Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus laevis. Isolation and partial characterization.

The relative levels of multiple RNA polymerases were determined in soybean (Glycine max L. var. Wayne) hypocotyl during various stages of development. The meristematic region of the hypocotyl contains more total polymerase activity per gram fresh weight and a greater proportion of polymerase I relative to II than the differentiated regions. The fully elongated tissue comprising the lower half of the hypocotyl contains mainly RNA polymerase II. The hook region contains a polymerase activity peak which is completely sensitive to a-amanitin and partially sensitive to rifamycin SV. This peak is not detectable in other regions of the hypocotyl. Polymerase I is reproducibly separated into a major and a minor component, both being resistant to a-amanitin. The two components elute at salt concentrations of 0.2 M and 0.23 M KCI, respectively, while the a-amanitin-sensitive polymerase (II) elutes at 0.3 M KCI. The polymerase activity peak which is detectable only in the hook region elutes at approximately 0.5 M KCI. Polymerase levels were also determined in water-stressed tissue and in tissue which was harvested after three days of growth instead of the usual four days. Studies on multiple RNA polymerases in soybean hypocotyl revealed occasional unpredictable changes in total activity, as well as in the relative proportions of the multiple enzymes (8). Because such changes may be related to changes in developmental processes, we determined the levels of multiple RNA polymerases in tissues representing different developmental stages during hypocotyl growth. In particular, we determined the relative levels of the multiple enzymes in different regions of the hypocotyl and in tissue that was partially water stressed. Making serial sections of the hypocotyl provides tissue of various developmental stages for the determination of enzyme levels as a function of maturation. This approach has led to several observations concerning the relative levels of multiple RNA polymerases during development. MATERIALS AND METHODS Chemicals. Nucleoside triphosphates, calf thymus DNA, dithiothreitol, 2-mercaptoethanol, and EDTA were all purchased 1 This work was supported by a Grant 5 F02 AM53070-02 from National Institutes of Health to P.J.R. and by a Grant G343458 from the National Science Foundation. Journal Paper 5651 of the Purdue Agricultural Experiment Station. from Sigma; 3H-UTP from Schwartz-Mann; DEAE-Sephadex A-25 from Pharmacia; rifamycin SV from Calbiochem; and a-amanitin from Henley and Co. (New Jersey). Plant Materials. Soybean seeds (Glycine max L. var. Wayne) were imbibed in deionized water for 6 hr and sown in moist vermiculite as described earlier (4). Chromatin Isolation. "Crude chromatin" (8) was prepared by a modification of the Huang and Bonner (6) procedure. The modification consists essentially of eliminating the centrifugation through dense sucrose, which results in a preferential loss of RNA polymerase 11 (8). Solubilization of RNA Polymerase. RNA polymerase was solubilized by the method of Hardin and Cherry (4), in which the polymerase is removed from the DNA by a 0.5-hr incubation at 37 C in the presence of 0.5 M ammonium sulfate. In addition, the post 100,OOOg supernatant containing the solubilized enzyme was passed through one layer of Miracloth. This step removes most of the yellow particulate material which fails to sediment. In all cases an equal volume of glycerol was added to the polymerase preparation and the samples were stored in liquid nitrogen. Assay of RNA Polymerase. Assay conditions were the same as described previously for column assays (8), in which the unlabeled UTP was omitted and the 'H-UTP was reduced from 22.5 Ci per assay tube. Chromatography on DEAE-Sephadex. Solubilized RNA polymerase preparations were thawed after storage in liquid nitrogen, and diluted 5-fold with TGMED (50 mM tris, pH 8, 25% glycerol (v/v), 5 mm MgCl2, 0.1 mm EDTA, 0.5 mM dithiothreitol) to reduce the (NH4)2SO, concentration to 0.05 M. The sample was pumped onto a 0.9 x 10 cm DEAE-Sephadex A-25 column previously equilibrated with TGMED buffer at a flow rate of 38 ml/hr. The column was washed with 20 ml of TGMED containing 0.15 M KCl and the enzymes were eluted with an 80-ml linear gradient ranging from 0.15 M to 0.6 M KCl in TGMED buffer (38 ml/hr). Fractions of 1.5 ml were collected and aliquots of 0.1 ml or 0.05 ml were assayed for polymerase activity as quickly as possible.