Better conditions for mammalian in vitro splicing provided by acetate and glutamate as potassium counterions
Better conditions for mammalian in vitro splicing provided by acetate and glutamate as potassium counterions
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DOI：
10.1093/nar/28.2.416
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发表时间：
2000-01-15
影响因子：
14.9
通讯作者：
Moore, MJ
中科院分区：
文献类型：
--
作者：
Reichert, V;Moore, MJ
我们在这里证明，用乙酸钾（KAc）或谷氨酸钾（KGlu）代替氯化钾（KCl）通常会提高从HeLa细胞核提取物中进行的体外剪接反应中获得的RNA中间体和产物的产率。用多种剪接底物可重复地观察到这种效应。提高的产率至少部分是由于KAc和KGlu反应中剪接前体和产物的稳定化。这种相对于KCl反应的稳定性在KGlu的情况下最大，并且在扩展的钾浓度范围内观察到。RNA稳定性差异不能归因于KCl的重金属污染，因为该盐的超纯制剂产生了类似的结果。在测试了各种改变盐的方法后，我们发现在剪接反应中用KAc或KGlu取代KCl，用MgAc 2取代MgCl 2是最简单和最有效的。由于这里定义的条件更接近模拟体内离子浓度，它们可能允许研究更弱的剪接底物，以及促进剪接体结构和功能的更详细的分析。
We demonstrate here that replacing potassium chloride (KCl) with potassium acetate (KAc) or potassium glutamate (KGlu) routinely enhances the yield of RNA intermediates and products obtained from in vitro splicing reactions performed in HeLa cell nuclear extract. This effect was reproducibly observed with multiple splicing substrates. The enhanced yields are at least partially due to stabilization of splicing precursors and products in the KAc and KGlu reactions. This stabilization relative to KCl reactions was greatest with KGlu and was observed over an extended potassium concentration range. The RNA stability differences could not be attributed to heavy metal contamination of the KCl, since ultrapure preparations of this salt yielded similar results. After testing various methods for altering the salts, we found that substitution of KAc or KGlu for KCl and MgAc2 for MgCl2 in splicing reactions is the simplest and most effective. Since the conditions defined here more closely mimic in vivo ionic concentrations, they may permit the study of more weakly spliced substrates, as well as facilitate more detailed analyses of spliceosome structure and function.