FIGURE 2.

Representative data of the time courses of GAL and GUS synthesis reactions in vitro. Progress of the synthesis reaction was detected by the hydrolysis of fluorescent substrate (TG-bgal and TG-GlcU) present in the IVTT system. GAL (A) and GUS (B) synthesis reactions with different DNA concentrations (GAL: 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, and 0 nm; GUS: 0.15, 0.12, 0.1, 0.8, 0.6, 0.5, and 0 nm). The time courses shown are those after subtracting that of no DNA. Fluorescence signals began to be seen earlier with higher DNA concentrations. Excitation and emission wavelengths used for A and B were 492/610 and 492/516 nm, respectively. C, the second derivative of the time course data (A and B) with respect to time gave the relative tetramer synthesis rate. The synthesis rate of GAL at 24 min (gray circles) and GUS at 35 min (●) was plotted against DNA concentration added. Curves fitted with Log(d²FI/dt²) = Log A + n Log[DNA] are shown, where A is a constant and n is the reaction order. We obtained n = 0.94 ± 0.05 and n = 3.7 ± 0.3 (from two independent experiments) with GAL and GUS, respectively. Note that similar trends were observed at different time points (at time points where the fluorescence signals were detectable and the effect of substrate depletion could be neglected).