Abstract
A polyclonal antiserum was raised against highly purified RNA polymerase II from soybean embryos. Pure RNA polymerase II was fractionated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose sheets, incubated with the immune antiserum and then with iodinated protein A. Autoradiograms showed that the immune antiserum recognized all subunits of RNA polymerase II. Subunits 42, 27 and 16 kdalton were particularly reactive. Application of this transfer technique to protein extracts from soybean embryos or from cultured soybean cells allowed the identification of subunits of RNA polymerase II in the extracts. Analysis of the staining of the bands on the autoradiograms for increasing amounts of pure RNA polymerase II demonstrated that the transfer was quantitative, so that standard curves could be drawn to estimate the unknown amounts of enzyme in the extracts.
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Abbreviations
- DEAE:
-
diethylaminoethyl
- SDS:
-
sodium dodecyl sulfate
References
Buhler, J.M., Huet, J., Davies, K.E., Sentenac, A., Fromageot, P. (1980) Immunological studies of yeast nuclear RNA polymerases at the subunit level. J. Biol. Chem 255, 9949–9954
Guilfoyle, T.J. (1980) Auxin-induced deoxyribonucleic acid dependent ribonucleic acid polymerase activities in mature soybean hypocotyl. Biochemistry 19, 6112–6118
Guilfoyle, T.J., Hagen, G., Malcolm, M. (1984) Immunological studies on plant DNA-dependent RNA polymerases with antibodies raised against individual subunits. J. Biol. Chem. 259, 640–648
Huet, J., Sentenac, A., Fromageot, P. (1982) Spot-immunodetection of conserved determinants in eukaryotic RNA polymerases. Study with antibodies to yeast RNA polymerase subunits. J. Biol. Chem. 257, 2613–2618
Jendrisak, J.J., Burgess, R.R. (1975) A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II. Biochemistry 14, 4639–4645
Krämer, A., Bautz, E.K.F. (1981) Immunological relatedness of subunits of RNA polymerase II from insects and mammals. Eur. J. Biochem. 117, 449–455
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685
Link, G., Richter, G. (1975) Properties and subunit composition of RNa polymerase II from plant cell cultures. Biochim. Biophys. Acta 395, 337–346
Miassod, R., Got, C., Torreton, J.-P. (1984) Immunological estimation of changes in the absolute amounts of nuclear RNA polymerases in strictly auxin-requiring, cultured soybean cells upon addition of 2,4-dichlorophenoxyacetic acid. Planta 162, 434–440
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Van der Meer, J., Dorssers, L., Zabel, P. (1983) Antibodylinked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis. EMBO J. 2, 233–237
Weeks J.R., Coulter, D.E., Greenleaf, A.L. (1982) Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme. J. Biol. Chem. 257, 5884–5891
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Miassod, R., Got, C. An immunological approach to quantitate RNA polymerases in plant cell extracts. Planta 162, 427–433 (1984). https://doi.org/10.1007/BF00393455
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DOI: https://doi.org/10.1007/BF00393455