Paddock G V
Dept. of Microbiology and Immunology, Medical University of South Carolina, Charleston 29425.
Biotechniques. 1989 Sep;7(8):856-65.
In studies of the effects of changes in mRNA structure and sequence on the initiation of protein synthesis, we used a generally applicable approach to transcribe reconstructed genes for duck alpha A globin by the bacteriophage SP6 RNA polymerase promoter in a pGEM-2 plasmid vector. The genes were reconstructed such that the first nucleotide to be transcribed, the 5' adenosine, was placed directly adjacent to the SP6 promoter sequence. The 3' ends of the genes were constructed such that cleavage with Ssp 1 endonuclease yielded a template that directed the synthesis of mRNA terminating in a poly A tail containing 56 adenosines and a single 3' uridine. Special conditions using a Mn++ buffer were developed to enable the SP6 RNA polymerase to initiate at the 5' adenosine and synthesize the A-start transcription product. The mRNA could be capped and was subsequently used as an effective template for in vitro translation and synthesis of duck alpha A globin.
在关于mRNA结构和序列变化对蛋白质合成起始影响的研究中，我们采用了一种普遍适用的方法，通过噬菌体SP6 RNA聚合酶启动子在pGEM - 2质粒载体中转录鸭αA珠蛋白的重组基因。这些基因经过重组，使得第一个被转录的核苷酸，即5'腺苷，直接与SP6启动子序列相邻。基因的3'端构建成用Ssp 1核酸内切酶切割后可产生一个模板，该模板指导合成终止于含有56个腺苷和一个单一3'尿苷的聚A尾巴的mRNA。开发了使用Mn++缓冲液的特殊条件，以使SP6 RNA聚合酶能在5'腺苷处起始并合成A起始转录产物。该mRNA可以加帽，随后用作体外翻译和合成鸭αA珠蛋白的有效模板。
