Virtanen A, Sharp P A
Center for Cancer Research, Massachusetts Insitute of Technology, Cambridge 02139.
EMBO J. 1988 May;7(5):1421-9. doi: 10.1002/j.1460-2075.1988.tb02959.x.
We have developed an in vitro system for polyadenylation of RNA substrates in cell-free nuclear extracts prepared from murine cells of lymphoid origin. RNA substrates containing the adenovirus L3, murine immunoglobulin (IgM) secreted and membrane polyadenylation sites were accurately polyadenylated in these extracts. Kinetic analysis showed that the rate of polyadenylation in vitro responds proportionally to the substrate concentration. Quantitation of the initial rate of polyadenylation at the three sites permitted comparison of the activities of extracts prepared from HeLa cells, B cells (Wehi 231) and plasmacytoma cells (P9.37.11). From this analysis, we concluded that in all three extracts the polyadenylation activity at the L3 site was higher than that of either of the IgM sites. In contrast to the preferential utilization of the secreted site in vivo in plasmacytomas, this site was not selectively processed in plasmacytoma as compared to B cell extracts. The efficiency of polyadenylation at both IgM sites in the plasmacytoma extract was significantly lower than that in the B cell extract. The common low activity at the IgM sites in the plasmacytoma cell extract suggests that the rate-limiting step for polyadenylation at these two sites differs from that at the L3 site.
我们已经开发出一种体外系统，用于在从淋巴样来源的鼠细胞制备的无细胞核提取物中对RNA底物进行多聚腺苷酸化。含有腺病毒L3、鼠免疫球蛋白（IgM）分泌型和膜型多聚腺苷酸化位点的RNA底物在这些提取物中被准确地多聚腺苷酸化。动力学分析表明，体外多聚腺苷酸化的速率与底物浓度成比例响应。对这三个位点多聚腺苷酸化初始速率的定量，使得能够比较从HeLa细胞、B细胞（Wehi 231）和浆细胞瘤细胞（P9.37.11）制备的提取物的活性。通过该分析，我们得出结论，在所有三种提取物中，L3位点的多聚腺苷酸化活性均高于任一IgM位点。与浆细胞瘤体内对分泌型位点的优先利用相反，与B细胞提取物相比，该位点在浆细胞瘤中并未被选择性加工。浆细胞瘤提取物中两个IgM位点的多聚腺苷酸化效率均显著低于B细胞提取物。浆细胞瘤细胞提取物中IgM位点普遍的低活性表明，这两个位点多聚腺苷酸化的限速步骤与L3位点不同。
