Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

Abstract

We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phage T7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and lysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 30 degrees . Positive complementation results in a 100-fold increase in phage titer. Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9(-), 10(-)), within head structures present in an extract that contains no phage DNA (gene 5(-)). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5(-), 9(-), 10(-)), which complements gene 19(-)-infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.