REGULATION OF PROTEIN-SYNTHESIS IN TETRAHYMENA - ISOLATION AND CHARACTERIZATION OF POLYSOMES BY GEL-FILTRATION AND PRECIPITATION AT PH 5.3
REGULATION OF PROTEIN-SYNTHESIS IN TETRAHYMENA - ISOLATION AND CHARACTERIZATION OF POLYSOMES BY GEL-FILTRATION AND PRECIPITATION AT PH 5.3
复制标题
DOI：
10.1093/nar/10.6.2145
复制
发表时间：
1982-01-01
影响因子：
14.9
通讯作者：
GOROVSKY, MA
中科院分区：
文献类型：
--
作者：
CALZONE, FJ;ANGERER, RC;GOROVSKY, MA
在嗜热四膜虫中，核糖体负载在多核糖体上的比例约为95%，而在饥饿细胞中约为4%。从这两种生理状态的细胞的胞质提取物被用来开发用于纯化多聚核糖体的柱色谱方法。发现Bio-Gel A 1.5 m可将总细胞质核糖体与许多可溶性蛋白质（包括RNA酶）分离，而多核糖体大小分布无可检测的变化。通过Bio-Gel A 15 μ m层析，可以将多聚体与单体和非多聚体mRNA分离，而无需大小选择。这些方法可以很容易地适用于大规模制备多聚核糖体，甚至是从其中一小部分核糖体在多聚核糖体上的细胞中制备。描述了一种从pH 5.3的稀溶液中可逆沉淀多核糖体和单体的方法，该方法极大地促进了多核糖体的分离。3 H标记的polyU与从柱级分中分离的RNA的杂交已被用于证明EDTA释放的多核糖体mRNA的纯化可以使用本文所述的柱色谱法进行。这些方法已被用来证明大多数细胞质mRNA在对数期Tetrahymenais加载到多聚核糖体上，而大多数mRNA在饥饿细胞中以非多聚核糖体形式存在。
The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growingTetrahymenathermophila, and about 4% in starved cells. Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes. Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution. Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection. These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes. A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation. Hybridization of3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here. These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phaseTetrahymenais loaded onto polyribosomes while most of the mRNA in starved cells exists in a non-polysomal form.