.
