Pirtle I L, Shortridge R D, Pirtle R M
Gene. 1986;43(1-2):155-67. doi: 10.1016/0378-1119(86)90019-3.
A bacteriophage lambda clone containing a 15.4-kb human DNA fragment was isolated and found to contain a glycine tRNA gene and, 758 bp away, a pseudogene, both with an anticodon of GCC. The nucleotide (nt) sequence of a 1362-bp segment of this clone, encompassing the gene, pseudogene, and their flanking regions, was determined. The gene and pseudogene have an identical sequence of eight nt (5'-CAGCTGGA-3') in their 5'-flanking regions immediately preceding the coding regions, as well as characteristic transcription termination sites of five consecutive T nt in the 3'-flanking regions. Neither of these genes has intervening sequences. Only one of the two genes was efficiently transcribed in vitro by RNA polymerase III in a HeLa cell-free system. During the course of transcription, primary transcripts of one gene were processed to yield mature-sized products. In contrast, the level of transcription of the second gene was significantly less than that of the first, and no mature-sized products could be detected. The nt sequence of the inefficiently transcribed gene has two base substitutions compared to the sequence of the efficiently transcribed gene, and the DNA sequence predicted from the human placental tRNAGlyGCC sequence. One of these nt substitutions is a C to T transition in the TTCG sequence within the B block of the characteristic internal split promoter sequence. The precursor-product relationships of the tRNA transcripts were established by comparing the RNase T1 and RNase A fingerprints of the precursors and products.
分离出一个含有15.4 kb人类DNA片段的λ噬菌体克隆，发现它含有一个甘氨酸tRNA基因，且在758 bp之外有一个假基因，二者的反密码子均为GCC。测定了该克隆一个1362 bp片段的核苷酸（nt）序列，该片段包含该基因、假基因及其侧翼区域。该基因和假基因在编码区之前的5'侧翼区域有一段相同的8 nt序列（5'-CAGCTGGA-3'），在3'侧翼区域还有特征性的由五个连续T nt组成的转录终止位点。这两个基因均无间隔序列。在HeLa无细胞体系中，RNA聚合酶III仅能有效地转录这两个基因中的一个。在转录过程中，一个基因的初级转录本被加工产生成熟大小的产物。相比之下，第二个基因的转录水平明显低于第一个，且未检测到成熟大小的产物。与有效转录基因的序列以及从人胎盘tRNAGlyGCC序列预测的DNA序列相比，低效转录基因的nt序列有两个碱基替换。其中一个nt替换是在特征性内部裂解启动子序列B区的TTCG序列中发生了C到T的转换。通过比较前体和产物的核糖核酸酶T1和核糖核酸酶A指纹图谱，确定了tRNA转录本的前体-产物关系。
