The in vitro transcripts of phage SP6 RNA polymerase usually contain extraneous 5' and 3' plasmid sequences in addition to the cloned gene sequence. Template plasmids were constructed to eliminate these extraneous sequences. The 5 S RNA gene of Xenopus borealis somatic cell was inserted at the transcription initiation site (BamHI site) of PCKSP6 so that the transcription starts from the 5' end of the gene. The restriction enzyme DraI site was inserted at the 3' end of 5 S RNA structural gene by oligonucleotide-directed site-specific mutagenesis. Now, when the recombinant DNA is digested with DraI, the resulting in vitro run-off transcripts of the linearized template using phage SP6 RNA polymerase contain the authentic 5' and 3'ends. Thus, the large quantity of authentic 5 S RNA and any mutant RNAs, which could easily be radioactively labeled, can be produced for structure-function relationship studies, RNA folding studies, and other biophysical studies that have been limited by sample quantities.
기존의 전사용 벡타를 사용한 파아지 SP6 RNA 중합효소의 전사반응 전사반응에서 얻어진 전사체 RMA는 그 5' 과 3' 끝에 플라스미드 염기들이 붙게된다. SP6 promoter 직후에 BamHI 제한효소 위치가 오도록 만들어진 pCKSP6에 Xenopus borealis somatic 5 S rRNA 유전자를 넣어서 5' 의 불필요한 염기들을 제거하였다. 또한 5 S rRNA 유전자의 3' 끝 직후에 DraI 제한효소자리를 oligonucleotide-directed site-specific mutagenesis에 의해 만들어 주었다. 위의 재조합 플라스미드를 DraI 제한효소에 의해 전사시켜 정확한 5'-과 3'- 말단 염기열을 갖는 Xenopus borealis 5 S rRNA를 다량 얻을 수 있었다.
