FIG. 3.

The selected UL26 activation domain is active both from a promoter-proximal position and from a remote enhancer position. HeLa cells were transfected with 10 μg of β-globin reporter plasmids, 1 μg of expression vectors encoding the GAL4 fusion proteins, and 1 μg of the reference plasmid OVEC-Ref. β-Globin RNA transcribed from the reporter plasmids was isolated from HeLa cells, hybridized to a 93-nt S1 probe covering position −18 to position +75 of a noncoding region of the β-globin reporter gene and mapped by using S1 nuclease. “βinit” indicates the correctly initiated transcripts of the reporter gene, and “ref” indicates transcripts of the reference plasmid OVEC-Ref, which was cotransfected as a control for transfection variation. Lanes: 1 and 11, S1 nuclease probe; 2 and 12, molecular mass markers (sizes [in nucleotides] are indicated on the left); 3 to 6, transfection with a β-globin reporter plasmid containing five GAL4-binding sites upstream of a minimal β-globin promoter (promoter proximal position); 7 to 10, transfection with a β-globin reporter plasmid containing five GAL4-binding sites downstream of the β-globin gene (remote position); 13 to 15, transfection with a β-globin reporter plasmid without GAL4-binding sites. In lanes 3, 7, and 13, cotransfection was performed with a vector expressing the GAL4 DNA-binding domain. In lanes 4, 8, and 14, cotransfection was performed with plasmid pHM275, encoding a GAL4-UL26 fusion protein. In lanes 5, 9, and 15, cotransfection was performed with plasmid pHM274, encoding a fusion of exon 3 sequences of IE2 to GAL4. In lanes 6, 10, and 16, cotransfection was performed with a plasmid encoding a fusion of GAL4 to the herpes simplex virus type 1 VP16 activation domain, which served as a positive control.