Enhancement of Escherichia coli plasmid and chromosomal recombination by the Ref function of bacteriophage P1.

The Ref activity of phage P1 enhances recombination between two defective lacZ genes in the Escherichia coli chromosome (lac- x lac- recombination). Plasmid recombination, both lac- x lac- and tet- x tet-, was measured by transformation of recA strains, and was also assayed by measurement of beta-galactosidase. The intracellular presence of recombinant plasmids was verified directly by Southern blotting. Ref stimulated recombination of plasmids in rec+ and rec(BCD) cells by 3-6-fold, and also the low level plasmid recombination in recF cells. RecA-independent plasmid recombination, either very low level (recA cells) or high level (recB recC sbcA recA cells), was not stimulated. Ref stimulated both intramolecular and intermolecular plasmid recombination. Both normal and Ref-stimulated lac- x lac- chromosomal recombination, expected to be mostly RecBC-dependent in wild-type bacteria, were affected very little by a recF mutation. We have previously reported Ref stimulation of lac- x lac- recombination in recBC sbcB bacteria, a process known to be RecF-dependent. Chromosomal recombination processes thought to involve activated recombination substrates, e.g., Hfr conjugation, P1 transduction, were not elevated by Ref activity. We hypothesize that Ref acts by unknown mechanisms to activate plasmid and chromosomal DNA for RecA-mediated recombination, and that the structures formed are substrates for both RecF-dependent (plasmid, chromosomal) and Rec(BCD)-dependent (chromosomal) recombination pathways.

Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.

It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.