Detection of stop codon readthrough in stably transformed Arabidopsis plants.

(A) Reporter gene constructs introduced into the Arabidopsis nuclear genome. The strong constitutive p045 promoter (open arrow) was used to drive expression of NAN-GUS dual reporter constructs containing either the wild-type TMV leaky stop codon region (p045:TMV-TAG) or the NRStop region (p045:TMV-NRStop) in stably transformed plants. (B) Histochemical analysis of progeny of single insertion transgenic Arabidopsis lines. Each panel contains six seedlings, 3 stained for NAN activity (left half) and 3 stained for GUS activity (right half). NAN activity stains blue, GUS activity magenta. Seedlings expressing a p045:GUS control construct show, as expected, strong GUS activity but no NAN activity (top panel). Seedlings expressing the p045:TMV-NRStop construct show strong NAN activity but negligible GUS activity (bottom panel), while seedlings expressing p045:TMV-TAG show strong NAN activity and low, but significant GUS activity (middle panel). (C) GUS/NAN activity ratios of seedlings of three individual transgenic lines expressing the p045:TMV-TAG (lines 7, 10, 11) or the p045:TMV-NRStop (lines 8, 11, 12) construct. NAN and GUS activity was quantified in total protein extracts from two pools of twelve seedlings of each line.