本研究係利用含插入序列基因之M13 mp18噬菌體當做模板進行核酸酶SI分析以研究小白鼠胸核苷激酶基因的轉錄起始點。
首先自電泳後之低融點洋菜糖膠上切取含第一表現序列至5'-端側翼核苷酸片段(約145 bp)之膠體，直接鍵結至Sma I處理過的M13mp18載體上。經轉染作用後，將無色溶菌斑取出培養。進而利用Sanger氏列序法之變法找出所要的單鏈模板。由此單鏈摸板再進一步合成含35S之放射性互補鏈DNA片段當作探針，經純化後，令此探針與Poly (A)+RNA之混合物進行雜合反應(58℃, 2~3小時)，經核酸酶S1作用以除去未配對單鏈DNA及RNA。最後再利用聚丙烯醯胺膠體電泳法測定殘留DNA片段之長度，由此發現小白鼠胸核苷激酶基因至少有一轉錄作用之起始點在轉譯起始點上游第42位的"A"。 In this study, a fragment (FSA) of mouse cytosolic thymidine kinase (tk) gene which containing the first exon and 5’-flanking region (ca 145 bp) was cut out from a cosmid (pMtk 116), and then cloned into Sma I-treatcd M13 mp18 vector. Two uncoding-strand-containing subclones (M13/FSAu) were isolated by checking the nucleotide sequence with modified Sanger's dideoxy method. The 35S-labelled coding strand (ca 200 bp) was prepared by a modified M13 sequencing procedure. The single-stranded hot DNA fragment thus obtaincd was further used as a probe for the study of the transcription initiation site(s) of tk gene by modified S1-mapping technique. From this study, at least one transcription initiation site which located at 42th nucleotide upstream from the translation initiation site was found, the result was further discussed.
關聯:
嘉南學報 15 : p.A78-A88 轉載 Journal of the Chinese agricultural chemical society 26(1),p.81-91 (1988)
