Plant Molecular Biology Manual by Heather Knight, Anthony J. Trewavas, Marc R. Knight (auth.),

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By Heather Knight, Anthony J. Trewavas, Marc R. Knight (auth.), Stanton B. Gelvin, Robbert A. Schilperoort (eds.)

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GUS activity staining - a powerful tool in plant molecular biology JEONG HEE LEE & FRITZ SCHOFFL Universitiit Tiibingen, Lehrstuhl fiir Allgemeine Genetik, Auf der Morgenstelle 28. 72076 Tiibingen, Germany Abbreviations: ATHSFl, A. thaliana HSFl; GUS, glucuronidase; HS, heat stress (shock); HSF, heat shock transcription factor; MU, methyl umbelliferone; MUG, 4-methyl umbelliferyl glucuronide; PAGE, polyacrylamide gel electrophoresis; RT, room temperature (25 0C); SDS, sodium dodecyl sulphate; uidA, gene encoding GUS; WT, wild type (untransformed); X-Gluc, 5-bromo-4-chloro-3indolyl glucoronide.

B Figure 1. Activity staining GUS in protein gels using the fluorescence substrate MUS (A) or X-Gluc as a substrate (B). Serial dilutions of total protein isolated from transgenic Arabidopsis plants expressing a heat-induced GUS gene (Schoff! , 1993; Prandl et aI. , 1994) were subjected to native PAGE, the gel was immediately incubated with the respective substrates and the staining was recorded by photography, in (A) following transillumination on a UV-screen. uct (MU) and a broadening of the bands.

Upon hs two additional intermediate size complexes of 340 kD and 440 kD appear and the monomeric bands disappear almost entirely. , 1995). The two hs-dependent heterotrimers are composed from one or two molecules of not staining ATHSFI (running as a 80 kD protein in SDS) and one or two molecules HSF-GUS or GUS-HSF (each one running as 180 kD protein in SDS). , 1995). The detection of HSF-GUS protein in DNA retardation bands exemplified another novel and very powerful application of the new technique.

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