PROCEDURE FOR ISOLATION OF MAMMALIAN MESSENGER RIBONUCLEIC-ACID
PROCEDURE FOR ISOLATION OF MAMMALIAN MESSENGER RIBONUCLEIC-ACID
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DOI：
10.1021/bi00754a027
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发表时间：
1972-01-01
期刊：
影响因子：
2.9
通讯作者：
MENDECKI, J
中科院分区：
文献类型：
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作者：
BRAWERMAN, G;LEE, SY;MENDECKI, J
乔治布拉沃曼，f约瑟夫门德基。摘要：在哺乳动物细胞多聚核糖体RNA的异质性快速标记组分中，已证实存在一个大的poly（A）片段。Poly（A）的独特性质使这种RNA种类的分离成为可能，它具有mRNA的各种特征。在Tris-HCl（pH7.6）和多聚体蛋白或甲基化白蛋白存在下，在苯酚提取期间，poly-（A）片段导致这些RNA分子转移到非水相。用pH9.0的Tris缓冲液反萃取回收。这种行为的关键因素似乎是苯酚萃取过程中的离子浓度。Poly（A）片段还能使RNA分子在pJL时有效地与硝酸纤维素膜滤器（Millipore滤器）结合。在从rRNA中分离动物细胞mRNA的方法的发展过程中，由于明显缺乏结合物理特性而受到阻碍。在大多数细胞中，mRNA分子的大小分布是这样的，它们的沉降值与核糖体组分的沉降值重叠。只有在网织红细胞中，血红蛋白的10 S信使代表了主要的种类，才有可能通过区带沉降法分离这种RNA成分（Lockard和Lingrel，1969）。我们希望报道一种分离多聚核糖体RNA分子的方法，这些RNA分子似乎代表mRNA，基于这些分子中存在大的poly（A）序列。一段时间以来，人们已经知道，通过差别酚提取，可以从动物细胞中获得显著富集具有DNA样碱基组成的组分的RNA组分。在低温下用苯酚水溶液处理大鼠肝细胞或腹水细胞显示优先释放rRNA，而在高温下再提取产生的RNA组分具有与DNA相似的核苷酸组成（Georgiev和Mantieva，1962）。分离的基础被认为是DNA样RNA在细胞核中的定位。通过用中性和碱性Tris-HCl缓冲液连续进行苯酚萃取获得类似的分离（Brawerman等人，1963年）。在后一种情况下，观察到“DNA样”RNA特别富含腺苷酸。随后的研究表明，大鼠肝细胞质中含有快速标记的、富含腺苷酸的、对苯酚不敏感的DNA样RNA
George Brawerman, f Jozef Mendecki. t and Se Yong Lee § abstract: The presence of a large poly (A) segment has been demonstrated in the heterogeneous rapidly labeled compo-nents of polysomal RNA from mammalian cells. Unique prop-erties of poly (A) permit the isolation of this RNA species, which possesses various characteristics of mRNA. The poly-(A) segment causes the transfer of these RNA moleculesto the nonaqueous phase during phenol extraction in the presence of Tris-HCl (pH 7.6) and of polysomal proteins or methylated albumin. They are recovered by reextractionwith pH 9.0 Tris buffer. The critical factor for this behavior appears to be the ionic concentration during phenol extraction. The poly (A) segment also causes the effective binding of the RNA mole-cules to nitrocellulose membrane filters (Millipore filters) at pJL rogress in the development of procedures for the separa-tion of animal cell mRNA from rRNA has been hampered by the apparent lack of distinguishingphysical features. The size distribution of mRNA molecules in most cells is such that their sedimentation values overlap those of the ribosomal components. Only in the case of reticulocytes, in which the 10S messenger for hemoglobin represents the major species, has it been possible toisolate this RNA component by zone sedimentation (Lockard and Lingrel, 1969). We wish to report on a procedure for the isolation of polysomal RNA molecules that appearto represent mRNA, based oh the occurrence in these molecules of a large poly (A) sequence. It has been knowh for some time that RNA fractions con-siderably enriched in components with a DNA-like base com-position could be obtained from animal cells by differential phenol extraction. Treatment of rat liver or ascites cells with aqueous phenol at low temperature was shown to release preferentially rRNA, while reextraction at elevated temperature yielded RNA components with a nucleotide composition similar to that of DNA (Georgiev and Mantieva, 1962). The basis of the fractionation was thought to be the localization of the DNA-like RNA in the nucleus. A similar separation was obtained by sequential phenol extractions with neutral and alkaline Tris-HCl buffers (Brawerman et al., 1963). In this latter case, the “DNA-like” RNA was observed to be par-ticularly rich inadenylic acid. Subsequent studies with this technique showed that rat liver cytoplasm containedrapidly labeled, adenylate-rich, DNA-like RNA refractory to phenol