DNA and RNA synthesis in leaves during TYMV replication
1. Small scale HAP column chromatography has been developed as a rapid and reproducible method for the isolation of TYMV-specific DS-RNA. 2. A procedure for the isolation of an outer chloroplast membrane fraction from healthy and diseased Chinese cabbage has been developed. This membrane system from diseased chloroplasts has been shown to contain most of the TYMV-specific DS-RNA. It is capable of incorporating 3H-UTP into DS-RNA in vitro. 3. In vivo and in vitro labelling studies, in conjunction with HAP column chromatography, have established the existence of DS-RNA in the nuclei fraction of diseased cells. No DS-RNA (as measured by chromatographic behaviour on HAP) has been found in any fraction of healthy Chinese cabbage leaf tissue. 4. The rate of total and nuclear DS-RNA synthesis is maximal before virus particles are detected. 5. The asymmetry of synthesis of viral-specific DS-RNA has been followed during the development of infection in a systemically infected leaf. Minus strand synthesis was predominant before virus particles were detected and late in the infection when virus synthesis was virtually complete. Plus strand synthesis was the predominant reaction during the period of rapid virus production. 6. DNA from the nuclei plus chloroplast fraction of healthy Chinese cabbage leaf has been fractionated into two components by HAP column chromatography: Component A-DNA which represents the bulk DNA of the cell, and component B-DNA, a minor component. 7. In healthy tissue component B-DNA appears to be metabolically distinct from component A-DNA. This distinction has been established by differential labelling rates of the two components. 8. Physical and biochemical studies indicate that component B-DNA consists of two species: (i) SS-DNA and (ii) DNA-RNA hybrid. 9. The labelling rate of component A-DNA is unchanged by TYMV synthesis. The apparent labelling rate of the two species of component B-DNA is increased by virus synthesis. 10. Detergent-solubilized nuclei plus chloroplasts from Chinese cabbage contain RNase-sensitive, actinomycin D-insensitive DNA synthesis activity. This activity is increased by TYMV infection.
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