Transcription in Isolated Wheat Nuclei

Nuclei isolated from embryos of wheat (var. Yamil) incorporated I3HIUTP into a trichioroacetic acki-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously publsed reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [IH1yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 mililmlar (NH.)2SO4, 100 millilar KCO), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloiddal silica suspension (Percoll). These nuclei were elpsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with I'HIyeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activty. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro. The mechanism by which gene transcription and other nuclear events are controlled by factors in the cytoplasm and outside the cell are poorly understood. One approach to the study of specific interactions between nucleus and cytoplasm requires the isolation of active nuclei capable of initiating and completing the synthesis of intact RNA molecules. In such an in vitro transcription system, the effect of cytoplasmic components on gene expression can be ascertained. Although RNA synthesis has been studied in isolated plant nuclei by measuring the incorporation of precursors into an undefined product, there have been few reports on the size characterization of the RNA synthesis in vitro (7, 15, 17). In a recent paper (15), RNA synthesized by maize nuclei was heterodisperse with little RNA synthesized in size classes greater than 18S. In our preliminary studies on the characterization of RNA synthesized in isolated wheat nuclei, we did not detect the synthesis of RNA greater than 20S. One possible cause of our inability to detect specific high mol wt classes of RNA in plant nuclei is the 1 This research was supported by the Science and Education Administration of the United States Department of Agriculture under Grant 59010410-8-0196-0 from the Competitive Research Grants Office, and by the United States Public Health Service under Grant GM 19247. 'Present address: Department of Biochemistry, P.O. Drawer BB, Mississippi State University, Mississippi State, Mississippi 39762. 'To whom reprint requests should be addressed. presence ofendogenous nucleases. This report verifies that isolated wheat nuclei contain active RNases. An assay is described to detect the presence ofRNase and a method is outlined to eliminate RNase contamination in isolated wheat nuclei so that large mol wt RNA can be recovered. MATERIALS AND METHODS Chemicals. Ultrapure sucrose and SDS were purchased from Bio-Rad, Percoll from Pharmacia, and the nylon meshes from Henry Simon Ltd., Cheshire, England. Isotopes were obtained from New England Nuclear and all other biochemicals were purchased from Sigma. Solutions were filter-sterilized or autoclaved, and glassware was heated in 1% (w/v) sodium lauryl sulfate for 15 min at 60 C and rinsed with sterile distilled H20 prior to use. Preparation of Plant Material. Wheat embryos were mechanically isolated from dry, mature wheat seeds (Triticum aestivum L., var. Yamhill) using a combination of blending and sieving followed by separation on a sucrose gradient (unpublished results). The whole, intact embryo fraction was imbibed in germination medium (3) containing penicillin G (200,ug/ml) and streptomycin sulfate (100 fig/ml) for 3 h at 27 C. Isolation of Nuclei. Three methods were used to isolate nuclei from purified wheat embryos. All procedures were done at 4 C. In the first method (method I) wheat embryos were homogenized (tissue to buffer, 1:2) for 30 s in a chilled mortar and pestle in a modified Honda buffer (5) containing 0.44 M sucrose, 2.5% (w/v) Ficoll (mol wt 400,000), 5.0o (w/v) Dextran 40, 25 mm Tris-HCl (pH 7.6), 10 mm MgCl2, 10 mLm /-mercaptoethanol, and 0.5% (v/ v) Triton X-100. After homogenization, 5 volumes of buffer were added and the homogenate was sequentially filtered through four layers of cheesecloth, one layer of Miracloth, and two nylon meshes (80 and 61 ,um). The filtrate was centrifuged at 5,850g for 5 min and the supernatant was discarded. The nuclear pellet was gently suspended in NRB4 containing 50 mm Tris-HCl (pH 7.8), 5 mm MgCl2, 10 mm, -mercaptoethanol and 20%1o glycerol. These nuclei will be referred to as the crude nuclear pellet or nuclei prepared by method I. The second nuclear isolation procedure (method II) was identical to the first with three exceptions: (a) nuclei were homogenized in Honda containing 2 mM spermine (8); (b) the nuclear pellet was dissolved in Honda without spermine; and (c) the nuclear suspension was more extensively purified by centrifugation in a discontinuous gradient of Percoll (11). The Percoll gradient contained 5-ml layers of 40, 60, 80%o (v/v) Percoll on a 5-ml layer of 2 M sucrose cushion. The Percoll contained 0.44 M sucrose, 25 mm Tris-HCl (pH 7.5), and 10 mM MgCl2. The gradients were centrifuged at 4,080g in a Sorvall HB4 swinging bucket rotor for 30 min. Most of the nuclei banded in the 80%o Percoll, just above the 2 M sucrose cushion. They were removed with a Pasteur pipette, 4Abbreviation: NRB: nuclear resuspension buffer. 305 www.plantphysiol.org on October 23, 2 17 Published by Downloaded from Copyright © 1980 American Society of Plant Biologists. All rights reserved.