Analysis of DNA inversions in the shufflon of plasmid R64

The shufflon, a multiple DNA inversion system in the plasmid R64, consists of four DNA segments flanked and separated by seven 19-bp repeat sequences. Site-specific recombinations mediated by the rci product occur between each inverted repeat sequence, resulting in inversions of the four segments independently or in groups. The seven 19-bp repeat sequences are classified into four types (repeat-a, -b, -c, and -d), according to their 3-bp variable sequences. We individually cloned A, B, and C segments of the R64 shufflon and determined the in vivo inversion frequency of each segment. The inversion frequencies of three segments differed greatly. The inversion frequency declined in the following order: segments A, B, and C. Synthetic 19-mer oligonucleotides corresponding to both strands of repeat-a, -b, -c, and -d sequences were inserted into appropriate sites of pBR322. The rci-mediated DNA inversion occurred between two synthetic inverted repeats, indicating that the 19-bp inverted repeat sequences are the sole elements required in cis for the shufflon system. The inversion frequencies of DNA segments flanked by various sequences indicate that the four types of repeat sequences determine the inversion frequency of the four DNA segments of the R64 shufflon. Deletion of a DNA segment flanked by direct repeat sequences could not be detected.