RecA-independent high-frequency deletion of recombinant cosmid DNA in Escherichia coli.
Segments of DNA were deleted from recombinant cosmid DNAs during propagation in Escherichia coli hosts in liquid culture. DNAs of more than 1000 cosmids propagated in various E. coli hosts were analysed by agarose gel electrophoresis (AGE). The effects of vectors, insert DNAs and host genetic characters on the formation of deletions were examined. The probability of deletion and the pattern of deletion bands observed by AGE differed from clone to clone, and after extensive culture the deletion band patterns remained almost constant during further culture. Most recombinant clones eventually showed deletion during prolonged liquid culture. Mutations in the recA gene of E. coli hosts, including a deletion mutation, did not prevent deletion. Most deletions occurred in the insert portions of cosmid DNAs. Nucleotide sequence analysis of six deletion junctions in test cosmid cMB15 demonstrated that deletions occurred between two short complete direct repeats of about 4-10 bp, irrespective of whether the cosmid was propagated in a recA host or a rec+ host. Some deletions occurred at the same sites either in a recA host or a rec+ host. These results suggest that the deletion events are mainly mediated by a recA-independent recombination system(s) of E. coli host cells.
Department of Cell Biology, National Institute for Basic Biology, Aichi, Japan.
M
M
Ishiura
N
N
Hazumi
H
H
Shinagawa
A
A
Nakata
T
T
Uchida
Y
Y
Okada
D001483:Base Sequence; D002872:Chromosome Deletion; D003360:Cosmids / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000235:genetics*
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D011938:Rec A Recombinases