Scheer U, Trendelenburg F, Franke W W
J Cell Biol. 1975 Apr;65(1):163-79. doi: 10.1083/jcb.65.1.163.
The effect of actinomycin D(AMD) on the association of the nascent ribonucleo-protein (RNP) fibrils containing the precursors of ribosomal RNA (pre-rRNA) with their template deoxyribonucleoprotein (rDNP) strands has been studied in lampbrush stage oocytes from Triturus alpestris. Ovary pieces were incubated in vitro either in media containing radioactive ribonucleosides and then, for various times, in solutions containing 25 mug/ml AMD, or were directly exposed to the drug. The ultrastructure of the nucleoli and the nuclear periphery was studied by electron microscopy of thin sections and positively stained spread preparations of isolated nuclear contents, and by light and electron microscope autoradiography. The fate of the labeled pre-rRNA was followed by gel electrophoresis of RNA extracted from manually isolated nuclei. Our results show that the growing fibrils which contain the nascent pre-rRNA progressively detach from the DNP strands, the majority being released between 45 and 180 min after application of the drug. The release pattern seems to be random and does not show preference for regions close to the initiator or terminator sites of the transcribed rDNP units. There is a pronounced tendency to removal of groups of adjacent mascent fibrils. The effect of the drug is very heterogeneous. Even after 3 h of treatment with AMD the nucleoli exhibit several individual transcriptional units which appear almost completely covered with lateral fibrils. Autoradiography revealed that most of this released RNP remains within the confinements of the nucleoli which show some foci of aggregation and condensation of fibrillar components but no clear "segregation" phenomenon. In the gel-electrophoretic analysis, a significant but moderate decrease of labeled pre-rRNA was noted only in the first stable pre-rRNA component, whereas pre-rRNA classes of lower molecular weight are very stable under these conditions. The results are discussed in relation to the stability of rDNA transcription complexes and as a basis for an explanation of the ultrastructural changes which are generally observed in nucleoli of AMD-treated cells. It is postulated that inhibition of transcription results in a slow but progressive release of the arrested incomplete RNP fibrils from the template.
在高山螈灯刷期卵母细胞中，研究了放线菌素D（AMD）对含有核糖体RNA前体（前体rRNA）的新生核糖核蛋白（RNP）纤维与其模板脱氧核糖核蛋白（rDNP）链结合的影响。将卵巢组织块在含有放射性核糖核苷的培养基中体外培养，然后在含有25μg/ml AMD的溶液中培养不同时间，或者直接暴露于该药物。通过对薄切片的电子显微镜检查以及对分离的核内容物进行正染色铺展制片，利用光学显微镜和电子显微镜放射自显影技术研究核仁及核周边的超微结构。通过对人工分离细胞核中提取的RNA进行凝胶电泳，追踪标记的前体rRNA的命运。我们的结果表明，含有新生前体rRNA的正在生长的纤维逐渐从DNP链上脱离，大多数在施加药物后45至180分钟之间释放。释放模式似乎是随机的，对靠近转录rDNP单元起始位点或终止位点的区域没有偏好。存在明显的去除相邻新生纤维群的趋势。药物的作用非常不均一。即使在用AMD处理3小时后，核仁仍显示出几个单独的转录单元，这些单元几乎完全被侧向纤维覆盖。放射自显影显示，大部分释放的RNP仍保留在核仁范围内，核仁显示出纤维状成分的一些聚集和浓缩焦点，但没有明显的“分离”现象。在凝胶电泳分析中，仅在第一个稳定的前体rRNA组分中观察到标记的前体rRNA有显著但适度的减少，而在这些条件下，较低分子量的前体rRNA类别非常稳定。结合rDNA转录复合物的稳定性对结果进行了讨论，并作为解释在AMD处理细胞的核仁中普遍观察到的超微结构变化的基础。据推测，转录抑制导致被阻滞的不完全RNP纤维从模板上缓慢但逐渐释放。
