An electrophoretic analysis of tobacco mosaic virus biosynthesis.

Abstract 1. 1. A detailed electrophoretic analysis of the time-course of tobacco mosaic virus biosynthesis in nutrient-cultured tobacco leaf blade tissue is described. The mobility of the TMV formed (av.: −9.7 × 10 −5 cm 2 /v./sec.) is fairly constant during the time-course, and agrees with the mobility of the inoculum. 2. 2. Calculations of the concentration of TMV from the areas of scanning peaks of the electrophoretic patterns give results comparable with independent determination of TMV concentration by microanalysis. Tobacco mosaic virus appears at 72 hr. after inoculation, increases in concentration until 200 hr., and remains constant thereafter. 3. 3. A new electrophoretic component, B , appears in infected tissue when the TMV content is about one-third maximum. It increases in concentration thereafter in parallel with TMV. The properties of component B are consistent with those of a protein. It has an average mobility of −3.4 × 10 −5 cm. 2 /v./sec., is soluble between pH 7.0 and 4.6, and does not appear to have a high molecular weight. 4. 4. Both uninfected and infected leaf tissue contain a heterogeneous electrophoretic component, ` A , which appears to be variable in composition, and has an average mobility of −6.9 × 10 −5 cm. 2 /v./sec. These proteins precipitate between pH 5.5 and 4.6 and appear to account for the major part of extractable nonvirus protein. The concentration of group A calculated from the scanning peak areas is variable with time, indicating that the proteins, in contrast with TMV and component B , are readily metabolized. TMV biosynthesis in infected leaf tissue is associated with the development of a transitory excess in the concentration of group A as compared with uninfected controls. 5. 5. Most of the group A proteins are of low molecular weight, but in all samples this group also includes a high-molecular-weight component of S 20 = 19–23. The concentration of the high-molecular-weight component is proportional to that of group A and to that of the total protein content of leaf extracts. 6. 6. Low concentrations of thiouracil block TMV biosynthesis. The TMV formed in partially inhibitory concentrations of thiouracil has identical mobility with TMV formed normally. In 0.0001 M thiouracil, leaf tissue forms 40% of the TMV concurrently present in untreated tissue, but component B does not appear. The excess in group A associated with TMV formation is greater in thiouracil-inhibited tissue than in untreated tissue. In 0.001 M thiouracil, no TMV is formed, and the group A concentration is at a minimum. In higher concentrations of thiouracil the group A concentration in infected tissue approaches that of the uninfected controls. 7. 7. Thiouracil alters the concentration of group A in uninfected tissue. The concentration is at a minimum in about 0.001 M thiouracil, but in higher concentrations of the inhibitor is above that of untreated tissue. It is likely that the inhibitory effect of thiouracil on TMV biosynthesis is associated with its effect on the group A proteins. 8. 8. The effect of thiouracil on group A was confirmed by pH fractionation of tobacco leaf tissue extracts. 9. 9. The possibility that component B represents a TMV precursor, and that some part of the group A complex is involved in TMV biosynthesis, is discussed. The data available at present do not permit final conclusions on these questions.