Identification of mild-cold responsive element (MCRE) in the cirp gene. (A) Sequence comparison of the cirp genomic fragments. Note the presence of common octanucleotide (bold). (B) Enhancer activity of MCRE. The indicated DNA fragments (Enhancer) were placed upstream of the SV40 promoter in pCAT promoter vector. HEK293 cells were co-transfected with the constructs and plasmids expressing β-galactosidase (LacZ). Cells were maintained at 37°C or transferred to 32°C on the following day, and 36 hours later cell extracts were assayed for CAT level and LacZ activity. Values are expressed as CAT protein level normalized to LacZ activity, and the means ± SE of three determinations are shown. a.u., arbitrary unit. *, CAT level normalized to LacZ at 32°C divided by that at 37°C. MCREx3, 3 copies of MCRE flanked on its 5′ and 3′ sides by T and G, respectively, and joined together. (C) Activities of mutant MCRE. Three copies of the indicated octanucleotide (Element) each with a mutation (bold) were assayed for the activity as in (B). **, normalized CAT level at 32°C divided by that at 37°C, and expressed as relative to the value obtained with pCAT promoter vector having no enhancer (none). The means ± SE of results from two independent transfections are shown.