Shih T Y, Young H A, Parks W P, Scolnick E M
Biochemistry. 1977 May 3;16(9):1795-801. doi: 10.1021/bi00628a005.
The in vitro transcription of viral specific DNA sequences in nuclei and chromatin isolated from mouse cells chronically infected with Moloney murine leukemia virus (Mo-MuLV) has been studied. The in vitro RNA synthesized by Escherichia coli RNA polymerase has been isolated by sulfhydryl affinity column following reaction in the presence of 5-mercuriuridine triphosphate. By comparison of the Crt curves of the in vitro RNA with that of 70S viral RNA, the content of viral sequences is found to be 1.3% in nuclei product and 0.24% in chromatin product which is lower than the 2.5% found in chromatin associated RNA. This latter value, however, is very close to the in vivo viral RNA content in pulse-labeled [3H]RNA of the infected cells. Unexpectedly, it is observed that over 20% of the chromatin associated RNA prelabeled in vivo with [5-3H]uridine is elongated and tagged with Hg atoms during RNA synthesis catalyzed by the exogenous E. coli RNA polymerase in the presence of Hg-UTP. The elongation reaction is dependent on the presence of all four nucleotide triphosphates and appears to be due to E. coli RNA polymerase per se. It is suggested that most of the viral specific sequences observed in the in vitro RNA products are very likely initiated and derived from the chromatin associated species. The implication of the present findings for in vitro RNA synthesis in nuclei and chromatin as related to regulation of gene expression is discussed.
对从长期感染莫洛尼鼠白血病病毒（Mo-MuLV）的小鼠细胞中分离出的细胞核和染色质中病毒特异性DNA序列的体外转录进行了研究。在5-巯基尿苷三磷酸存在下反应后，通过巯基亲和柱分离了由大肠杆菌RNA聚合酶合成的体外RNA。通过比较体外RNA与70S病毒RNA的Crt曲线，发现细胞核产物中病毒序列的含量为1.3%，染色质产物中为0.24%，低于染色质相关RNA中发现的2.5%。然而，后一个值与感染细胞脉冲标记的[3H]RNA中的体内病毒RNA含量非常接近。出乎意料的是，观察到在体内用[5-3H]尿苷预标记的超过20%的染色质相关RNA在Hg-UTP存在下由外源大肠杆菌RNA聚合酶催化的RNA合成过程中被延长并标记上汞原子。延长反应依赖于所有四种三磷酸核苷酸的存在，并且似乎是由于大肠杆菌RNA聚合酶本身。有人提出，在体外RNA产物中观察到的大多数病毒特异性序列很可能起始于并来源于染色质相关物种。讨论了本研究结果对细胞核和染色质中体外RNA合成与基因表达调控的意义。
