FIG. 1.

Construction of 73.KAN, 73.STOP, and 73.MR viruses. (A) γHV68-BAC (1) was used to generate the 73.KAN, 73.STOP, and 73.MR mutants. The WT BAC was mutagenized by RecA-mediated recombination to include a kanamycin resistance cassette in ORF 73. The Kan cassette was replaced by allelic exchange with the ORF 73 coding sequence containing a translation stop codon 30 amino acids into the coding frame. In addition, immediately following the introduced stop codon an extra base was added, resulting in a reading frameshift. The sum of these two mutations generates a unique XbaI site in ORF 73. To control for spurious mutations generated during insertion of the Kan cassette, 73.MR was created by replacing the Kan cassette in ORF 73 in the 73.KAN virus with the WT ORF 73 sequence. (B) Southern blot analyses of 73.STOP, 73.MR, and WT virus. Insertion of the STOP/XbaI cassette resulted in a unique band of 1,708 bp in the 73.STOP lane. The rescue of the kanamycin resistance cassette insertion is demonstrated by comparison of the PstI digests of 73.STOP and 73.MR. The probe contains sequence from genomic coordinates bp 101,654 to 105,377. B, BamHI; P, PstI; and X, XbaI.