|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1990 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1989 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Shufflon, or clustered inversion region, is a novel type of DNA rearrangement found in the IncI1 plasmid R64. The shufflon of R64 consists of four DNA segments, designated A, B, C and D, which differ in DNA length and sequence. The four DNA segments are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any two inverted repeats results in a complex DNA rearrangement. An analysis of open reading frames revealed that the shufflon is a biological switch to select one of the seven C-termini of a gene, pilV.
By constructing a series of deletion-derivatives of plasmid R64, a region responsible for the conjugal transfer was located within a 54-kb DNA region to the right of replication region. We determined the nucleotide sequence of a 19-kb DNA region responsible for the formation of thin pilus, and found that 18 open reading frames (ORFs) are present in the same orientation. The pliv gene is the last one of the 18 ORFs. By introducing deletion and inser
tion mutations into all the 18 ORFs, we demonstrated that most of the 18 ORFs are required for the formation of thin pilus and for the conjugal transfer. To reveal the role of the DNA rearrangement in the shufflon region, we constructed R64 derivatives where the C-termini of the pilv gene are fixed. Donor cells carrying R64 derivative plasmids with the seven fixed pilv C-termini were mated with one of several recipient bacterial strains including E. Coli K-12, B and C, Salmonella typhimurium LT2 and Shigella flexneri. The results indicated that the transfer frequencies of liquid mating dramatically change according to the combination of recipient strains and variable C-terminiof the pilv gene in the donors, suggesting that the shufflon determines the recipient specificity in liquid mating in plasmid R64. We also sequenced the oriT region located at the other end of transfer region of R64 and found two open reading frames, designated nikA and nikB. NikA and nikB were found indispensable for the transfer of R64 and were demonstrated to function together as an oriT-specific nickase. Less