Figure 3.—

Transcriptional enhancer activities of the ZRS and pZRS regions. (A) The canine ZRS or pZRS sequence with or without the PPD mutation was inserted 269 bp upstream of the luciferase gene under the control of an SV40 promoter. The full-length insert includes a 1917-bp region containing pZRS and ZRS. The asterisks represent the mutational positions. The ZRS region (1218–1917 bp) is a highly conserved region among humans, dogs, and mice in which the core ZRS (1571–1750 bp) is located. The boxes with a solid line (left) represent the sizes of inserts with or without mutations (*). The broken line is used as a reference to designate the size of the full-length insert. After transfection of P19 embryonal carcinoma cells with the appropriate constructs, luciferase activities were determined, standardized with β-galactosidase, and shown as relative values calculated by setting the activity of an empty vector (pGL2, promoter only) as one. The solid bars (right) represent expressions from the constructs with mutations. Data were obtained in triplicate. (B) Transcriptional activity of constructs containing a human ZRS with or without mutations. The constructs with mutations contain nucleotide changes for Dutch-C/G, Belgian1-A/T, Belgian2-T/C, and Cuban-G/A. Data were obtained in triplicate.