MOLECULAR-CLONING AND DNA SEQUENCING OF THE RADC GENE OF ESCHERICHIA-COLI K-12

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::Kn(R)) has a 10.5-kb EcoRI/KpnI DNA fragment spanning the region from pyrE to the insertion mutation fpg-1::Kn(R). 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::Kn(R) 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 M(r) 11,500. The order of the genes in this region of the E. coli chromosome is: fpg - rpmBG - radC - pyrE.