A DISCONTINUOUS HEADFUL PACKAGING MODEL FOR PACKAGING LESS-THAN HEADFUL LENGTH DNA-MOLECULES BY BACTERIOPHAGE-T4

Bacteriophage T4 and other double-stranded DNA-containing bacteriophag es package DNA by the classical headful packaging mechanism. In this m echanism, the packaging machinery cuts a DNA concatemer and packages a single unit length genome within the viral capsid. The length of the packaged DNA molecule is determined by the size of the viral capsid. S urprisingly, during large DNA cloning experiments, we observed that th e in vitro phage T4 packaging system can package and transduce DNA mol ecules that are much smaller than the T4 headful size. We analyzed thi s phenomenon by using defined plasmid DNAs as substrates for in vitro packaging. The data showed that phage T4 can successfully package and transduce 4 to 29 kb plasmid DNA molecules. When two plasmid DNAs with different antibiotic markers were added to the packaging reaction mix ture, transductants that are resistant to both the antibiotics were ob tained, suggesting that both the plasmid DNAs are packaged within the same head. Analysis of the transducing particles by equilibrium CsCl d ensity-gradient centrifugation showed that the particles have the same density as the wild-type phage. That the less than headful length mol ecules were not converted to T4 headful length prior to packaging was established by a number of independent approaches. Finally, unit lengt h plasmid DNA molecules of appropriate size were isolated from the in vitro packaged particles. Based on these data, we propose a discontinu ous headful packaging model for packaging less than headful length mol ecules. In this model, the packaging machinery packages the first avai lable less than headful length DNA molecule and generates a partially full head. The partially full head then reinitiates packaging on a sec ond DNA molecule. This process continues until the head is filled with DNA. (C) 1996 Academic Press Limited