Felzenszwalb I, Boiteux S, Laval J
Groupe 'Réparation des Lésions Chimio et Radioinduites', UA158 CNRS, U140 INSERM, Institut Gustave Roussy, Villejuif, France.
Mutat Res. 1992 May;273(3):263-9. doi: 10.1016/0921-8777(92)90088-k.
The radC102 mutation that sensitizes E. coli K-12 cells to ultraviolet light, ionizing radiations and alkylating agents was localized between the fpg and pyrE genes at 81.7 min on the bacterial chromosome. E. coli strain BH20 (radC+, fpg-1::KnR) has a 10.5-kb EcoRI/KpnI DNA fragment spanning the region from pyrE to the insertion mutation fpg-1::KnR. The proximity of the radC gene to this insertion mutation provided a strategy to isolate the radC+ gene based on the cloning of radC+ and fpg-1::KnR on the same DNA fragment using the resistance to kanamycin as a selector. A library of EcoRI/KpnI DNA fragments of E. coli strain BH20 was inserted into pUC19. One recombinant plasmid conferring resistance to kanamycin was selected and named pRCV10. The pRCV10 plasmid partially restores the resistance to UV-radiation when transformed into SR1187 (radC102), but sensitizes the wild-type strain to the same treatment. The radC102 complementing region was localized on a 1.2-kb BglII/BglII DNA fragment which was sequenced. The DNA sequence complementing the radC102 mutation contained an ATG translation start codon with an open reading frame of 297 base pairs which encodes a polypeptide of Mr 11,500. The order of the genes in this region of the E. coli chromosome is: fpg--rpmBG--radC--pyrE.
使大肠杆菌K-12细胞对紫外线、电离辐射和烷化剂敏感的radC102突变位于细菌染色体上81.7分钟处的fpg和pyrE基因之间。大肠杆菌菌株BH20（radC +，fpg-1::KnR）有一个10.5 kb的EcoRI/KpnI DNA片段，跨越从pyrE到插入突变fpg-1::KnR的区域。radC基因与该插入突变的接近性提供了一种策略，可基于使用卡那霉素抗性作为选择器，将radC +和fpg-1::KnR克隆到同一DNA片段上来分离radC +基因。将大肠杆菌菌株BH20的EcoRI/KpnI DNA片段文库插入pUC19中。选择了一个赋予卡那霉素抗性的重组质粒并命名为pRCV10。当将pRCV10质粒转化到SR1187（radC102）中时，它部分恢复了对紫外线辐射的抗性，但使野生型菌株对相同处理敏感。radC102互补区域定位在一个经测序的1.2 kb BglII/BglII DNA片段上。与radC102突变互补的DNA序列包含一个ATG翻译起始密码子，其开放阅读框为297个碱基对，编码一个Mr 11,500的多肽。大肠杆菌染色体该区域的基因顺序为：fpg--rpmBG--radC--pyrE。
