A discontinuous headful packaging model for packaging less than headful length DNA molecules by bacteriophage T4.
Bacteriophage T4 and other double-stranded DNA-containing bacteriophages package DNA by the classical headful packaging mechanism. In this mechanism, 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. Surprisingly, during large DNA cloning experiments, we observed that the in vitro phage T4 packaging system can package and transduce DNA molecules that are much smaller than the T4 headful size. We analyzed this 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 mixture, transductants that are resistant to both the antibiotics were obtained, suggesting that both the plasmid DNAs are packaged within the same head. Analysis of the transducing particles by equilibrium CsCl density-gradient centrifugation showed that the particles have the same density as the wild-type phage. That the less than headful length molecules were not converted to T4 headful length prior to packaging was established by a number of independent approaches. Finally, unit length plasmid DNA molecules of appropriate size were isolated from the in vitro packaged particles. Based on these data, we propose a discontinuous headful packaging model for packaging less than headful length molecules. In this model, the packaging machinery packages the first available less than headful length DNA molecule and generates a partially full head. The partially full head then reinitiates packaging on a second DNA molecule. This process continues until the head is filled with DNA.
Leffers
G
G
Department of Biology, Catholic University of America, Washington, DC 20064, USA.
Rao
V B
VB
D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004261:DNA Replication; D004274:DNA, Recombinant / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C030894:terminase