FIG. 1.
HCMV genome and structure of UL128-to-UL150 mutant TRΔ4. (A, lines 1 and 2) Schematic diagrams of the HCMV AD169 and TR genomes. Indicated are the unique long (UL) and unique short (US) segments and inverted terminal and internal repeats (gray and black rectangles). The open rectangle represents the UL128-to-UL150 genes. AD169 lacks most of the UL128-to-UL150 genes and has mutations in others (35). (Line 3) The UL128-to-UL150 region of the TR genome. Hatched rectangles represent 60-bp sequences used to mediate homologous recombination with TR-BAC sequences. The positions of EcoRI restriction sites (Ec) and the predicted fragment sizes (10 kb and 22.3 kb) are indicated. (Line 4) Schematic diagram of UL128-to-UL150 genes in TRΔ4 replaced with LacZ/Ampr sequences (wide horizontal black rectangle). (B) Southern blot analysis of viral DNA. Eight ampicillin-resistant BAC clones were screened for the replacement of UL128-to-UL150 genes with LacZ/Ampr sequences. DNA was digested with EcoRI and subjected to electrophoresis and Southern blot analysis with a probe directed against Ampr gene sequences. W.T., parental (wild type) TR. A 15-kb fragment was expected for recombinants in which UL128 to UL150 were replaced with LacZ/Ampr sequences.