Orozco E M, Hallick R B
J Biol Chem. 1982 Mar 25;257(6):3258-64.
The locations of transfer RNA genes with respect to the restriction endonuclease cleavage map of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. Purified chloroplast tRNAs were treated with snake venom phosphodiesterase to remove the 3'-CCA terminus, and radioactively labeled by the action of Escherichia coli tRNA nucleotidyltransferase in the presence of [alpha-32P]CTP. Chloroplast DNA was treated individually and with combinations of the enzymes Bal I, Bam HI, Eco RI, Pst I, Pvu II, Sal I, and Xho I. The location of tRNA genes with respect to the cleavage sites for these enzymes was determined by hybridization of the 32P-labeled tRNAs to membrane filter blots of the chloroplast DNA restriction nuclease fragments following gel electrophoresis. The 145-kilobase pair genome was resolved into nine areas of strong tRNA hybridization, separated by areas of weak or no tRNA hybridization. The loci of tRNA genes are within the Eco RI fragments Eco A, B, G, H, I, J', P, Q, and V.
已经确定了纤细裸藻（克氏裸藻）Z普林斯海姆株叶绿体DNA的限制性内切酶切割图谱上转运RNA基因的位置。纯化的叶绿体tRNA用蛇毒磷酸二酯酶处理以去除3'-CCA末端，并在[α-32P]CTP存在下通过大肠杆菌tRNA核苷酸转移酶的作用进行放射性标记。叶绿体DNA分别用Bal I、Bam HI、Eco RI、Pst I、Pvu II、Sal I和Xho I酶单独处理或组合处理。通过凝胶电泳后将32P标记的tRNA与叶绿体DNA限制性核酸酶片段的膜滤杂交，确定了tRNA基因相对于这些酶切割位点的位置。145千碱基对的基因组被解析为九个tRNA强杂交区域，由弱或无tRNA杂交区域隔开。tRNA基因的位点位于Eco RI片段Eco A、B、G、H、I、J'、P、Q和V内。
