FIG. 2.

Disruption of ORF39 in the MHV-68 BAC. (A) We generated a gM-deficient MHV-68 BAC by inserting an oligonucleotide with multiple stop codons and an internal EcoRI restriction site into a PmlI restriction site midway through ORF39. (B) BAC DNA was digested with EcoRI, electrophoresed, transferred to nylon membranes, and probed with a BamHI-SacI genomic fragment as indicated in panel A. The predicted bands were 9,215 bp for the wild type (WT) and revertant (REV) and 4,343 and 4,872 bp for the gM mutant (gM⁻). (C) The same samples were digested with PmlI. Because the oligonucleotide insertion disrupted the PmlI site at genomic coordinate 56407, the predicted band for the mutant BAC was 11,420 bp, with 7,542 and 3,878 bp for the wild type and revertant.