Infante A A, Heilmann L J
Biochemistry. 1981 Jan 6;20(1):1-8. doi: 10.1021/bi00504a001.
We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.
我们利用无细胞翻译和二维凝胶电泳技术，研究了海胆卵和胚胎中多核糖体及细胞质非多核糖体[核糖核蛋白（游离RNP）]信使核糖核酸（mRNA）群体的复杂性。我们发现，通过这种方法检测到的所有mRNA种类在多核糖体和游离RNP中均有呈现；基本上所有存在于多核糖体中的信息也存在于游离RNP组分中。然而，细胞质分布明显是非随机的，因为一些模板相对集中在游离RNP中，而另一些则主要存在于多核糖体中。在未受精卵mRNA指导下合成的多肽，在质量上与使用胚胎mRNA合成的多肽没有区别，这表明从卵到早期发育阶段，丰富类mRNA的复杂性保持不变。然而，特定mRNA的丰度发生了很大变化，并且在发育过程中检测到一些mRNA在多核糖体/游离RNP分布上的变化。多核糖体和游离RNP中特定mRNA相对丰度的差异以及发生的发育变化表明，mRNA在细胞质中进行翻译时存在显著的选择。在翻译产物中鉴定出三种不同形式的肌动蛋白（称为α、β和γ）。这三种肌动蛋白的信息在未受精卵和早期卵裂胚胎中均存在，但γ形式优先位于多核糖体中，而α和β形式则位于游离RNP中。在发育过程中，这三种肌动蛋白的相对浓度及其在多核糖体和游离RNP中的相对分布都发生了很大变化。对体内标记蛋白质的检测在很大程度上支持了体外研究结果。结果表明，肌动蛋白mRNA的合成在发育过程中大幅增加，并且在早期发育阶段，肌动蛋白mRNA的表达部分受翻译水平的控制。
