A recB recC sbcB recJ host prevents recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli.
Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.
Ishiura
M
M
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Hazumi
N
N
Koide
T
T
Uchida
T
T
Okada
Y
Y
D001426:Bacterial Proteins / Q000235:genetics*; D002872:Chromosome Deletion; D003360:Cosmids; D004274:DNA, Recombinant / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005838:Genotype; D011938:Rec A Recombinases / Q000235:genetics*; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011938:Rec A Recombinases