Suter B, Altwegg M, Choffat Y, Kubli E
Arch Biochem Biophys. 1986 May 15;247(1):233-7. doi: 10.1016/0003-9861(86)90552-7.
The nucleotide sequence of the two major Drosophila melanogaster tRNATyr isoacceptors was determined to be pC-C-U-U-C-G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3 U-A-G-A-G-C-m2(2)G-G-psi-G-G-A-C-U-G/Q-psi-A-m1G-A-Um-C-C-A-U-A-G-m7 G-D-C-G-C-U-G-G-U(T)-psi-C-A-m1A-A-U-C-C-G-G-C-U-C-G-A-A-G-G-A-A-C-C-AOH . The two isoacceptors differ by the presence of a G or a Q in the wobble position. Both contain a partial modification in position 54 (U/T). Thus, these tRNAs are transcribed from a single gene (or many genes with identical sequences). A fast and sensitive postlabeling method for sequencing tRNA anticodons is described. Nuclease S-1-treated tRNA is labeled with 5[32P]-pCp using T-4 RNA ligase. The tRNA fragments are then separated on 7 M urea/20% PAA gels. After autoradiography the RNA is eluted and digested with T-2 RNase. The nature of the labeled nucleotides is determined by two-dimensional thin-layer chromatography. The same method can be used to determine the 5' sequence of a tRNA by 3' labeling 5' tRNA halves with 5[32P]-pCp and subsequent chemical sequencing.
已确定两种主要的黑腹果蝇tRNATyr同工受体的核苷酸序列为：pC-C-U-U-C-G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3 U-A-G-A-G-C-m2(2)G-G-psi-G-G-A-C-U-G/Q-psi-A-m1G-A-Um-C-C-A-U-A-G-m7 G-D-C-G-C-U-G-G-U(T)-psi-C-A-m1A-A-U-C-C-G-G-C-U-C-G-A-A-G-G-A-A-C-C-AOH。这两种同工受体在摆动位置上存在一个G或一个Q而有所不同。两者在第54位（U/T）都有部分修饰。因此，这些tRNA是从单个基因（或许多具有相同序列的基因）转录而来的。本文描述了一种快速且灵敏的tRNA反密码子测序的后标记方法。用T-4 RNA连接酶将核酸酶S-1处理过的tRNA用5[32P]-pCp进行标记。然后将tRNA片段在7M尿素/20%聚丙烯酰胺凝胶上分离。放射自显影后，洗脱RNA并用T-2核糖核酸酶消化。通过二维薄层层析确定标记核苷酸的性质。同样的方法可用于通过用5[32P]-pCp对5' tRNA半体进行3'标记并随后进行化学测序来确定tRNA的5'序列。
