Abstract
Here we describe an as yet neglected application of GUS fusion proteins, the highly sensitive GUS activity staining of protein bands in gels following protein electrophoresis. The protocols have been developed for the analysis of the activation of multimeric heat-shock transcription factor (HSF) complexes ofArabidopsis thaliana. Both, C- and N-terminal fusions between GUS and HSF were active in both moieties of the fusion proteins. The increased molecular masses of the GUS-tagged proteins allowed the detection of an interaction between the recombinant protein and wild-type HSF ofA. thaliana. We show that following PAGE, both fluorometric and histochemical substrates can be used with equal efficiency for the detection of GUS activities as stained bands in as little as 5 ng total protein isolated from transgenicA. thaliana plants. The same sensitivity of the activity staining was obtained using 50 pM (121 pg) of affinity-purified recombinant HSF-GUS protein isolated after expression inE. coli. Photometric detection of GUS activity of commercial GUS enzyme was approximately as sensitive as was activity staining. Further applications of GUS activity staining;e.g., for the analysis of cross linking and DNA-protein interaction, will be discussed.
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Abbreviations
- ATHSF1:
-
Arabidopsis thaliana heat-shock transcription factor 1
- GUS:
-
β-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°C)
- SDS:
-
sodium dodecyl sulphate
- uidA :
-
gene encoding GUS
- WT:
-
wild type (untransfromed)
- X-gluc:
-
5-bromo-4-chloro-3-indolyl glucuronide
References
Gallagher, S.R. 1992.GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression. Academic Press, New York.
Hübel, A., J.H. Lee, C. Wu, and F. Schöffl. 1995.Arabidopsis heat shock factor is constitutively active inDrosophila and human cells. Mol. Gen. Genet. 248:136–141.
Hübel, A. and F. Schöffl. 1994.Arabidopsis heat shock factor: Isolation and characterization of the gene and the recombinant protein. Plant Mol. Biol. 26:353–362.
Jefferson, R.A. 1987. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Reptr. 5:387–405.
Jefferson, R.A., T.A. Kavanagh, and M. W. Bevan. 1987. GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901–3907.
Lee, J.H., A. Hübel, and F. Schöffl. 1995. Derepression of the activity of genetically engineered heat shock transcription factor causes constitutive synthesis for heat shock proteins and increased thermotolerance in transgenicArabidopsis. 8:603–642.
Prändl, R., E. Kloske, and F. Schöffl. 1995. Developmental regulation and tissue-specific differences of heat shock gene expression in transgenicArabidopsis plants. Plant Mol. Biol. 28:73–82.
Raikhel, N. 1994. Nuclear targeting in plants. Plant Physiol. 100:1627–1632.
Schöffl, F., G. Schröder, M. Kliem, and M. Rieping. 1993. An SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants. Transgenic Res. 2:93–100.
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Lee, J.H., Schöffl, F. GUS activity staining in gels: A powerful tool for studying protein interactions in plants. Plant Mol Biol Rep 13, 346–354 (1995). https://doi.org/10.1007/BF02669190
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DOI: https://doi.org/10.1007/BF02669190