32P-Inkorporation in die Chloroplasten-RNS von Tabakblättern. Ausschluß der Beteiligung von Bakterien-Verunreinigungen

Abstract

1. The contribution of bacteria to the in vivo labelling pattern of tobacco chloroplast ribonucleic acid was investigated. It was shown that the contaminating bacteria do not markedly contribute to the incorporation of 32P into the RNA of tobacco leaves. Moreover, we tested other possibilities to eliminate eventual bacterial contaminations during RNA synthesis in leaves and in chloroplasts. 2. The RNA synthesis in non-specified bacteria isolated from the surface of tobacco leaves was inhibited nearly completely after 60 min preincubation with 50 μg Rifamycin SV per mi. In the same concentration the drug inhibited less than 10 per cent of the synthesis of total RNA and chloroplast RNA in tobacco leaves. Yet after long term (10 h) preincubation the leaves treated with high concentrations (300 μg Rif. per ml) showed a strong inhibition of RNA synthesis in chloroplasts but not in nuclei. 3. Contaminating bacteria could be quantitatively separated from the chloroplasts by sucrose gradient centrifugation. 4. Chloroplast RNA free of bacterial RNA can be prepared after treatment of a mixture of chloroplasts and bacteria with 1—3 per cent Triton X-100 and subsequent low-speed centrifugation, since the detergent solubilizes the chloroplast structures but does not affect the bacteria. 5. Processing and entire synthesis of rRNA is much more rapid in bacteria than in chloroplasts. After 60 min labelling with 32 P no ribosomal precursor RNA was found in bacteria, whereas chloroplasts had accumulated more radioactivity in the precursors than in the mature rRNA. 6. The MAK column elution pattern of bacterial RNA is different from that of chloroplast RNA. The .10 6 chloroplast rRNA and its precursor is eluted at higher NaCl concentration than the concentrations necessary for the elution of the bacterial RNA fractions. This analytical method seems suitable to distinguish between chloroplast rRNA and bacterial rRNA.