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Plant Physiology 65:305-308 (1980)
© 1980 American Society of Plant Biologists

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Articles

Transcription in Isolated Wheat Nuclei

I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY 1

Dawn Sywassink Luthe2 and Ralph S. Quatrano3

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [3H]UTP into a trichloroacetic acid-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 published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [3H]yeast 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 millimolar (NH4)2SO4, 100 millimolar KCl), 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 colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [3H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. 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.

2 Present address: Department of Biochemistry, P. O. Drawer BB, Mississippi State University, Mississippi State, Mississippi 39762.

3 To whom reprint requests should be addressed.

1 This research was supported by the Science and Education Administration of the United States Department of Agriculture under Grant 5901-0410-8-0196-0 from the Competitive Research Grants Office, and by the United States Public Health Service under Grant GM 19247.




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