Partial purification of a developmentally regulated messenger RNA from Dictyostelium discoideum by thermal elution from poly(U)-sepharose.
Partial purification of a developmentally regulated messenger RNA from Dictyostelium discoideum by thermal elution from poly(U)-sepharose.
复制标题
通过聚(U)-琼脂糖热洗脱来部分纯化盘基网柄菌的发育调节信使RNA。
DOI：
10.1016/0022-2836(81)90554-4
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发表时间：
1981
影响因子：
5.6
通讯作者：
Jacobson,A
中科院分区：
文献类型：
--
作者：
Palatnik,CM;Storti,RV;Jacobson,A
我们已经证明，从聚（U）-琼脂糖凝胶的热洗脱可用于将新合成的信使RNA与从盘基网柄藻的营养细胞分离的稳态mRNA分离（Palatniket等人，1979年，1980年）。因为我们能够富集10 - 60倍的新转录本，我们认为这种技术可能适用于发育调控mRNA的纯化。然而，该技术的实用性将取决于特定mRNA合成的差异速率。例如，在植物生长的Dictyosteliumamoeacum中，新合成的mRNA中翻译活性的相对分布与稳态mRNA中发现的相似（Palatniket等人，1979），并且该技术不导致任何特定mRNA种类的实质性纯化。为了测试该方法的适用性，我们已经研究了，作为一个模型系统，肌动蛋白mRNA合成的变化，在早期D。盘状体发育研究表明，在网柱藻发育过程中，肌动蛋白mRNA的相对浓度发生了变化。RNA合成抑制剂的实验表明，这种mRNA浓度的变化是在转录水平上介导的。这一结论得到了本文实验的支持：从生长和发育细胞中新合成的含poly（A）的RNA在mRNA依赖的网织红细胞裂解物中翻译，并通过双向聚丙烯酰胺凝胶电泳分析翻译产物。在两个小时的发展，当肌动蛋白mRNA的相对浓度是在其峰值，我们发现，它的翻译活性也大大丰富的部分RNA是新合成的。在发育5小时后，可翻译的肌动蛋白mRNA总量的百分比开始下降，肌动蛋白在这一RNA片段编码的翻译产物中所占的百分比要小得多。因此，新合成的mRNA组分的物理分离和表征提供了证据，表明在Dictyostelium发育过程中肌动蛋白mRNA浓度的变化发生在信使RNA合成的水平上。此外，结果显著地说明了从聚（U）-琼脂糖凝胶热洗脱用于富集由发育或代谢状态的改变诱导的mRNA的有用性。
We have demonstrated that thermal elution from poly(U)-Sepharose is useful for separating newly synthesized messenger RNAs from steady-state mRNAs isolated from vegetative cells ofDictyostelium discoideum(Palatniket al., 1979,1980). Because we were able to enrich between ten- and 60-fold for new transcripts, we suggested that this technique might be applicable to the purification of developmentally regulated mRNAs. The utility of the technique would, however, depend on the differential rate of synthesis of the particular mRNAs. For example, in vegetatively growingDictyosteliumamoebae the relative distribution of translation activities in newly synthesized mRNA is similar to that found in steady-state mRNA (Palatniket al., 1979), and the technique does not lead to substantial purification of any particular mRNA species. In order to test the applicability of the method, we have studied, as a model system, changes in actin mRNA synthesis during earlyD. discoideumdevelopment. It has been shown that the relative concentration of actin mRNA changes duringDictyosteliumdevelopment. Experiments with inhibitors of RNA synthesis have suggested that changes in the concentration of this mRNA are mediated at the transcriptional level. This conclusion is supported by the experiments of this paper: newly synthesized poly(A)-containing RNA from growing and developing cells was translated in mRNA-dependent reticulocyte lysates and the translation products were analyzed by two-dimensional polyacrylamide gel electrophoresis. At two hours of development, when the relative concentration of actin mRNA is at its peak, we find that its translation activity is also substantially enriched in the fraction of RNA that is newly synthesized. At five hours of development, when the percentage of total translatable actin mRNA has begun to decline, actin represents a much smaller percentage of the translation products coded for by this RNA fraction. The physical isolation and characterization of the newly synthesized mRNA fraction thus provides evidence that changes in the concentration of actin mRNA duringDictyosteliumdevelopment occur at the level of messenger RNA synthesis. Furthermore, the results dramatically illustrate the usefulness of thermal elution from poly(U)-Sepharose for enriching for mRNAs induced by alterations in developmental or metabolic states.