Positive Autoregulation Delays the Expression Phase of Mammalian Clock Gene Per2
(A) Schematic representation of the Per1::luc and Per2::luc reporters. The Per1 promoter driving the luciferase reporter (Per1::luc) contains the 6.7-kb region upstream of the translation-initiation codon and includes five E-boxes (CACGTG), and the Per2 promoter driving the luciferase reporter (Per2::luc) contains 0.2-kb upstream of the first exon and includes two E-box like elements, E′ (CACGTT) and E* (CAGGTG). Filled boxes represent exons, and ellipses are E-boxes and E-box like elements. (B) Promoter activities of Per1::luc and Per2::luc. Per1::luc was activated 6.86±0.38 times and Per2::luc was activated 2.52±0.08 times by co-expression of CLOCK-BMAL1 with respect to their basal promoter activities, respectively. V indicates vector control and C/B indicates CLOCK and BMAL1 co-expression. Error bars indicate SEM determined from independent experiments in triplicate. (C) Initial velocities of Per1 and Per2 mRNA degradation in rat SCN-derived cultured cells. Cellular abundances of Per1 and Per2 mRNA were measured after actinomycin D treatment. The degradation slope of Per1 mRNA was −0.68 and the mRNA half-life was 44.1 min, whereas the degradation slope of Per2 mRNA was −0.60 and the mRNA half-life was 50.0 min. Error bars indicate SEM determined from independent experiments in quadruplicate, with the exception of the experiment for Per1 1 hour after treatment, which was performed in duplicate. See materials and methods for a detailed description of the experimental procedure.