[Localization with BrUTP labeling technique of RNA polymerase II transcription in meristematic cells of Allium cepa].
The technique of pulse-labeling of cells with 5-bromouridine-5'-triphosphate (BrUTP) and immunological detection of the incorporated BrUTP is a newly-developed experimental approach to localize nascent RNA polymerase II transcripts in the nucleus. This technique has been widely used since it was first reported in 1993, but most of the studies were carried out with fluorescence microscopy and animal cells, we are still limited in the knowledge about ultrastructural localization of RP II transcripts in the nucleus of eukaryotes, especially in that of plant cells. Attempts have been made in this paper to localize nascent RP II transcripts in the nucleus of meristematic cells of Allium cepa with the technique of pulse-labeling of BrUTP and immunoelectron microscopy. After labeling with BrUTP and detection with anti-BrdU antibody, a large number of gold particles representing RP II transcripts were observed in the periphery of chromatin domains, the gold particles in the periphery accounted for 92.70% of the total gold particles in the extranucleolar regions, and the gold density of the periphery was 75.42/micron 2, much higher than that of the center of the chromatin domains and the interchromatin domains which were only 5.89/micron 2 and 2.00/micron 2 respectively, indicating that RP II transcripts were being actively synthesized in the periphery of the chromatin domains. When the specimens were treated with alpha-amanitin which can specifically inhibits the activity of RP II, gold particles in the extranucleolar regions of the specimens were decreased dramatically and the gold density of the regions dropped strikingly from 44.60/micron 2 to 2.67/micron 2, confirming that the gold particles in the regions represent RP II transcripts. The distribution of nascent RP II transcripts in the nucleus of the intact plant cells is discussed.
Hu
B
B
Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024.
Xing
M
M
D000490:Allium / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D012319:RNA Polymerase II / Q000378:metabolism*; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D002843:Chromatin; C018009:5-bromouridine triphosphate; D012319:RNA Polymerase II; D014544:Uridine Triphosphate