Spd control of reporter gene expression through the 5′ UTR of spe2. (A) The lacZ reporter constructs, incorporating the 5′ UTRs of spe2, aru1, srm1, and sam1. (B) β-gal expression in the presence of 0, 10, and 1000 µM of Spd with (±) thiamine in Δspe2 cells transformed by empty reporter vector or the spe2 5′ UTR reporter plasmid. Statistical significance was calculated compared to controls using Student's t-test, (**) P < 0.01. The error bars are the standard deviation of three independent experiments. (C) β-gal expression measured in response to increasing doses of Spd without thiamine in Δspe2 cells that were transformed with the reporter plasmid containing the 5′ UTRs, as listed in A. Statistical significance was calculated compared to other controls using Student's t-test, (**) P < 0.01. The error bars are the standard deviation of three independent experiments. (D) The GFP reporter constructs with the 5′ UTRs of spe2. (E) Reporter activity of Δspe2 cells transformed with the spe2 5′ UTR GFP reporter plasmid, in response to 10 µM of Spd with (+) and without (−) thiamine (thi). GFP was detected by western blot. GFP expression is enhanced approximately fivefold by adding 10 µM of Spd, relative to no Spd normalized against actin.
