Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay

Abstract

A new physical method was developed to assay genetic recombination of phage T7 in vivo. The assay utilized T7 mutants that carry unique restriction sites and was based on the detection of a new restriction fragment generated by recombination. Using this assay, we reexamined the genetic requirements for recombination of T7 DNA. Our results were in total agreement with previous findings in that recombination required the products of genes 3 (endonuclease), 4 (primase), 5 (DNA polymerase), and 6 (exonuclease). Recombination was found to be independent of DNA ligase and of DNA packaging and maturation functions. The process of genetic recombination of bac

In vitro recombination of bacteriophage T7 DNA detected by a direct physical assay

“…Rationale of recombination assay. We have previously shown the feasibility of using restriction enzyme site polymorphisms to study genetic recombination of T7 DNA in vivo (5). In this study we used the same approach to assay T7 recombination in vitro.…”

“…As determined by control experiments, this assay was sensitive enough to detect as little as 1% recombination by using only ethidium bromide staining and UV illumination. The sensitivity of this assay could be increased another 10-fold by Southern hybridization with a 32p_ labeled DNA probe (5). However, the sensitivity could not be further enhanced by using 32plabeled probes of higher specific activity or longer exposures of Southern blots due to background noise from random DNA degradation by the crude extracts.…”

“…The mixture was incubated at 30°C for 60 min, and then the reaction was stopped by adding EDTA and Sarkosyl (5) along with 100 ,ug of proteinase K per ml. After 2 h at 42°C, the DNA was extracted with phenol, precipitated with ethanol, and digested with BglI, BstNI, EcoRI, and HindIll (5). One-half of the reaction mixture was then subjected to agarose gel electrophoresis (5).…”

“…After 2 h at 42°C, the DNA was extracted with phenol, precipitated with ethanol, and digested with BglI, BstNI, EcoRI, and HindIll (5). One-half of the reaction mixture was then subjected to agarose gel electrophoresis (5). In vitro recombination promoted by T7 mutant extracts.…”

“…The agarose gels were blotted onto nitrocellulose sheets (21) and hybridized with a nick-translated DNA probe in a solution containing 0.9 M NaCl, 0.05 M NaPO4 (pH 7.0), 5 mM EDTA, 0.3% sodium dodecyl sulfate, 30 ,ug of denatured calf thymus DNA per ml, and a heat-denatured 32P-labeled DNA fragment (2 x 105 cpm/ml). Hybridization was done at 65°C for 16 h, and the filter was washed as described elsewhere (5).…”