Primer-dependent copying of rabbit globin mRNA with Qβ replicase
Primer-dependent copying of rabbit globin mRNA with Qβ replicase
复制标题
使用 Qβ 复制酶对兔珠蛋白 mRNA 进行引物依赖性复制
作者：
G. Feix
通过在Mn2+存在下用Qβ复制酶孵育这些异种rna的互补链，已经实现了这些rna的合成（参考文献1,2）。Qβ复制酶已以这种方式用于没有复制酶可用的rna的体外复制。最近发现的Qβ复制酶e3的引物依赖性RNA合成提供了这种合成的进一步扩展。这种方法特别适用于具有3 '端聚(a)序列的真核rna （mrna）的复制。互补RNA合成从这些poly(A)序列开始，以oligo(rU)6为引物，继续进入mrna的异源部分。该方法的特别优点是模板RNA的特殊结构特征对合成起始的独立性，以及完全抑制通常发生在Qβ复制酶孵育过程中的任何干扰6S RNA合成的可能性。由于新合成的互补RNA具有明确的合成起点，因此它可能对模板RNA的序列测定非常有用。我在这里描述了使用兔珠蛋白mRNA作为真核生物mRNA的引物依赖复制的一个例子的实验。
THE synthesis of complementary strands of heterologous RNAs has been achieved by incubating these RNAs with Qβ replicase in the presence of Mn2+ (refs 1, 2). Qβ replicase has been used in this way for the in vitro copying of RNAs for which no replicating enzymes are available. A further extension of such synthesis is offered by the recently discovered primer-dependent RNA synthesis by Qβ replicase3. This approach is specially suited for the copying of eukaryotic RNAs (mRNAs) with a 3′-terminating poly(A) sequence. The complementary RNA synthesis is started at these poly(A) sequences with oligo(rU)6 as primer and continues into the heterologous part of the mRNAs. The particular advantages of this method are the independence of special structural features of the template RNA for the initiation of synthesis and the possibility to inhibit completely any disturbing 6S RNA synthesis which normally takes place in Qβ replicase incubations. Because of its defined start of synthesis, the newly synthesised complementary RNA may be very useful for sequence determinations of the template RNAs. I describe here experiments using rabbit globin mRNA as an example of the primer-dependent copying of eukaryotic mRNAs.