| Abstract: | An earlier technique for insertional mutagenesis of large viral genomes involved the insertion of the thymidine kinase (TK) gene at a specific target site, cotransfection of the fragment carrying the insertion with the intact viral genome, and selection of the progeny for viral recombinants expressing the TK gene. The inserted TK gene could then be replaced by cotransfection of the recombinant DNA with fragments carrying a foreign sequence or a deletion in the target sequence. To enable the probing of larger target domains and facilitate insertional mutagenesis, we extended this technique by insertion of a 2.2-kilobase-pair (kbp) herpes simplex virus 1 (HSV-1) chimeric alpha TK gene into the 7.5-kbp mini-Mu-phage (alpha TK-mini-Mu) and lysogenized Escherichia coli with the helper Mu phage and the alpha TK-mini-Mu. Induction of phage multiplication of the lysogenized E. coli after transformation with plasmids carrying HSV-1 DNA and subsequent infection of E. coli RecA+ lysogenized with Mu phage yielded plasmid populations carrying randomly inserted alpha TK-mini-Mu DNA. Application of this procedure for insertional mutagenesis of the BamHI B fragment, which spans the junction between the unique and reiterated sequences of the L component of viral DNA, yielded two types of recombinants. Viral recombinant designated RBMu1 contained the intact alpha TK-mini-Mu inserted into unique sequences of BamHI B fragment. In recombinant RBMu2, the alpha TK-mini-Mu was inserted at or in the repeated sequences, but approximately equal to 14 kbp comprising most of the internal reiterations of the viral genome in the prototype arrangement were deleted. |
