The orderly, in vitro emergence of DNA from bacteriophage φ29 particles

Abstract

φ29 DNA-containing 12−13− particles (produced by infecting nonsuppressor hosts of Bacillus subtilis with phage containing suppressible mutations in cistrons 12 and 13) can be complemented with lysates containing proteins p12* and p13 to yield infectious phage. Complementation of these particles with lysates containing p12* but not p13 or complementation with purified p12* in the absence of p13 produces a structure (called complex) which has a markedly different organization. Electron microscopy and sedimentation analysis after digestion with DNase I or proteinase K indicate that complex is composed of an intact phage head with a genome-sized linear DNA molecule attached at the collar-tail region. EcoRI digestion establishes that the DNA molecule has a unique orientation. Gel electrophoresis indicates that p12*, the neck appendage protein, is transferred to the particles when complex is formed. Complex can also be produced by incubation of 12−13− particles at 42°, by incubation at pH 6.0, or by incubation in the presence of 20 mM EDTA. Complex is also formed from DNA-containing 12− particles but to a lesser extent.