Identity of ISRE in PLSCR1 genomic sequence.

Four putative ISRE-like elements (filled boxes) located between −4120 bp and +60 bp of the 5′ flanking region and first untranslated exon of PLSCR1 gene are depicted in linear map (top): #4 = (−3815)gaaaagaGAATcc(−3800); #3 = (−2733)acaaaaaGAAAgc(−2721); #2 = (−2519aaaaacaGAAAcc(−2497); #1 = (+21)gggaaaagGAAAccg(+35). Arrow denotes transcription initiation site. Sequence spanning these various putative ISRE-like elements were selectively deleted by PCR and the truncated PLSCR1 DNA cloned into pGL3-luciferase reporter vector as described in “Materials and methods.” Daudi cells were then cotransfected with β-galactosidase-pSV (as transfection efficiency control) and these PLSCR1-pGL3-luciferase plasmids containing the following insertions of PLSCR1 5′ genomic DNA: −4120 bp to +60 bp (spanning #1-4); −3307 bp to +60 bp (spanning #1-3); −2277 bp to +60 bp (spanning #1 only); −4120bp to +18 bp (spanning #2-4); and pGL3 vector without insert (vector). After 24 hours of transfection, either 0 (solid bars) or 1000 IU/mL (open bars) IFN-α2a was added to the cell cultures, and 18 hours later, the cells were harvested for measurement of luciferase and β-galactosidase activities (see “Materials and methods”). Bar graph reports ratio of luciferase/β-galactosidase activities measured at 18 hours. Error bars denote mean ± SEM (n = 3). Data of single experiment, representative of 3 experiments so performed. The average IFN-induced increase (mean ± SD) obtained for each reporter construct from the combined data of all 3 experiments was 3.9 ± 0.4 (insert spanning #1-4); 5.3 ± 1.0 (insert spanning #1-3); 5.2 ± 0.8 (insert spanning #1); 0.8 ± 0.1 (insert spanning #2-4); 0.8 ± 0.2 (vector control).