| Abstract: | Antibodies raised against the 180-kDa subunit of cauliflower RNA polymerase II bind selectively to the largest subunit of RNA polymerase II purified from a variety of plant species. The selective binding of this antibody to the largest RNA polymerase II subunit has allowed us to probe for the size of this subunit in crude cell extracts, in fractions containing partially purified RNA polymerase II, and in isolated nuclei. Fractions containing RNA polymerase II were subjected to electrophoresis in the presence of sodium dodecyl sulfate, blotted onto nitrocellulose, and blots were probed with antibody. Immunoglobulin complexes were revealed with 125I-Protein A. Published purification procedures result in rapid conversion of a 220-kDa subunit to a 180-kDa polypeptide, but purification at high pH (pH 9.0) retards this proteolysis. RNA polymerase II associated with isolated nuclei is largely protected from proteolytic degradation, and a 240-kDa polypeptide as well as a 220-kDa polypeptide can be detected. These results suggest that the 180-kDa subunit of RNA polymerase II arises artificially during cell lysis and enzyme purification, and that even the 220-kDa polypeptide may be a degradation product of a 240-kDa polypeptide in plants. |
