The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA.
The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA.
复制标题
卵清蛋白 mRNA 的完整互补 DNA 转录本的合成和特性。
作者：
J. Monahan;S. Harris;S. Woo;D. Robberson;B. O’Malley
在不同盐、脱氧核糖核苷酸浓度、温度和时间条件下，研究了利用AMV RNA指导的DNA聚合酶合成鸡卵清蛋白mRNA的互补DNA拷贝（cDNA）。观察到在46 ℃下不存在单价阳离子的情况下，卵清蛋白mRNA的完整转录物可以受到酶的影响。完全合成所需的最低脱氧核糖核苷酸为dATP、dGTP和dCTP为35 μ M，dTTP为200 μ M。通过一些不同的实验标准，其中包括碱性蔗糖梯度沉降和聚丙烯酰胺凝胶电泳含有98%甲酰胺，直接电子显微镜可视化，并保护卵清蛋白[25 I]mRNA从核酸酶消化，它可以证明，相当一部分的完整的mRNA转录确实是合成的。cDNA/卵清蛋白mRNA杂合体在羟基磷灰石上的Tm为92 ℃，表明RNA转录物的合成具有高保真度。当这样一个完整的卵清蛋白[3 H]cDNA以10（8）cpm/mug的比活性合成并与过量的鸡DNA杂交时，杂交动力学表明该cDNA由非重复序列组成。
The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.