Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase.
Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase.
复制标题
酵母细胞质苏氨酰-tRNA 合成酶基因的克隆和表征。
DOI：
10.1093/nar/13.17.6171
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发表时间：
1985
影响因子：
14.9
通讯作者：
A. Tzagoloff
中科院分区：
文献类型：
--
作者：
L. Pape;A. Tzagoloff
将编码线粒体tRNAThr 1合成酶的酵母核基因MST 1的DNA片段用作探针，以筛选其他酵母苏氨酰-tRNA合成酶基因。在低严格性下，MST 1探针与具有同源基因的酵母基因组DNA的6.6kb EcoRI片段强烈杂交，并且另外与具有第二苏氨酰-tRNA合成酶基因（THS 1）的较小的3.6kb EcoRI片段更弱地杂交。为了克隆THS 1，通过连接到大小选择的（3- 4.5kb）基因组DNA的EcoRI片段的pUC 18构建文库。分离出几个含有3.6kb EcoRI片段的克隆。在克隆的DNA片段中鉴定了对应于THS 1的2，202个核苷酸长的开放阅读框。THS 1编码的蛋白与E.大肠杆菌苏氨酰-tRNA合成酶在后者的长度（642个氨基酸），是42%相同的预测MST 1产品超过其462个残基。原位破坏THS 1的染色体拷贝对细胞是致命的，表明该基因编码细胞质苏氨酰-tRNA合成酶。
A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes. At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1). To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA. Several clones containing the 3.6 kb EcoRI fragment were isolated. A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA. The predicted protein encoded by THS1 is 38% identical to the E. coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues. In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase.