AN EXTRACELLULAR DARWINIAN EXPERIMENT WITH A SELF-DUPLICATING NUCLEIC ACID MOLECULE
AN EXTRACELLULAR DARWINIAN EXPERIMENT WITH A SELF-DUPLICATING NUCLEIC ACID MOLECULE
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DOI：
10.1073/pnas.58.1.217
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发表时间：
1967-01-01
影响因子：
11.1
通讯作者：
SPIEGELMAN, S
中科院分区：
文献类型：
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作者：
MILLS, DR;PETERSON, RL;SPIEGELMAN, S
材料和方法-(a)酶、底物和测定：放射性核糖核苷酸三磷酸的合成和膜过滤器上标记RNA的液体闪烁计数先前已详细描述。 4 如前所述，从病毒中提取 Qt3 (ts-1) 温度敏感突变体的 RNA。 8 系列中的第一个反应以 0.2 gg/0.25 ml 标准反应的浓度开始。通过CsCl和蔗糖步骤9纯化的相同复制酶制剂用于将要描述的转移实验的所有步骤中。(b)产物的沉降分析：从各种反应中取出等分试样(0.01-0.10ml)并调节至相对于十二烷基硫酸钠(SDS)的0.2％(按重量计)。将每个样品在 TE 缓冲液（0.01 M Tris、pH 7.4 和 0.003 M EDTA）中稀释至最终体积 0.20 ml，然后分层置于 5 ml 线性梯度蔗糖（0.10 M Tris、pH 7.4 和 0.003 M EDTA 中 2-20%）上。这些梯度在 Spinco SW-39 转子中以 39,000 rpm、4VC 离心 5 小时。逐滴收集0.25ml级分，用10％三氯乙酸(TCA)沉淀，洗涤到硝酸纤维素膜过滤器上，并在Packard液体闪烁计数器中计数。 (c)凝胶电泳：未溶胀的二丙烯酸乙二酯交联聚丙烯酰胺凝胶(3.6％)和预溶胀N， 如前所述制备 N'-亚甲基双丙烯酰胺交联聚丙烯酰胺凝胶 (2.4%)。'0 电泳运行在室温下进行 90 分钟，对于直径 0.7 cm 的凝胶，以 5 ma/凝胶和 50 伏电压进行；对于直径 0.9 cm、长度 9 cm 的凝胶，以 10 ma/凝胶进行电泳。通过扫描每个凝胶进行凝胶的光密度测量。 在配有 266-miA 干涉滤光片的 Joyce 高分辨率“chromoscan”中用透射紫外光进行凝胶（转移到 0.5 cm 深的石英池中）。使用二氧化碳冷却的切片机将冷冻凝胶切成 0.5 毫米切片。'0 将连续的 0.5 毫米切片放入小瓶中，在 TE 或 SSC（0.015 M 氯化钠和 0.015 M 柠檬酸钠）缓冲液中洗脱，在 5℃ 下轻轻搅拌 12 小时。从每次洗脱中取出等分试样，用冷沉淀 10% TCA，洗涤到硝酸纤维素膜过滤器上，并在 Packard 液体闪烁计数器中计数。
Materials and Methods.-(a) Enzyme, substrates, and assays: Synthesis of radioactive ribo-nucleotide triphosphates and liquid scintillation counting of labeledRNA on membrane filters have been detailed previously. 4 RNA from a temperature-sensitive mutant of Qt3 (ts-1) was extracted from the virus as described previously. 8 The first reaction in the series was initiated at a concentration of 0.2, gg/0.25 ml of a standard5 reaction. The same replicase preparation purified through the CsCl and sucrose steps9 was used in allthe steps of the transfer experiments to be described.(b) Sedimentation analysis of products: Aliquots (0.01-0.10 ml) were withdrawn from various reactions and adjusted to 0.2%(by weight) withrespect to sodium dodecyl sulfate (SDS). Each sample was diluted to a final volume of 0.20 ml in TE buffer (0.01 M Tris, pH 7.4, and 0.003 M EDTA), then layered on a 5-ml linear gradient of sucrose (2-20% in 0.10 M Tris, pH 7.4, and 0.003 M EDTA). These gradients were centrifuged in a Spinco SW-39 rotor at 39,000 rpm at 4VC for 5 hr. Fractions of 0.25 ml were collected dropwise, precipitated with 10% trichloroacetic acid (TCA), washed onto cellulose nitrate membrane filters, and counted in a Packard liquid scintillation counter.(c) Gel electrophoresis: Unswollen ethylene diacrylate cross-linked polyacrylamide gels (3.6%) and preswollenN, N'-methylene-bis-acrylamide cross-linked polyacrylamide gels (2.4%) were prepared as described previously.'0 Electrophoresis runs were made at room temperature for 90 min, at 5 ma/gel and 50 volts for gels 0.7 cm in diameter and 10 ma/gel for gels 0.9 cm in diameter and 9 cm in length.Optical density measurements of gels were performed by scanning each gel (transferred to a quartz cell 0.5 cm in depth) with transmitted ultraviolet light in a Joyce high-resolution" chromoscan" equipped with a 266-miA interference filter. Frozen gels were sectioned in 0.5-mm slices with the use of a carbon dioxide-cooled microtome.'0 Successivepairs of 0.5-mm sections were placed in vials and eluted in TE or SSC (0.015 M sodium chloride and 0.015 M sodium citrate) buffers with gentle agitation for 12 hr at 5 C. Aliquots were removed from each elution, pre-cipitated with cold 10% TCA, washed onto cellulose nitrate membrane filters, and counted in a Packard liquid scintillation counter.