Komine Y, Adachi T, Inokuchi H, Ozeki H
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
J Mol Biol. 1990 Apr 20;212(4):579-98. doi: 10.1016/0022-2836(90)90224-A.
By using a set of 476 ordered DNA clones (in lambda phage vector) that covers the entire chromosome of Escherichia coli K12, we have made an exhaustive survey of tRNA genes in the E. coli genome. Ultraviolet-irradiated bacteria were separately infected with each of the 476 clones and the RNA molecules produced upon infection were labeled with 32P. The labeled tRNAs were separated by gel electrophoresis and then characterized by fingerprinting analysis. Fifty-nine of the 476 clones produced tRNAs, including adjacent overlapping ones that share the same tRNA genes. The products of all the previously mapped tRNA genes (about 60, to date) were detected according to their expected positions, and 19 more tRNA genes were newly elucidated. These new tRNA genes were identified by sequencing the DNA from relevant regions of the clones; the DNA sequences were scanned for the stretches that could be folded into the familiar cloverleaf structure and the transcription units were deduced by predicting the promoters and terminators. The total complement of the tRNA genes in E. coli K12 was 78 for 45 tRNA (or 41 anticodon) species, distributed in 40 different transcription units throughout the chromosome. In addition, a gene for selenocysteine tRNA was detected by hybridization and mapped to a specific DNA segment. A comprehensive tRNA gene map of E. coli was constructed, including the selenocysteine tRNA gene. All the tRNA genes encode the 3' CCA, and in several cases the terminal 19 nucleotides (including the 3' CCA) of a tRNA gene is repeated several times. Finally, in the present study the sites for a long inversion (approx. 800 x 10(3) base-pairs, around the oriC region) in Kohara's library was determined to be within the 23 S-5 S regions in rrnD and rrnE, revealing the exchange of combinations of spacer and distal tRNA genes between these two ribosomal RNA operons.
我们使用了一组覆盖大肠杆菌K12整个染色体的476个有序DNA克隆（λ噬菌体载体），对大肠杆菌基因组中的tRNA基因进行了详尽的调查。用这476个克隆分别感染经紫外线照射的细菌，并将感染后产生的RNA分子用32P进行标记。标记的tRNA通过凝胶电泳分离，然后通过指纹分析进行鉴定。476个克隆中有59个产生tRNA，包括共享相同tRNA基因的相邻重叠克隆。根据预期位置检测到了所有先前定位的tRNA基因（迄今为止约60个）的产物，另外还新阐明了19个tRNA基因。通过对克隆相关区域的DNA进行测序来鉴定这些新的tRNA基因；扫描DNA序列以寻找可折叠成常见三叶草结构的片段，并通过预测启动子和终止子来推导转录单元。大肠杆菌K12中tRNA基因的总数为78个，对应45种tRNA（或41种反密码子），分布在染色体上40个不同的转录单元中。此外，通过杂交检测到一个硒代半胱氨酸tRNA基因，并将其定位到一个特定的DNA片段上。构建了一张包括硒代半胱氨酸tRNA基因的大肠杆菌全面tRNA基因图谱。所有tRNA基因都编码3' CCA，在几种情况下，一个tRNA基因的末端19个核苷酸（包括3' CCA）会重复多次。最后，在本研究中确定了小原文库中一个长倒置（约800×10³碱基对，围绕oriC区域）的位点位于rrnD和rrnE的23 S - 5 S区域内，揭示了这两个核糖体RNA操纵子之间间隔区和远端tRNA基因组合的交换。
