Preparation of messenger RNA from mature skeletal and cardiac muscle.
Preparation of messenger RNA from mature skeletal and cardiac muscle.
复制标题
从成熟骨骼肌和心肌中制备信使 RNA。
DOI：
10.1139/o82-072
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
G. Jones
中科院分区：
文献类型：
--
作者：
P. Rogers;G. Jones
信使RNA （poly(A) + RNA）是从成年大鼠的成熟骨骼肌中通过几种方法制备的。这些程序包括肌肉多核糖体（方法1）的寡聚纤维素层析，用CsCl梯度离心分离肌肉多体的多体RNA（方法2），用CsCl梯度离心分离肌肉总RNA（方法3），或用苯酚提取制备肌肉总RNA（方法4）。在十二烷基硫酸钠-蔗糖梯度上，每一种方法都能产生大量的RNA，并在宽波段（从4S到大于28S）内沉积。此外，来自每个过程的poly (A) + RNA刺激了小麦无生殖细胞系统中的蛋白质合成。用方法2从多体RNA中制备poly (A) + RNA，获得了产量和信使活性的最佳组合。这种多聚(A) + RNA制备刺激了小麦胚芽系统中许多假定的肌原纤维蛋白的无细胞合成，包括肌球蛋白重链和肌动蛋白。通过适当反应混合物的DNase I亲和层析证实了后一种蛋白在无细胞产物中的存在。用方法2也从大鼠心肌多体RNA中分离到Poly(A) + RNA。该RNA还指导小麦胚芽系统中肌原纤维蛋白的合成。已经比较了在骨骼肌和心肌聚(A) + RNA存在下合成的蛋白质的相对数量。结果表明，该方法适用于从成熟哺乳动物肌肉中分离具有信息活性的RNA。
Messenger RNA (poly(A) + RNA) has been prepared from mature skeletal muscle of adult rats by several procedures. These procedures involved oligodT-cellulose chromatography of either muscle polyribosomes (method 1), polysomal RNA isolated by CsCl gradient centrifugation of muscle polysomes (method 2), total muscle RNA isolated by CsCl gradient centrifugation (method 3), or total muscle RNA prepared by phenol extraction (method 4). Each procedure produced RNA in significant yields which sedimented in a broad band (from 4S to greater than 28S) on sodium dodecyl sulfate--sucrose gradients. In addition, poly (A) + RNA from each procedure stimulated protein synthesis in the wheat germ cell-free system. The best combination of yield and messenger activity was obtained for poly (A) + RNA prepared from polysomal RNA by method 2. This poly(A) + RNA preparation stimulated the cell-free synthesis of a number of presumptive myofibrillar proteins, including myosin heavy chain and actin, in the wheat germ system. The presence of the latter protein among the cell-free products was confirmed by DNase I affinity chromatography of appropriate reaction mixtures. Poly(A) + RNA was also isolated from rat cardiac muscle polysomal RNA by method 2. This RNA also directed the synthesis of myofibrillar proteins in the wheat germ system. The relative amounts of the proteins synthesized in the presence of skeletal and cardiac muscle poly(A) + RNA have been compared. The data indicate that the method described is suitable for the isolation of RNA with message activity from mature mammalian muscle.