FIG. 2.
Construction of the ToledoΔ15kb clone. (A) The 15-kb fragment containing ORFs UL136 through UL150 (in the dashed-line box) was deleted from ToledoBAC and replaced by a zeocin resistance gene to generate ToledoΔ15kb BAC. (B) The parental ToledoBAC (B lanes) and ToledoΔ15kb (Δ lanes) clones were confirmed by PCR analysis. The specific ORFs (indicated above the lane designations) in the 15-kb region were amplified, and the PCR products were observed on an agarose gel. (C) The ToledoBAC (lane B), ToledoΔ15kb (lane Δ), and Toledo (lane T) strains were digested with EcoRI and run on an agarose gel. Southern blots were performed using 32P-labeled probes for UL56, UL147, and the zeocin resistance gene (Zeocin). (D) HFF cells were infected with the ToledoBAC and ToledoΔ15kb viruses. Total RNAs were isolated from the infected cells and used to synthesize cDNAs. ToledoBAC-infected cDNA was labeled with Cy3 (green), and ToledoΔ15kb-infected cDNA was labeled with Cy5 (red). Both cDNAs were mixed and used for hybridization in an HCMV microarray. A partial microarray image shows the levels of expression of the ORFs in the 15-kb region from both viruses. Yellow signals indicate that the RNAs are detected from both pools of virally infected cells, and green signals indicate that the RNAs were detected from ToledoBAC transcripts and not from ToledoΔ15kb.