Figure 5.

Stability of HD^–145/CAT and HD^–145(A-125T)/CAT mRNA in wheat germ translation extracts. (A) ³²P-labeled HD^–145/CAT and HD^–145(A-125T)/CAT mRNA were translated in wheat germ extracts and, at the indicated time points, an aliquot was removed (10 µl), incubated with 50 µg of Proteinase K at 37°C for 15 min, after which the sample was phenol/chloroform extracted. Following ethanol precipitation, RNA samples were fractionated on a 1.4% agarose/formaldehyde gel. The gel was stained with SYBR gold (Molecular Probes) to ensure equal recovery of rRNA from the translation extracts (data not shown), dried and exposed to X-Omat film (Kodak) at –70°C with an intensifying screen. The time at which mRNA was extracted is shown above the panel. (B) Summary of three experiments measuring the relative stability of HD^–145/CAT and HD^–145(A-125T)/CAT in wheat germ extracts. Following fractionation on 1.0% formaldehyde agarose gels, the radioactivity associated with each mRNA was quantitated on a Fujix BAS2000 with a Fuji imaging screen. For each mRNA species, the amount of radiolabel present at the 0 min time point is taken as 100%. All gels were stained with SYBR gold (Molecular Probes) to ensure equal recovery of rRNA from the translation extracts (data not shown). The nature of the mRNA species being assayed is indicated above the panel.