De zuivering en de eigenschappen van de replicatieve vorm van het RNA van cowpea-mozaïekvirus
Description:... The yield of RF was 10 micro-g per 100 g of leaf material. A modified isolation procedure was worked out for the determination of the sedimentation coefficient of the RF. To overcome breakdown due to the effect of the RNase incubation, the singlestranded RNA was precipitated in high salt. For this the RNA solution was brought to 2 M NaCl and frozen at -20° C. After slowly thawing at 4° C the precipitated singlestranded RNA was centrifuged off. The resulting supernatant, containing the doublestranded RNA, s-RNA and some contaminating single-stranded RNA, was subjected to gel filtration on a Sephadex G 200 column (2-5 x 35 cm). The peak eluting just after the void volume of the column was collected and centrifuged on a 5 to 20% linear sucrose gradient. After centrifugation the gradient was fractionated and each fraction was tested for RNase resistance. The RF sedimented with a peak at 15 S and a shoulder at 18 to 19 S. Studier's (1965) formula (S 20, w = 0.0882 x M 0.346) permits the calculation of the molecular weight of double-stranded RNA from its sedimentation coefficient. The molecular weights calculated were 2.8 x 10 6dalton and 5.0 x 10 6dalton. This suggested that there are two RFs in plants infected by CPMV: one for the middle component RNA and one for the bottom component RNA. , Electron microscopy provided independent data on the length distribution of the double-stranded RNA. Again the single-stranded RNA was precipitated from the RNA solution by treatment with high salt as described above. After gel filtration the peak eluting just after the void volume of the column was collected and subjected to equilibrium centrifugation in a Cs 2 SO 4 gradient with a starting density of 1.60 g/cm 3. After equilibrium had been reached, the material banding at a density between 1.58 g/cm 3and 1.62 g/cm 3was collected and used for electron microscopy. Electron microscopy was carried out according to the spreading method of Kleinschmidt et al. (1962). The frequency distribution of the lengths of 397 molecules showed that molecules varied in length from 0.1 micro-to 2.4 micro-. No molecules longer than 2.4 micro-were found. The relative frequency distribution of the length of the double-stranded RNA, i.e. the amount of RNA (number of molecules x length) having a certain length, indicated that a large amount of the double-stranded RNA consisted of molecules of the expected length of 1.48 micro-and 2.45 micro-respectively, which was calculated from the base translation of 3.17 Å published by Granboulan and Franklin (1966). This again suggested that both middle component RNA and bottom component RNA each induce their own replicative structure.
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