Klemenz R, Geiduschek E P
Nucleic Acids Res. 1980 Jun 25;8(12):2679-89. doi: 10.1093/nar/8.12.2679.
The 5' terminus of Saccharomyces cereviasiae 35S pre rRNA was mapped on the rDNA using two methods: 1) Suitable restriction endonuclease fragments were hybridized to total high molecular weight RNA and extended with reverse transcriptase to the 5' end of the RNA template. 2) Other restriction fragments spanning the 5' terminus of 35S pre rRNA and radioactively labeled at their ends were hybridized to high molecular weight RNA and the non hybridized nucleic acids were digested with S1 nuclease. On the basis of these experiments, the 5' terminus of 35S pre rRNA was placed approximately 670 nucleotides upstream from the 17S rRNA coding region. The exact position was determined by reverse transcription as above, but in the presence of dideoxyribonucleoside triphosphates, which served as a way of sequencing the 5' terminal region. 35S pre rRNA synthesis is initiated at a site in EcoRI restriction fragment B which is 48 nucleotides upstream from the EcoRI cleavage site in the coding strand.
采用两种方法将酿酒酵母35S前体rRNA的5'末端定位在rDNA上：1）将合适的限制性内切酶片段与总的高分子量RNA杂交，并用逆转录酶延伸至RNA模板的5'末端。2）跨越35S前体rRNA 5'末端并在其末端进行放射性标记的其他限制性片段与高分子量RNA杂交，未杂交的核酸用S1核酸酶消化。基于这些实验，35S前体rRNA的5'末端位于17S rRNA编码区上游约670个核苷酸处。确切位置如上述通过逆转录确定，但在双脱氧核糖核苷三磷酸存在的情况下进行，双脱氧核糖核苷三磷酸用作对5'末端区域进行测序的一种方法。35S前体rRNA的合成起始于EcoRI限制性片段B中的一个位点，该位点在编码链上位于EcoRI切割位点上游48个核苷酸处。
