Jackson D A, Hassan A B, Errington R J, Cook P R
Sir William Dunn School of Pathology, University of Oxford, UK.
EMBO J. 1993 Mar;12(3):1059-65. doi: 10.1002/j.1460-2075.1993.tb05747.x.
HeLa cells were encapsulated in agarose microbeads, permeabilized and incubated with Br-UTP in a 'physiological' buffer; then sites of RNA synthesis were immunolabelled using an antibody that reacts with Br-RNA. After extending nascent RNA chains by < 400 nucleotides in vitro, approximately 300-500 focal synthetic sites can be seen in each nucleus by fluorescence microscopy. Most foci also contain a component of the splicing apparatus detected by an anti-Sm antibody. alpha-amanitin, an inhibitor of RNA polymerase II, prevents incorporation into these foci; then, using a slightly higher salt concentration, approximately 25 nucleolar foci became clearly visible. Both nucleolar and extra-nucleolar foci remain after nucleolytic removal of approximately 90% chromatin. An underlying structure probably organizes groups of transcription units into 'factories' where transcripts are both synthesized and processed.
将HeLa细胞包封在琼脂糖微珠中，使其通透化，并在“生理”缓冲液中与溴尿苷三磷酸（Br-UTP）一起孵育；然后使用与溴化核糖核酸（Br-RNA）反应的抗体对RNA合成位点进行免疫标记。在体外将新生RNA链延伸少于400个核苷酸后，通过荧光显微镜在每个细胞核中可看到大约300 - 500个局灶性合成位点。大多数位点还含有通过抗Sm抗体检测到的剪接装置成分。α-鹅膏蕈碱是RNA聚合酶II的抑制剂，可阻止其掺入这些位点；然后，使用稍高的盐浓度，大约25个核仁位点变得清晰可见。在通过核酸酶去除约90%的染色质后，核仁位点和核仁外位点仍然存在。一种潜在的结构可能将转录单元组组织成“工厂”，在那里转录本既能合成又能加工。
