A HIGHLY SENSITIVE SYSTEM FOR THE IN-VITRO ASSEMBLY OF BACTERIOPHAGE-PHI-29 OF BACILLUS-SUBTILIS

A sensitive system for the assay of bacteriophage phi 29 assembly in v itro was developed using 12 recombinant proteins and synthetic pRNA. T his system detected in vitro assembled infectious phages up to 10(7) p laque forming units (PFU) per milliliter without any background. phi 2 9 DNA-gp3 concentration dependence in phage assembly was found to be f irst order, while the DNA-packaging protein gp16 dependence was higher order. The requirement for specific phi 29 pRNA for phi 29 DNA packag ing was confirmed by the finding that no plaques were formed when only Escherichia coil RNAs were present. The activity of a mutant pRNA, wi th 10(5)-fold reduction in DNA packaging efficiency, was also demonstr ated. Additionally, the tail proteins were found to have dual roles, o ne acting as phage tails and the other stabilizing the DNA-gp3 filled capsids. (C) 1994 Academic Press, Inc.