Transcription in Isolated Wheat Nuclei: II. CHARACTERIZATION OF RNA SYNTHESIZED IN VITRO.
Transcription in Isolated Wheat Nuclei: II. CHARACTERIZATION OF RNA SYNTHESIZED IN VITRO.
复制标题
分离的小麦细胞核中的转录：II。
作者：
Dawn S. Luthe;R. Quatrano
通过不连续Percoll梯度离心，从吸收3小时的小麦（Yamhill品种）胚胎中分离出无RNase活性的细胞核。在这些细胞核中观察到的最大RNA合成速率约为每毫克DNA每分钟5皮摩尔[(3)H]UTP。在15毫摩尔MgCl(2)或2毫摩尔MnCl(2)存在下测量时，发现两个单价阳离子最优；15和75毫摩尔（nh4）(2)SO(4)。在高单价阳离子最适条件下，RNA合成速率在孵育前10 ~ 15分钟呈线性，3小时后仍在增加。体外合成的RNA（30分钟脉冲后30分钟追逐）显示出明显的18和26S RNA峰，分别占总放射性的13%和17%。体外合成的RNA总体模式与体内模式相似。大约40 - 50%的RNA合成被α -amanitin抑制，浓度为每毫升4微克。新合成的6 ~ 10S RNA似乎被α - amantin选择性抑制。
Nuclei free of RNase activity were isolated from 3-hour-imbibed wheat (var. Yamhill) embryos by centrifugation through a discontinuous gradient of Percoll. The maximum rate of RNA synthesis observed in these nuclei was approximately 5 picomoles [(3)H]UTP per milligram DNA per minute. Two monovalent cation optima were found when measured in the presence of 15 millimolar MgCl(2) or 2 millimolar MnCl(2); 15 and 75 millimolar (NH(4))(2)SO(4). At the high monovalent cation optimum, the rate of RNA synthesis was linear for the first 10 to 15 minutes of incubation and still increasing after 3 hours. RNA synthesized in vitro (30-minute pulse followed by a 30-minute chase) showed distinct 18 and 26S RNA peaks comprising 13 and 17% of the total radioactivity, respectively. The over-all pattern of RNA synthesized in vitro was similar to the in vivo pattern. Approximately 40 to 50% of the RNA synthesized was inhibited by alpha-amanitin at 4 micrograms per milliliter. The newly synthesized 6 to 10S RNA appeared to be selectively inhibited by alpha-amanitin.