REARRANGEMENT OF CONFORMATION OF ESCHERICHIA COLI 55 RNA
REARRANGEMENT OF CONFORMATION OF ESCHERICHIA COLI 55 RNA
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DOI：
10.1073/pnas.61.1.292
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发表时间：
1968-01-01
影响因子：
11.1
通讯作者：
MONIER, R
中科院分区：
文献类型：
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作者：
AUBERT, M;SCOTT, JF;MONIER, R
材料和方法-5S RNA的制备：从E.coliRNase iLO-细胞中提取5S RNA，并按前面所述进行纯化，只需一次修饰。为了在提取时尽可能防止RNA的降解，将冷冻细胞(25克)在2-40℃的TurMix混合器中与25毫升Macalid(American Tansul Co.，Houston，Texas)悬浮液(6.25克加入500毫升0.05M Tris-HCl中，pH 7.6，0.01M氯化镁)混合。加入150ml90%(w/v)的苯酚水溶液和125ml0.01M醋酸钠缓冲液，pH 5.0，0.01M醋酸镁，以最大搅拌速度搅拌2min。C‘4.用同样的方法从含0.4%(w/v)葡萄糖和250mC/L CL-尿嘧啶(47.4mC/mMole，CEA，Saclay)的63型培养基上生长的细胞制备尿嘧啶标记的5S RNA。变性处理：尿素处理：5S RNA按所需浓度溶解于0.25M Tris-HCl缓冲液中，pH 8.9，含7M尿素(新鲜重结晶)和0.01M EDTA。将溶液在230℃保持45分钟，然后在盐-冰混合物中快速冷却。加入十分之一体积的冷的20%(w/v)醋酸钠溶液，调节到pH 5.0，通过加入2.5vol95%的乙醇沉淀RNA，预先在-10℃冷却。热处理：5S RNA在所需的浓度0.15M的氯化钠，0.015的柠檬酸钠，pH 7.0中溶解。将溶液在600℃加热4分钟，然后在29伏的冷蒸馏水中稀释，迅速冷却。用乙醇沉淀法提取RNA。
Materials and Methods.-Preparation of 5S RNA: 5S RNA was extracted from E. coli RNase ILo-cells and purified as previously described,'0 with only one modification. In order to prevent the degradation of RNA as much as possible at the time of extraction, frozen cells (25 gm) were mixed at 2-40C in a Turmix mixer with 25 ml of a Macaloid (American Tansul Co., Houston, Texas) suspension (6.25 gm in 500 ml of 0.05 M tris-HCl, pH 7.6, 0.01 M MgCl2). Then 150 ml of a 90%(w/v) aqueous phenol solution and 125 ml of 0.01 M sodium acetate buffer, pH 5.0, 0.01 M magnesium acetate were added, and the suspension was mixed for 2 min at the maximum mixer speed. C'4. uracil-labeled 5S RNA was prepared by the same procedure from cells grown on type-63 me-dium,'3 supplemented with 0.4%(w/v) glucose and 250 Mc/liter of CL-uracil (47.4 mc/mmole, CEA, Saclay). Denaturation treatments: Urea treatment: 5S RNA was dissolved at the required con-centration in 0.25 M tris-HCl buffer, pH 8.9, containing 7 M urea (freshly recrystallized) and 0.01 M EDTA. The solution was kept at 230C for 45 min and then quickly cooled in a salt-ice mixture. One-tenth volume of a cold 20%(w/v) sodium acetate solution adjusted at pH 5.0 was added and the RNA was precipitated by adding 2.5 vol of 95% ethanol, previously cooled at-10CC.Heat treatment: 5S RNA was dissolved at the required concentration in 0.15 M NaCl, 0.015 sodium citrate, pH 7.0. The solution was heated at 600C for 4 min and then quickly cooled by dilution in 29 vol of cold distilled water. The RNA was recovered by ethanol precipitation.