De zuivering en de eigenschappen van de replicatieve vorm van het RNA van cowpea-mozaiekvirus
Purified infectious preparations of cowpea mosaic virus (CPMV) consist of three centrifugal components with sedimentation coefficients of 58, 95 and 115 S . These are referred to as top (T), middle (M) and bottom (B) component and contain 0, 24 and 33% RNA respectively (Van Kammen, 1967). All three components are isometric particles with a diameter of 28 mp and have serologically similar capsids. Van Kammen (1968) demonstrated that both RNA- containing components of the virus are necessary for infection.This thesis deals with the isolation and properties of the replicative form (RF) of CPMV-RNA. Some properties of the virus and its RNA are studied to provide reference values for the isolation of the RF.After the virus had been separated into its components by means of centrifugation in a zonal rotor, the buoyant densities of middle and bottom component were determined in a CsCl gradient. They proved to be 1.412 g/cm 3and 1.469 g/cm 3respectively. From both middle and bottom component the RNA was extracted with phenol. This RNA was homogeneous in the analytical ultracentrifuge and had sedimentation coefficients of 26.4 S and 33.5 S respectively. By applying Spirin's (1963) relationship between the sedimentation coefficient of an RNA and its molecular weight (M = 1550 x S 2.1) molecular weights were calculated as 1.5 x 10 6dalton and 2.5 x 10 6dalton for middle and bottom component RNA. In a Cs 2 SO 4 gradient the mixture of both RNAs showed only one band with a mean density of 1.628 g/cm 3.The RF was isolated from CPMV-infected primary leaves of Vigna unguiculata . After homogenization of the leaves the fraction sedimenting at 15,000 x g was used for extraction of the RNA. This fraction consisting mainly of chloroplasts, membranes, nuclear fragments and nucleoli was suspended in a small volume of a buffer solution composed of 0.1 M glycine + NaOH pH 9.5, 0.1 M NaCl, 0.005 M Na 3 EDTA, 1% sodiumdodecylsulphate and 3% diethylpyrocarbonate. This mixture was deproteinized by three extractions with phenol. The nucleic acid was precipitated as the quaternary ammonium salt by adding 0.33% cetyltrimethylammoniumbromide. The precipitate was collected by centrifugation and by repeated washing with 0.1 M sodiumacetate in 70% alcohol converted into the sodium salt. This treatment removed even small traces of protein from the nucleic acid preparation. DNA was broken down by DNase and the single-stranded RNA was hydrolyzed by incubation with RNase A and RNase T 1 .Subsequently the solution was passed through a Sephadex G 200 column (2.5 x 35 em) to separate the breakdown products from the remaining high molecular weight material. The sharp frontrunning peak was collected. The nucleic acid in this peak proved to have the properties expected for the RF of CPMV- RNA. It was RNase resistent in 1 x SSC. It showed a sharp helix-coil transition curve in 1 x SSC with a T m of 94° C as measured bij the temperature dependent RNase resistance. Its buoyant density proved to be 1.60 g/cm 3.The yield of RF was 10 μg 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 μto 2.4 μ. No molecules longer than 2.4 μwere 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 μand 2.45 μrespectively, 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.The double-stranded RNA has been used in molecular hybridization experiments with viral RNA and RNA of the separate components. A saturation curve was determined to gain information on the amount of single-stranded 32P-labeled RNA necessary to replace the homologous RNA completely from the double-stranded structure. Two micrograins of double-stranded RNA were heated for 20 minutes in a closed tube with increasing amounts of 32P-labeled CPMV-RNA. to 105° C to get separation of the strands of the double-stranded structure. Thereafter the tube with the hybridisation mixture was kept at 70° C for 2 hours to obtain annealing of the complementary strands. The tube was cooled on ice, opened and the mixture was incubated with 100 μg RNase A and 150 U RNase T 1 to break down the single-stranded RNA. The RNase-resistent RNA was precipitated with TCA and collected on millipore filters. The amount of annealed exogeneous RNA was determined from the radioactivity of the RNase- resistent RNA. With 2 μg double-stranded RNA, saturation was reached at 50 pg of virus-RNA.The hybridization product was subjected to equilibrium centrifugation in a Cs 2 SO 4 gradient and was shown to have a density of 1.58 g/cm 3. This proved that a true hybrid was formed during the annealing process.Hybridization was also carried out with separate middle and bottom component RNAs. The preliminary results show that with middle component RNA alone 85% of the maximum hybridization level was reached, and with bottom component RNA alone 14%. Turnip yellow mosaic virus RNA did not give any hybridization. These results suggest that M-RNA and B-RNA hybridized independently with the minus strands of the double-stranded structures, which indicates that no overlap occurs in base sequence between M-RNA and B-RNA. This means that M-RNA and B-RNA do not have any genes in common.The final conclusion is that the cowpea mosaic virus genome consists of two different pieces of RNA, both carrying a different part of the genetic information of the virus and both inducing their own replicative structure in the host cell.
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| Format: | Doctoral thesis biblioteca |
| Language: | Dutch |
| Subjects: | biological properties, cowpea mosaic virus, cowpeas, plant diseases, plant viruses, purification, rna, vigna, biologische eigenschappen, koebonenmozaïekvirus, plantenvirussen, plantenziekten, vignabonen, zuiveren, |
| Online Access: | https://research.wur.nl/en/publications/de-zuivering-en-de-eigenschappen-van-de-replicatieve-vorm-van-het |
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