BETA-GLUCURONIDASE ACTIVITY IN TRANSGENIC AND NONTRANSGENIC TOBACCO CELLS - SPECIFIC ELIMINATION OF PLANT INHIBITORS AND MINIMIZATION OF ENDOGENOUS GUS BACKGROUND

Bacterial beta-glucuronidase is often introduced into plants as a repo rter gene fused to constitutive or inducible promoters, However, the p resence of both endogenous inhibitors of GUS activity and endogenous G US enzymes in transgenic plants could lead to an underestimation of GU S. In this paper, a decrease of the V-m values and a greater affinity (K-m) of the GUS enzyme for its substrate (p-NPG) has been recorded wh en increasing amounts of protein from untransformed tobacco cells has been added to the pure beta-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extract s are passed through Sephadex G-25 spin columns prior to the assays. A fter such a treatment, the activity of E. coli GUS in transgenic tobac co cells (constitutive or inducible systems) was stimulated by a facto r 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method wa s also effective in suppressing the endogenous GUS or GUS-like activit y which can interfere with the activity originating from the introduce d GUS gene.