RELATIONSHIP BETWEEN ESCHERICHIA-COLI GROWTH AND DELETIONS OF CTG-CENTER-DOT-CAG TRIPLET REPEATS IN PLASMIDS

Instabilities that are intrinsic to natural repetitive DNA sequences p roduce high frequencies of length changes ill vivo. Triplet repeats cl oned in plasmids in Escherichia coli undergo expansions and deletions, and this instability is affected by multiple factors. We show that CT G . CAG repeats in plasmids can influence the growth of E. coli, which affects the observed stabilities. At extended growth periods, the obs erved frequencies of deletions were dramatically increased if the cell s passed through stationary phase before subculturing. Deletions were particularly pronounced for a plasmid containing the longest repeat, 5 25 bp in total, with the CTG sequence as the lagging strand template f or replication. Measurements of cell growth showed that the lag phase associated with E. coil growth was increased for cultures containing p lasmids with long CTG . CAG repeats, particularly when the CTG-contain ing strand was the lagging template. High frequencies of deletions wer e observed because of a growth advantage of cells containing plasmids with deleted tripler repeats. Incubation conditions that reduced the b acterial growth-rate produced a decreased extent of deletions, presuma bly because they alleviated the growth advantage of cells harboring pl asmids with deleted tripler repeats. The experimental observations wer e simulated by a model in which shorter triplet repeats provided a gro wth advantage due to a shorter lag phase. We demonstrate that the accu mulation of deletions within repeating sequences during growth of E. c oli can be prevented, and discuss these findings in relation to the st udies of repetitive DNA sequences. These are the first observations to show a direct influence between a plasmid-based DNA sequence or struc ture and factors controlling bacterial growth. (C) 1996 Academic Press Limited