Relationship between Escherichia coli growth and deletions of CTG.CAG triplet repeats in plasmids.
Instabilities that are intrinsic to natural repetitive DNA sequences produce high frequencies of length changes in vivo. Triplet repeats cloned in plasmids in Escherichia coli undergo expansions and deletions, and this instability is affected by multiple factors. We show that CTG-CAG repeats in plasmids can influence the growth of E. coli, which affects the observed stabilities. At extended growth periods, the observed frequencies of deletions were dramatically increased if the cells passed through stationary phase before subculturing. Deletions were particularly pronounced for a plasmid containing the longest repeat, 525 bp in total, with the CTG sequence as the lagging strand template for replication. Measurements of cell growth showed that the lag phase associated with E. coli growth was increased for cultures containing plasmids with long CTG-CAG repeats, particularly when the CTG-containing strand was the lagging template. High frequencies of deletions were observed because of a growth advantage of cells containing plasmids with deleted triplet repeats. Incubation conditions that reduced the bacterial growth-rate produced a decreased extent of deletions, presumably because they alleviated the growth advantage of cells harboring plasmids with deleted triplet repeats. The experimental observations were simulated by a model in which shorter triplet repeats provided a growth advantage due to a shorter lag phase. We demonstrate that the accumulation of deletions within repeating sequences during growth of E. coli can be prevented, and discuss these findings in relation to the studies of repetitive DNA sequences. These are the first observations to show a direct influence between a plasmid-based DNA sequence or structure and factors controlling bacterial growth.
Bowater
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Albert B. Alkek Institute of Biosciences and Technology Texas A and M University Center for Genome Research, Department of Biochemistry and Biophysics Texas Medical Center, Houston 77030, USA.
Rosche
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WA
Jaworski
A
A
Sinden
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RR
Wells
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RD
D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000254:growth & development*; D005798:Genes, Bacterial; D030342:Genetic Diseases, Inborn / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011938:Rec A Recombinases / Q000235:genetics; D017384:Sequence Deletion; D018911:Trinucleotide Repeats
D004269:DNA, Bacterial; D011938:Rec A Recombinases