Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes.
Incorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and thus must be catalysed by RNA polymerase I. Immunofluorescence and confocal microscopy shows that incorporation occurs in a pattern consisting of many small foci distributed widely through the dense fibrillar component of the nucleoli. Immunogold labelling using silver-enhanced Nanogold probe at the electron microscopic level confirms the sites of transcription as small foci approximately 200 nm in diameter. Simultaneous fluorescence in situ hybridization with a probe to the external transcribed spacer (ETS) region of the pre-rRNA shows that the structures revealed by this probe and the BrUTP immunofluorescence labelling are very similar. A probe to the transcribed portion of the rDNA (18S) also shows a good correlation to the sites of BrUTP incorporation within the nucleolus. On the other hand a probe to the non-transcribed intergenic spacer region (NTS) shows very little coincidence with the sites of BrUTP incorporation, and double fluorescence in situ labelling with both 18S and NTS probes confirms this difference in localization. These results suggest that most BrUTP foci correspond to single transcribed genes.
Thompson
W F
WF
Department of Cell Biology, John Innes Centre, Colney, Norwich, UK.
Beven
A F
AF
Wells
B
B
Shaw
P J
PJ
D002466:Cell Nucleolus / Q000378:metabolism; D004275:DNA, Ribosomal / Q000235:genetics*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017343:Genes, Plant; D017404:In Situ Hybridization, Fluorescence; D018532:Pisum sativum / Q000166:cytology / Q000235:genetics*; D018517:Plant Roots / Q000378:metabolism; D014158:Transcription, Genetic
D004275:DNA, Ribosomal; D003856:Deoxyuracil Nucleotides; C032549:bromodeoxyuridine triphosphate