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Genetic recombination of bacteriophage T7 DNA in vitro.

We have demonstrated recombination of bacteriophage T7 DNA in vitro. An extract of Escherichia coli B cells infected with wild-type T7(T7+) is incubated with mature DNA extracted from T7 phage. Packaging of the exogenous DNA within the phage head appears to be preceded by recombination of exogenously added DNA with DNA present in the extracts. In order to detect the recombination, we used an exogenous DNA bearing a marker (ss-) such that progeny phage which have packaged this marker are able to plate on Shigella sonnei D2 571-48, whereas T7+ phage present in the extracts do not. The recombinational process bears many of the characteristics of in vivo recombination. The exogenous DNA is not packaged intact but undergoes fragmentation to a length of about 3000 base pairs before being incorporated into a mature DNA molecule. If ss- DNA bearing an amber mutation is used in the assay, the frequency of amber+ progeny produced varies with the distance of the amber marker from the ss- marker. When DNA bearing three mutations is used in the reaction, phage heterozygous for the unselected marker are readily detected. Finally the products of phage genes 4 (DNA replication protein), 5(DNA polymerase), and 6(exonuclease), genes previously implicated in recombination in vivo, are required for the in vitro reaction.