Ribonucleic Acid Turnover in Tobacco Leaves infected with Tobacco Mosaic Virus

Abstract

A LARGE number of investigations using different techniques have shown that the nucleus and the chloroplasts of infected leaf cells are implicated in the synthesis of tobacco mosaic virus (TMV). The source of materials for the synthesis of virus protein and ribonucleic acid (RNA) has been shown to be low molecular weight residues which are in metabolic sequence with carbon dioxide, ammonia and inorganic phosphate¹. Recently, however, Reddi² reported that the uridine, but not the phosphate, component of TMV–RNA was derived from the degradation of ribosomal RNA, which occurred as a result of infection. This communication describes some results of a study of the effect of TMV synthesis on the RNA content and turnover of mainly two fractions of tobacco leaves. Half-leaves of Nicotiana tabacum var. White Burley, about 20 cm long, were inoculated with TMV, the opposite leaves serving as control, except when the sampling was to continue for more than three days after inoculation when separate plants were used. Leaves were sampled by taking leaf pieces of equal wet weight for each sample in such a way that each part of the leaf was represented in each sample. Incubation of the leaf pieces in phosphorus-32 was achieved by infiltrating them with 1–5 µc./ml. of carrier-free phosphorus-32 in distilled water at pH 6.5, and leaving them at 23.5° C under illumination. The leaf tissue was ground in 25 mM tris-hydrochloric acid at pH 7.6, containing 5 mM magnesium chloride, 1 mM calcium chloride, 0.5 M sucrose, and 10 mM β-mercaptoethanol at 0°–4° C. Debris was removed by centrifugation at 100g for 30 sec. The supernatant was fractionated into a nuclear-chloroplast fraction (2,000g for 5 min), containing also starch grains and some cell wall material; a mitochondrial fraction (42,000g for 12 min), containing also cell membranes, some green material and, in the case of infected leaves, some virus; and a ribosomal fraction (140,000g for 90 min) containing ribosomes and the remaining virus. Ribosomes and virus were separated by dissolving the ribosomal pellet in 0.3 ml. tris buffer, 25 mM, at pH 7.6, containing 5 mM magnesium chloride, and centrifuging the solution at 120,000g for 70 min on a 5–20 per cent sucrose density gradient column prepared in 25 mM tris buffer containing 5 mM magnesium chloride. The column was sampled by collecting drops; the drops constituting the ribosomal zone were pooled and the concentration determined by spectrophotometry. RNA was extracted with phenol and an equal volume of 25 mM tris buffer at pH 7.6 by shaking in the cold for 10 min. The water phase was re-extracted with phenol and excess phenol was removed with two ether extractions. Ribonucleic acid was precipitated with three volumes of ethanol and three drops of 3 M sodium acetate overnight at −17° C, and the precipitate was dissolved in distilled water. Precipitation with ethanol was repeated twice. When RNA free of radioactive impurities was required, the ethanol precipitations were followed by three cycles of solution in 6 M guanidine chloride and 10 mM ethylenediamine tetraacetic acid at pH 7.2 and precipitation with 1.2 volumes ethanol in the cold. Finally, the RNA was washed with 70 per cent ethanol, defatted, and hydrolysed in 0.2 ml. 1 N potassium hydroxide at 30° C for 16 h. The hydrolysate was adjusted to pH 3.5 with perchloric acid, and the nucleotides were separated by high voltage electrophoresis in 50 mM potassium citrate buffer at pH 3.50. Radioactivity was counted using a liquid scintillator in a ‘Packard Tricarb’ spectrometer.