Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host

Abstract

A genomic library of Physarum was constructed in the replacement vector EMBL3. Efficient propagation of the recombinant phages occurred only on the recBC-sbcB- host Escherichia coli CES200, which is deficient in the exonucleases I and V. Thirteen different recombinants with actin-related sequences were detected and 10 were purified from 90,000 plaques (the equivalent of 6 Physarum genomes) on strain CES200. Comparison of the plating efficiencies of the library and the actin-related isolates suggests that palindromic DNA sequences are responsible for the instability of Physarum DNA in E. coli. In one of these isolates, lambda PpA10, and in a 2.81-kilobase subclone of that isolate in plasmid pBR322, a deletion of 360 base pairs was detected that led to stable propagation of the recombinant DNA molecules in Rec+ E. coli. Electron microscopic analysis of the 2.81-kilobase fragment, after denaturation and self-hybridization, revealed secondary structures consistent with "foldback" structures. Restriction and DNA blot analysis of lambda PpA10 suggest that the unstable DNA segment is in close proximity to, if not part of, the previously defined actin-gene locus ardA.