Ryals J, Hsu R Y, Lipsett M N, Bremer H
J Bacteriol. 1982 Aug;151(2):899-904. doi: 10.1128/jb.151.2.899-904.1982.
A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.
本文描述了一种快速筛选和分类众多独立的近紫外辐射抗性大肠杆菌突变体的方法，这些突变体包括tRNA修饰和RNA合成控制突变体。发现其中一类突变体在硫胺素生物合成以及将tRNA中的尿苷修饰为4-硫尿苷（已知是近紫外辐射的靶点）的能力方面同时存在缺陷。这些突变体被发现无法合成噻唑，即硫胺素的前体。添加噻唑可恢复硫胺素缺乏，但不会使细胞对近紫外辐射敏感。对其中一个突变体的体外研究表明，它缺乏tRNA的4-硫尿苷修饰所需的蛋白质因子C（nuvC）。在P1转导中，噻唑标记与组氨酸标记共转导，这将噻唑标记定位在大肠杆菌染色体图谱上42至46分钟之间。87%的自发回复突变恢复了硫胺素产生和4-硫尿苷产生，表明是单点突变。我们的结果表明，我们分离出了nuvC突变体，并且nuvC多肽参与两种功能，即tRNA修饰和噻唑生物合成。
