Properties of Bacteriophage T4 Mutants Defective in Gene 30 (Deoxyribonucleic Acid Ligase) and the rII Gene

Abstract

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rIl double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.

Suppression of Amber Mutations of Bacteriophage T4 Gene 43 (DNA Polymerase) by Translational Ambiguity

“…At 10 min after infection, the infection mixtures were diluted 10-fold in medium containing 3H-thymidine (53 ACi of 3H per ml at a specific activity of 66.5 MCi of 3H per Ag of thymidine). Samples were withdrawn at 10-min intervals after dilution into isotope, and trichloroacetic acid-precipitable radioactivity was determined by using the methods previously described (31). gene 43 mutants, we assayed for polymerase activity in vitro.…”

The genome of bacteriophage T4.

“…generally are assumed to belong to the same functional category as the genes on which these suppressors act. Exceptions are some ligase (gene 30) defect suppressors, which appear to act by altering cell membrane properties (98). The genes listed at the far right are not known to fall into any ofthe functional categories shown.…”

Analysis of expression of the rII gene function of bacteriophage T4

“…Cells grown in M9 medium to a density of 2 x 108 ml were infected at MOI of 10 with amXC30 at 30 C. At various time intervals postinfection, 1-ml samples of infected culture were pulse-labeled with 1 UCi of "4C-labeled amino acids. Extracts of the isotopically labeled cells were then prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic assays by using the methods previously described (13). The figure shows a picture of an autoradiogram of a 7.5% gel and the results of densitometric tracings that were carried out on the rIIA and rIIB bands in the autoradiogram.…”

“…The media and methods that were used for assays of T4 DNA and protein synthesis and for characterization of T4-induced proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been described (13,14). [Methyl-3H]thymidine and l4Clabeled mixture of .-amino acids were purchased from New England Nuclear Corp., Boston, Mass.…”

“…appears that rII mutations result in decreased levels of DNase activity in infected cells and that this leads to decreased dependence on repair by ligase (13,16,23). T4 mutants that are defective in both gene 30 and the rII gene can utilize the host DNA ligase for growth (9).…”