Wu G J, Zubay G
Proc Natl Acad Sci U S A. 1974 May;71(5):1803-7. doi: 10.1073/pnas.71.5.1803.
A cell-free system for nuclear-directed transcription has been developed that gives prolonged synthesis in the presence of cytoplasm. The nuclear and cytoplasmic components have been prepared from Krebs II ascites tumor cells for most experiments but further observations indicate that components prepared from other cell types may be used. After an initial 5- to 10-min period of relatively rapid RNA synthesis a linear rate ensues for 2-3 hr. In the absence of cytoplasm no net RNA synthesis occurs after the initial 10-min period. Experiments with alpha-amanitin suggest that about half of the cell-free synthesized RNA is made by RNA polymerase II, the enzyme believed to be responsible for messenger synthesis in vivo.The conditions used for RNA synthesis were derived from conditions found to be optimal for protein synthesis that proceeds linearly for 2-3 hr. It has not yet been possible to demonstrate the synthesis of protein from cell-free synthesized RNA in this system. A major problem here is that isolated nuclei, even when carefully washed, contain a great deal of translatable RNA.
已开发出一种用于核定向转录的无细胞系统，该系统在有细胞质存在的情况下能进行长时间合成。大多数实验中，核和细胞质成分是从克雷布斯II腹水瘤细胞制备的，但进一步观察表明，也可使用从其他细胞类型制备的成分。在最初5至10分钟相对快速的RNA合成期之后，会有2至3小时的线性合成速率。在没有细胞质的情况下，最初10分钟后不会发生净RNA合成。用α-鹅膏蕈碱进行的实验表明，无细胞合成的RNA中约一半是由RNA聚合酶II合成的，该酶被认为在体内负责信使RNA的合成。用于RNA合成的条件源自被发现对能进行2至3小时线性合成的蛋白质合成而言最优的条件。在该系统中尚未能够证明从无细胞合成的RNA合成蛋白质。这里的一个主要问题是，即使经过仔细洗涤，分离的细胞核仍含有大量可翻译的RNA。
