Keiichi Tomaru

CHROMATOGRAPHY OF TOBACCO MOSAIC VIRUS, ITS CONSTITUENTS, AND NUCLEIC ACIDS EXTRACTED FROM INFECTED TOBACCO LEAF TISSUES.

Abstract Purified tobacco mosaic virus (TMV), TMV-ribonucleic acid (RNA), and aggregated TMV-protein were absorbed and eluted at specific NaCl concentrations by chromatography with methylated albumin kieselguhr columns. The A-protein, an alkaline hydrolyzate of the aggregated TMV-protein, was not absorbed. It is suggested that the specific elution of TMV and the aggregated TMV-protein were due to their quaternary structures. In the chromatography of the nucleic acids extracted from TMV-infected tobacco leaf tissues by the pyrophosphate-phenol method, infectious RNA was eluted later than ribosomal RNA as a single peak. There were no other differences between the chromatographs of nucleic acids from infected tissues and those from healthy tissues. Extraction by pyrophosphate and phenol directly from infected leaves yielded more infectious RNA than extraction by sodium dodecyl sulfate and phenol after homogenization.

Vacuum freeze-drying of Tobacco leaf tissues infected with Cucumber mosaic virus.

Abstract The preservation of cucumber mosaic virus (CMV) was studied by means of vacuum freeze-drying of infected leaf tissue. CMV was inoculated on leaves of tobacco seedlings ( Nicotiana tabacum L. var. Bright Yellow) and the plants were grown at 25°C. Eight to ten days after inoculation the systemically infected leaves were cut into pieces after removal of the midribs. They were then frozen in an acetone dry-ice bath and were vacuum-dried until the pressure was reduced to 10 −4 mm Hg. Dried tissues in both sealed and open ampules were stocked at 0–5°C in a refrigerator, the open ampules being kept in a desiccator with calcium dichloride. The activity of the virus was examined, throughout the trials, by counting local lesions on the primary leaves of cowpea on which extracts from the dried tissues were inoculated. The results obtained indicated that CMV maintained its activity fairly constantly for 425 days when preserved in this manner. CMV in dried tissue stocked in the open ampules showed slightly less activity after 26 days than that in ampules which were sealed at 10 −1 mm Hg. In extracting CMV from the tissue, incubation before maceration of the tissue for 30 minutes or 1 hour with phosphate buffer did not affect the virus activity, whereas activity was sharply reduced after more than 2 hours of incubation. More active inoculum was obtained by grinding the tissue with a mortar and pestle than by homogenizing with a homogenizer. This method preserved CMV very safely and made it practical to prepare standard inoculum of CMV for use in quantitative assays by means of local lesions.