A sedimentation analysis of DNA found in Escherichia coli infected with phage λ mutants

Abstract

A study was made of the phage DNA produced in Escherichia coli infected with mutants of λ unable to produce infectious particles because of defects in genes controlling head formation. Bacteria were irradiated with UV to eliminate host DNA synthesis and then infected with various λ sus mutants under conditions where subsequent radioactive labelling appeared exclusively in phage DNA. The intracellular DNA was extracted and examined by sedimentation in neutral and alkaline sucrose gradients. Mutants in genes A, B, C, D, and E cause blocks in DNA maturation which result in the accumulation of “rapidly sedimenting” species. Mutants in genes W and F accumulate much less of the rapidly sedimenting DNA and synthesize about 50–100% of the normal amounts of mature molecules as found in control infections. Intracellular phage DNA was purified from host material by density labelling and examined by electron microscopy. The “rapidly sedimenting” species appeared to be linear concatamers with lengths up to and exceeding four units. In an A mutant circular molecules were also found; these were both of monomer and multimer length (dimers and trimers). The proportion of immature DNA found by electron microscopy (long molecules) and by sedimentation (fast sedimenting DNA) agreed very well.